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Questions in Regards to Isolating Promotor Region for Recombinant DNA Techniques

Questions in Regards to Isolating Promotor Region for Recombinant DNA Techniques


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For a while now I have been trying to isolate the promotor region of this Protein.

Using sticky end PCR, I need to isolate this promotor region so it has paticular sticky ends for the purpose of placing it into a plasmid. However, I have a couple of questions on how to do this:

A. For inserting it into a plasmid, how precise does the sequence have to be? It is nearly impossible to find a restriction enzyme which lines up exactly to the start codon. How far can I deviate away, while still allowing the protein to be transcribed properly (on the plasmid)?

Thanks.


If you are performing sticky end PCR you do not need any restriction enzymes at all.

Design primers for your acceptor plasmid to 'linearize' it at the ATG and add a unique 4 nucleotide on the full length forward and another unique on on the full reverse primer. Design your promoter fragment primers to have compatible overhangs to that of the vector. And to answer your question: you will have very very poor yields if both are not perfectly complementary.

You will need to phosphorylate the full length primers for the promoter because primers are usually not phosphorylated at the 5' end. Do your PCR, anneal and ligation as per normal.

But if I were you I would switch over to Golden Gate cloning, our lab did a year ago and I have not regretted it one moment.

Edit: I have added a diagram below. There was one thing which I overlooked in my original answer: You can get a direct repeat of the overhangs when the two longest products of each annealing reaction combine in a blunt end ligation but that should not affect your expression too much. However I still think that sticky end PCR is not the best way out of this. Blunt end ligation, gibson or my favourite: Golden Gate are all much more powerful techniques


Recombinant DNA and gene isolation

Although a formidable array of approaches fall under the broad umbrella of molecular genetics, the basic technology is elegant and conceptually simple (Watson et al. 1983, Roberts et al. 1992). It has enabled a reductionist approach to be applied to the study of genes and their sequences, as well as the transcripts that underlie organ development and function. Cardiovascular research has, in the past, utilized the systemic and integrative approaches that are inherent in the study of an organ system. Molecular genetics and recombinant DNA offer the cardiologist a complementary focus, as both normal and abnormal heart functions can be defined in terms of the underlying genetic complement and its regulation. One can approach heart function in terms of defining the basic components that participate in the developmental and functional processes of the heart at different developmental times. A future challenge will be to integrate the information regarding these different components with the heart's function in an intact biological system.


9.3: Cloning and Recombinant Expression

To accomplish the applications described above, biochemists must be able to extract, manipulate, and analyze nucleic acids. To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base). The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus of eukaryotic organisms is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases.

Unlike DNA in eukaryotic cells, RNA molecules leave the nucleus. Messenger RNA (mRNA) is analyzed most frequently because it represents the protein-coding genes that are being expressed in the cell.

DNA isolation techniques have been described in section 5.1 and are the first step used to study or manipulate nucleic acids. RNA can also be extracted and is studied to understand gene expression patterns in cells. RNA is naturally very unstable because enzymes that break down RNA are commonly present in nature. Some are even secreted by our own skin and are very difficult to inactivate. During RNA extraction, RNase inhibitors and the special treatment of glassware are used to reduce the risk of destroying the sample during isolation

Gel Electrophoresis

Because nucleic acids are negatively charged ions at neutral or alkaline pH in an aqueous environment, they can be moved by an electric field. Gel electrophoresis is a technique used to separate charged molecules on the basis of size and charge. The nucleic acids can be separated as whole chromosomes or as fragments. The nucleic acids are loaded into a slot at one end of a gel matrix, an electric current is applied, and negatively charged molecules are pulled toward the opposite end of the gel (the end with the positive electrode). Smaller molecules move through the pores in the gel faster than larger molecules this difference in the rate of migration separates the fragments on the basis of size. The nucleic acids in a gel matrix are invisible until they are stained with a compound that allows them to be seen, such as a dye. Distinct fragments of nucleic acids appear as bands at specific distances from the top of the gel (the negative electrode end) that are based on their size (Figure 5.15). A mixture of many fragments of varying sizes appear as a long smear, whereas uncut genomic DNA is usually too large to run through the gel and forms a single large band at the top of the gel.

Figure 5.15 DNA Gel Electrophoresis. Shown are DNA fragments from six samples run on a gel, stained with a fluorescent dye and viewed under UV light. (credit: modification of work by James Jacob, Tompkins Cortland Community College)

Polymerase Chain Reaction (PCR)

The details of PCR are discussed in section 5.1. This technique is used in DNA cloning to rapidly increase the number of copies of specific regions of DNA.

Cloning

In general, cloning means the creation of a perfect replica. Typically, the word is used to describe the creation of a genetically identical copy. In biology, the re-creation of a whole organism is referred to as &ldquoreproductive cloning.&rdquo Long before attempts were made to clone an entire organism, researchers learned how to copy short stretches of DNA&mdasha process that is referred to as molecular cloning.

Molecular cloning allows for the creation of multiple copies of genes, expression of genes, and study of specific genes. To get the DNA fragment into a bacterial cell in a form that will be copied or expressed, the fragment is first inserted into a cloning vector.

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector therefore contains features that allow for the convenient insertion or removal of a DNA fragment to or from the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that cuts the DNA. DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined together by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

There are many types of cloning vectors, but the most commonly used ones are genetically engineered plasmids. Cloning is generally first performed using Escherichia coli, and cloning vectors in E. coli include plasmids, bacteriophages (such as phage &lambda), cosmids, and bacterial artificial chromosomes (BACs). Some DNA, however, cannot be stably maintained in E. coli, for example very large DNA fragments. For these studies, other organisms such as yeast may be used. Cloning vectors in yeast include yeast artificial chromosomes (YACs).

Figure 5.16 Example of a Common Cloning Vector.

All commonly used cloning vectors in molecular biology have key features necessary for their function, such as a suitable cloning site with restriction enzymes and a selectable marker. Others may have additional features specific to their use. For reasons of ease and convenience, cloning is often performed using E. coli. Thus, the cloning vectors used often have elements necessary for their propagation and maintenance in E. coli, such as a functional origin of replication (ori). The ColE1 origin of replication is found in many plasmids. Some vectors also include elements that allow them to be maintained in another organism in addition to E. coli, and these vectors are called shuttle vectors.

Cloning site

All cloning vectors have features that allow a gene to be conveniently inserted into the vector or removed from it. This may be a multiple cloning site (MCS) or polylinker, which contains many unique restriction sites. The restriction sites in the MCS are first cleaved by restriction enzymes, then a PCR-amplified target gene also digested with the same enzymes is ligated into the vectors using DNA ligase. The target DNA sequence can be inserted into the vector in a specific direction if so desired. The restriction sites may be further used for sub-cloning into another vector if necessary.

Other cloning vectors may use topoisomerase instead of ligase and cloning may be done more rapidly without the need for restriction digest of the vector or insert. In this TOPO cloning method a linearized vector is activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topoisomerase and forming a circular vector in the process. Another method of cloning without the use of DNA digest and ligase is by DNA recombination, for example as used in the Gateway cloning system. The gene, once cloned into the cloning vector (called entry clone in this method), may be conveniently introduced into a variety of expression vectors by recombination.

Restricition Enzymes

Restriction enzymes (also called restriction endonucleases) recognize specific DNA sequences and cut them in a predictable manner they are naturally produced by bacteria as a defense mechanism against foreign DNA.

As the name implies, restriction endonucleases (or restriction enzymes) are &ldquorestricted&rdquo in their ability to cut or digest DNA. The restriction that is useful to biochemists is usually a palindromic DNA sequence. Palindromic sequences are the same sequence forwards and backwards. Some examples of palindromes: RACE CAR, CIVIC, A MAN A PLAN A CANAL PANAMA. With respect to DNA, there are 2 strands that run antiparallelel to each other. Therefore, the reverse complement of one strand is identical to the other.

Like with a word palindrome, this means the DNA palindromic sequence reads the same forward and backward. In most cases, the sequence reads the same forward on one strand and backward on the complementary strand. REs often cut DNA into a staggered pattern. When a staggered cut is made in a sequence, the overhangs are complementary (Figure 5.17).

Figure 5.17 Restriction Enzyme Recognition Sequences. In this (a) six-nucleotide restriction enzyme recognition site, notice that the sequence of six nucleotides reads the same in the 5&prime to 3&prime direction on one strand as it does in the 5&prime to 3&prime direction on the complementary strand. This is known as a palindrome. (b) The restriction enzyme makes breaks in the DNA strands, and (c) the cut in the DNA results in &ldquosticky ends&rdquo. Another piece of DNA cut on either end by the same restriction enzyme could attach to these sticky ends and be inserted into the gap made by this cut.

Molecular biologists also tend to use these special molecular scissors that recognize palindromes of 6 or 8. By using 6-cutters or 8-cutters, the sequences occur throughout large stretches rarely, but often enough to be of utility.

EcoRI generates sticky of cohesive ends SmaI generates blunt ends

Figure 5.18 Restriction Enzymes. Restriction enzymes recognize palindromic sequences in DNA and hydrolyze covalent phosphodiester bonds of the DNA to leave either &ldquosticky/cohesive&rdquo ends or &ldquoblunt&rdquo ends. This distinction in cutting is important because an EcoRI sticky end can be used to match up a piece of DNA cut with the same enzyme in order to glue or ligate them back together. While endonucleases cut DNA, ligases join them back together. DNA digested with EcoRI can be ligated back together with another piece of DNA digested with EcoRI, but not to a piece digested with SmaI. Another blunt cutter is EcoRV with a recognition sequence of GAT | ATC.

Selectable marker

A selectable marker is carried by the vector to allow the selection of positively transformed cells. Antibiotic resistance is often used as marker, an example being the beta-lactamase gene, which confers resistance to the penicillin group of beta-lactam antibiotics like ampicillin. Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and kanamycin resistance gene. Shuttle vectors which are designed to be maintained in two different organisms may also require two selectable markers, although some selectable markers such as resistance to zeocin and hygromycin B are effective in different cell types. Auxotrophic selection markers that allow an auxotrophic organism to grow in minimal growth medium may also be used examples of these are LEU2 and URA3 which are used with their corresponding auxotrophic strains of yeast.

Another kind of selectable marker allows for the positive selection of plasmid with cloned gene. This may involve the use of a gene lethal to the host cells, such as barnase, Ccda, and the parD/parE toxins. This typically works by disrupting or removing the lethal gene during the cloning process, and unsuccessful clones where the lethal gene still remains intact would kill the host cells, therefore only successful clones are selected.

Reporter genes

Reporter genes are used in some cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified. Such features present in cloning vectors may be the lacZ&alpha fragment for &alpha complementation in blue-white selection, and/or marker gene or reporter genes in frame with and flanking the MCS to facilitate the production of fusion proteins. Examples of fusion partners that may be used for screening are the green fluorescent protein (GFP) and luciferase.

Figure 5.19 Reporter Genes. In this diagram, the green fluorescence protein is used as a reporter gene to study upstream regulatory sequences.

Elements for expression

If the expression of the targeted gene is desired, then a cloning vector also needs to contain suitable elements for the expression of the cloned target gene, including a promoter and ribosomal binding site (RBS). The target DNA may be inserted into a site that is under the control of a particular promoter necessary for the expression of the target gene in the chosen host. Where the promoter is present, the expression of the gene is preferably tightly controlled and inducible so that proteins are only produced when required. Some commonly used promoters are the T7 and lac promoters. The presence of a promoter is necessary when screening techniques such as blue-white selection are used.

Cloning vectors without promoter and RBS for the cloned DNA sequence are sometimes used, for example when cloning genes whose products are toxic to E. coli cells. Promoter and RBS for the cloned DNA sequence are also unnecessary when first making a genomic or cDNA library of clones since the cloned genes are normally subcloned into a more appropriate expression vector if their expression is required.

Types of cloning vectors

A large number of cloning vectors are available, and choosing the right vector may depend a number of factors, such as the size of the insert, copy number and cloning method. Large DNA inserts may not be stably maintained in a general cloning vector, especially for those with a high copy number, therefore cloning large fragments may require more specialized cloning vector.

Plasmids are autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the ones most commonly used. Most general plasmids may be used to clone DNA insert of up to 15 kb in size. Many plasmids have high copy number, for example pUC19 which has a copy number of 500-700 copies per cell, and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells.

Bacteriophage

The bacteriophages most commonly used for cloning are the lambda (&lambda) phage and M13 phage. There is an upper limit on the amount of DNA that can be packed into a phage (a maximum of 53 kb). The average lambda phage genome is roughly 48.5 kb (Figure 5.20). Therefore to allow foreign DNA to be inserted into phage DNA, phage cloning vectors may need to have some of their non-essential genes deleted to make room for the foreign DNA.

There is also a lower size limit for DNA that can be packed into a phage, and vector DNA that is too small cannot be properly packaged into the phage. This property can be used for selection - vector without insert may be too small, therefore only vectors with insert may be selected for propagation.

Figure 5.20 Lambda Phage. (A) Schematic representation of the circular genome of the lambda phage (B) Diagram of the Lambda Phage infectious particle and (C) Electron micrograph of the related bacteriophage, vibriophage VvAWI. The bar denotes 50 nm in length.

Cosmids are plasmids that incorporate a segment of bacteriophage &lambda DNA that has the cohesive end sites (cos) which contains elements required for packaging DNA into &lambda particles. It is normally used to clone large DNA fragments between 28 and 45 Kb.

Bacterial artificial chromosome

Insert size of up to 350 kb can be cloned in bacterial artificial chromosome (BAC). BACs are maintained in E. coli with a copy number of only 1 per cell. BACs have often been used to sequence the genome of organisms in genome projects, including the Human Genome Project. A short piece of the organism's DNA is amplified as an insert in BACs, and then sequenced. Finally, the sequenced parts are rearranged in silico, resulting in the genomic sequence of the organism. BACs have largely been replaced in this capacity with faster and less laborious sequencing methods like whole genome shotgun sequencing and now more recently next-gen sequencing.

Yeast artificial chromosome

Yeast artificial chromosome are used as vectors to clone DNA fragments of more than 1 mega base (1Mb = 1000kb = 1,000,000 bases) in size. They are useful in cloning larger DNA fragments as required in mapping genomes such as in human genome project. It contains a telomeric sequence, an autonomously replicating sequence( features required to replicate linear chromosomes in yeast cells). These vectors also contain suitable restriction sites to clone foreign DNA as well as genes to be used as selectable markers.

Human artificial chromosome

Human artificial chromosomes may be potentially useful as a gene transfer vectors for gene delivery into human cells, and a tool for expression studies and determining human chromosome function. It can carry very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have the problem of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector.

Animal and plant viral vectors that infect plant and animal cells have also been manipulated to introduce foreign genes into plant and animal cells. The natural ability of viruses to adsorb to cells , introduce their DNA and replicate have made them ideal vehicles to transfer foreign DNA into eukaryotic cells in culture. A vector based on Simian virus 40 (SV40) was used in first cloning experiment involving mammalian cells. A number of vectors based on other type of viruses like Adenoviruses and Papilloma virus have been used to clone genes in mammals. At present , retroviral vectors are popular for cloning genes in mammalian cells. In case of plant tranformation, viruses including the Cauliflower Mosaic Virus , Tobacco Mosaic Virus and Gemini Viruses have been used with limited success.

Summary of DNA Cloning

Figure 5.21 provides a summary of the basic cloning methods most widely used in biochemistry laboratories. Foreign DNA is isolated or amplified using PCR to obtain enough material for the cloning procedure. The DNA is purified and cut with restriction enzymes, and then mixed with a vector that has been cut with the same restriction enzymes. The DNA can then be stitched back together with DNA ligase. The DNA can then be transformed into a host system, often times bacteria, to grow large quantities of the plasmid containing the cloned DNA.

Restriction fragment patterning and DNA sequencing can be used to validate the cloned material.

Figure 5.21 Diagram Showing the Major Steps in Cloning.

For a Video Tutorial on DNA Cloning Visit: HHMI - BioInteractive

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they contain new combinations of genetic material. Proteins that are produced from recombinant DNA molecules are called recombinant proteins. Not all recombinant plasmids are capable of expressing genes. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins.

Reproductive Cloning

Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism. Most multicellular organisms undergo reproduction by sexual means, which involves the contribution of DNA from two individuals (parents), making it impossible to generate an identical copy or a clone of either parent. Recent advances in biotechnology have made it possible to reproductively clone mammals in the laboratory.

Natural sexual reproduction involves the union, during fertilization, of a sperm and an egg. Each of these gametes is haploid, meaning they contain one set of chromosomes in their nuclei. The resulting cell, or zygote, is then diploid and contains two sets of chromosomes. This cell divides mitotically to produce a multicellular organism. However, the union of just any two cells cannot produce a viable zygote there are components in the cytoplasm of the egg cell that are essential for the early development of the embryo during its first few cell divisions. Without these provisions, there would be no subsequent development. Therefore, to produce a new individual, both a diploid genetic complement and an egg cytoplasm are required. The approach to producing an artificially cloned individual is to take the egg cell of one individual and to remove the haploid nucleus. Then a diploid nucleus from a body cell of a second individual, the donor, is put into the egg cell. The egg is then stimulated to divide so that development proceeds. This sounds simple, but in fact it takes many attempts before each of the steps is completed successfully.

The first cloned agricultural animal was Dolly, a sheep who was born in 1996. The success rate of reproductive cloning at the time was very low. Dolly lived for six years and died of a lung tumor (Figure 5.22). There was speculation that because the cell DNA that gave rise to Dolly came from an older individual, the age of the DNA may have affected her life expectancy. Since Dolly, several species of animals (such as horses, bulls, and goats) have been successfully cloned.

There have been attempts at producing cloned human embryos as sources of embryonic stem cells. In the procedure, the DNA from an adult human is introduced into a human egg cell, which is then stimulated to divide. The technology is similar to the technology that was used to produce Dolly, but the embryo is never implanted into a surrogate mother. The cells produced are called embryonic stem cells because they have the capacity to develop into many different kinds of cells, such as muscle or nerve cells. The stem cells could be used to research and ultimately provide therapeutic applications, such as replacing damaged tissues. The benefit of cloning in this instance is that the cells used to regenerate new tissues would be a perfect match to the donor of the original DNA. For example, a leukemia patient would not require a sibling with a tissue match for a bone-marrow transplant.

Figure 5.22 Dolly the sheep was the first agricultural animal to be cloned. To create Dolly, the nucleus was removed from a donor egg cell. The enucleated egg was placed next to the other cell, then they were shocked to fuse. They were shocked again to start division. The cells were allowed to divide for several days until an early embryonic stage was reached, before being implanted in a surrogate mother.

Why was Dolly a Finn-Dorset and not a Scottish Blackface sheep?

Because even though the original cell came from a Scottish Blackface sheep and the surrogate mother was a Scottish Blackface, the DNA came from a Finn-Dorset.

Genetic Engineering

Using recombinant DNA technology to modify an organism&rsquos DNA to achieve desirable traits is called genetic engineering. Addition of foreign DNA in the form of recombinant DNA vectors that are generated by molecular cloning is the most common method of genetic engineering. An organism that receives the recombinant DNA is called a genetically modified organism (GMO). If the foreign DNA that is introduced comes from a different species, the host organism is called transgenic. Bacteria, plants, and animals have been genetically modified since the early 1970s for academic, medical, agricultural, and industrial purposes.

Watch this short video explaining how scientists create a transgenic animal.

Although the classic methods of studying the function of genes began with a given phenotype and determined the genetic basis of that phenotype, modern techniques allow researchers to start at the DNA sequence level and ask: &ldquoWhat does this gene or DNA element do?&rdquo This technique, called reverse genetics, has resulted in reversing the classical genetic methodology. One example of this method is analogous to damaging a body part to determine its function. An insect that loses a wing cannot fly, which means that the wing&rsquos function is flight. The classic genetic method compares insects that cannot fly with insects that can fly, and observes that the non-flying insects have lost wings. Similarly in a reverse genetics approach, mutating or deleting genes provides researchers with clues about gene function. Alternately, reverse genetics can be used to cause a gene to overexpress itself to determine what phenotypic effects may occur.

CRISPR Technology

CRISPR stands for clustered regularly interspaced short palindromic repeatsand represents a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.

5.23 Crystal structure of a CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target. CRISPR system Cascade protein subunits CasA, CasB, CasC, CasD, and CasE (cyan) bound to CRISPR RNA (green) and viral DNA (red) based on PDB 4QYZ and rendered with PyMOL.

Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.

Figure 5.24 Diagram of the CRISPR prokaryotic antiviral defense mechanism.

The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.


DESCRIPTION OF THE FIGURES

FIG. 1 shows the oligonucleotide fragment containing the factor Xa recognition sequence (FXRS) and an analogous fragment containing the thrombin recognition sequence, indicating the presence of restriction sites. Of particular importance is the StuI site 3′ to the Arg codon, which allows joining with the 5′ terminus of the hPTH coding sequence.

FIG. 2 shows schematically the strategy for recombinant hPTH production. The starting plasmid, pGH-L9, contains an E. coli tryptophan promoter and a nucleotide sequence coding for human GH which contains BglII and SalI restriction sites. The 35 nucleotide oligomer carrying the FXRS (see FIG. 1 ) was cloned into pGH-L9 at a BglII-SalI site. The resultant intermediate plasmid (pTGI, not shown), thus contained nucleotides coding for amino acids 1-138 of human GH linked to the FXRS sequence. This intermediate plasmid was treated with StuI to create a blunt end 3′ from the codon coding for Arg. The genetic sequence coding for hPTH was cut from the pPTHm124 plasmid with XbaI, blunt ended, and the XbaI site filled using the Klenow reagent, and the resultant sequence cloned into the pTGI intermediate plasmid. This procedure allowed the N-terminal codon of hPTH to abut the Arg codon of the FXRS fragment. This newly constructed plasmid was termed pGFP-1. The plasmid was subsequently expressed in E. coli , which produced the fusion protein containing hGH(1-138), the FXRS, and the hPTH(1-84) sequences. Cleavage with factor Xa released the intact hPTH(1-84) hormone in which the N-terminal serine residue was unmodified. The hPTH was then purified by HPLC.

FIG. 3 shows binding of recombinant hPTH(1-84) and of synthetic PTH(1-34) to opossum kidney cells.

FIG. 4 shows binding of recombinant hPTH(1-84) and of synthetic PTH(1-34) to UMR tumor cells.

FIG. 5 shows the cyclic AMP response of opossum kidney cells to stimulation with recombinant hPTH(1-84) and synthetic PTH(1-34).

FIG. 6 shows the cyclic AMP response of UMR tumor cells to stimulation with recombinant hPTH(1-84) and synthetic PTH(1-34).


AQA A LEVEL BIOLOGY - RECOMBINANT DNA TECHNOLOGY FULL SCHEME OF WORK

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Nine full lessons on the Recombinant DNA Technology topic for AQA A Level Biology. This topic is one of the most challenging due to the application and context-based nature. However, each lesson includes detailed and concise notes for some difficult to understand topics, along with relevant exam questions to help test skills, an essay prep, a practical, and fun and engaging activities to help consolidate learning.

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DNA Technology

Is it really possible to clone people? Another question is, should we clone people? Are scientific fantasies, such as depicted on TV shows such as Star Trek or in the movie GATTACA, actually a possibility? Who can really say? How, really, will science affect our future? The answers partially lie in the field of biotechnology.

Biotechnology is technology based on biological applications. These applications are increasingly used in medicine, agriculture and food science. Biotechnology combines many features of biology, including genetics, molecular biology, biochemistry, embryology, and cell biology. Many aspects of biotechnology center around DNA and its applications, otherwise known as DNA technology. There are many current applications of biotechnology however, we will focus on the applications towards medicine and the extension into the forensic sciences. First, though, we need to understand DNA technology.

DNA Technology

What is DNA technology? Is it using and manipulating DNA to help people? Is it using DNA to make better medicines and individualized treatments? Is it analyzing DNA to determine predispositions to genetic diseases? The answers to these questions, and many more, is yes. And the answers to many of these issues begin with the Human Genome Project.

The Human Genome Project

If we are all 99.9% genetically identical, what makes us different? How does that 0.1% make us tall or short, light or dark, develop cancer or not? To understand that 0.1%, we also need to understand the other 99.9%. Understanding the human genome is the goal of The Human Genome Project (HGP). This project, publicly funded by the United States Department of Energy (DOE) (Figure 1) and the National Human Genome Research Institute (NHGRI), part of the National Institutes of Health (NIH), may be one of the landmark scientific events of our lifetime.

Figure 1: The Human Genome Project logo of the DOE.

The goal of the HGP is to understand the genetic make-up of the human species by determining the DNA sequence of the human genome (Figure 2)and the genome of a few model organisms. However, it is not just determining the 3 billion bases it is understanding what they mean. Today, all 3 billion base pairs have been sequenced, and the genes in that sequence are in the process of being identified and characterized. A preliminary estimate of the number of genes in the human genome is around 22,000 to 23,000.

Figure 2: A depiction of DNA sequence analysis. Note the 4 colors utilized, each representing a separate base.

The sequence of the human DNA is stored in databases available to anyone on the Internet. The U.S. National Center for Biotechnology Information (NCBI), part of the NIH, as well as comparable organizations in Europe and Japan, maintain the genomic sequences in a database known as Genbank. Protein sequences are also maintained in this database. The sequences in these databases are the combined sequences of anonymous donors, and as such do not yet address the individual differences that make us unique. However, the known sequence does lay the foundation to identify the unique differences among all of us. Most of the currently identified variations among individuals will be single nucleotide polymorphisms, or SNPs. A SNP (pronounced ”snip”) is a DNA sequence variation occurring at a single nucleotide in the genome. For example, two sequenced DNA fragments from different individuals, GGATCTA to GGATTTA, contain a difference in a single nucleotide. If this base change occurs in a gene, the base change then results in two alleles: the C allele and the T allele. Remember an allele is an alternative form of a gene. Almost all common SNPs have only two alleles. The effect of these SNPs on protein structure and function, and any effect on the resulting phenotype, is an extensive field of study.

Gene Cloning

You probably have heard of cloning. Whereas cloning of humans has many ethical issues associated with it, the cloning of genes has been ongoing for well over 30 years, with cloning of animals occurring more recently. Gene cloning, also known as molecular cloning, refers to the process of isolating a DNA sequence of interest for the purpose of making multiple copies of it. The identical copies are clones. In 1973, Stanley Cohen and Herbert Boyer developed techniques to make recombinant DNA, a form of artificial DNA.

Recombinant DNA is engineered through the combination of two or more DNA strands, combining DNA sequences which would not normally occur together. In other words, selected DNA (or the DNA of ”interest”) is inserted into an existing organismal genome, such as a bacterial plasmid DNA or some other sort of vector. The recombinant DNA can then be inserted into another cell, such as a bacterial cell, for amplification and possibly production of the resulting protein. This process is called transformation, the genetic alteration of a cell resulting from the uptake, incorporation, and expression of foreign genetic material. Recombinant DNA technology was made possible by the discovery of restriction endonucleases.

Restriction Enzyme Digestion and Ligation

In the classical restriction enzyme digestion and ligation cloning protocols, cloning of any DNA fragment essentially involves four steps: 1. isolation of the DNA of interest (or target DNA)
2. ligation
3. transfection (or transformation)
4. a screening/selection procedure.

Isolation of DNA

Initially, the DNA fragment to be cloned needs to be isolated. This DNA of interest may be a gene, part of a gene, a promoter, or another segment of DNA, and is frequently isolated by the Polymerase Chain Reaction (PCR) or restriction enzyme digestion. A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA at a specific sequence (Table 1). The enzyme makes two incisions, one through each strand of the double helix, without damaging the nitrogenous bases. This produces either overlapping ends (also known as sticky ends) or blunt ends.

A. EcoRI digestion produces overlapping ”sticky” ends: The enzyme cleaves between the G and A on both strands. B. SmaII restriction enzyme cleavage produces ”blunt” ends. The enzyme cleaves between the G and C on both strands.

The 1978 Nobel Prize in Medicine was awarded to Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleases. The first practical use of their work was the manipulation of E. coli bacteria to produce human insulin for diabetics.

Once the DNA of interest is isolated, a ligation procedure is necessary to insert the amplified fragment into a vector to produce the recombinant DNA molecule. Restriction fragments (or a fragment and a plasmid/vector) can be spliced together, provided their ends are complementary. Blunt end ligation is also possible.

The plasmid or vector (which is usually circular) is digested with restriction enzymes, opening up the vector to allow insertion of the target DNA. The two DNAs are then incubated with DNA ligase, an enzyme that can attach together strands of DNA with double strand breaks. This produces the recombinant DNA molecule. Figure 3 depicts a plasmid with two additional segments of DNA ligated into the plasmid, producing the recombinant DNA molecule. Figure 4 depicts DNA before and after ligation.

Figure 3: This image shows a line drawing of a plasmid. The plasmid is drawn as two concentric circles that are very close together, with two large segments and one small segment depicted. The two large segments (1 and 2) indicate antibiotic resistances usually used in a screening procedure, and the small segment (3) indicates an origin of replication. The resulting DNA is a recombinant DNA molecule.

Figure 4: Sticky ends produced by restriction enzyme digestion can be joined with the enzyme DNA ligase.

Transfection and Selection

Following ligation, the recombinant DNA is placed into a host cell, usually bacterial, in a process called transfection or transformation. Finally, the transfected cells are cultured. Many of these cultures may not contain a plasmid with the target DNA as the transfection process is not usually 100% successful, so the appropriate cultures with the DNA of interest must be selected. Many plasmids/vectors include selectable markers - usually some sort of antibiotic resistance (Figure 3). When cultures are grown in the presence of an antibiotic, only bacteria transfected with the vector containing resistance to that antibiotic should grow. However, these selection procedures do not guarantee that the DNA of insert is present in the cells. Further analysis of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of a process known as PCR (see below) or restriction fragment analysis, both of which need to be followed by gel electrophoresis and/or DNA sequencing (DNA sequence analysis).

DNA sequence analysis (the analysis of the order of the nitrogenous bases that make up the DNA), PCR, or restriction fragment analysis will all determine if the plasmid/vector contains the insert. Restriction fragment analysis is digestion of isolated plasmid/vector DNA with restriction enzymes. If the isolated DNA contains the target DNA, that fragment will be excised by the restriction enzyme digestion. Gel electrophoresis will separate DNA molecules based on size and charge. Examples are shown in Figure 5.

Figure 5: Agarose gel following agarose gel electrophoresis on UV light box: In the gel with UV illumination (left), the ethidium bromide stained DNA glows pink Right, photo of a gel. Far left: DNA ladder of fragments of known length. Lane 1: A PCR product of just over 500 bases. Lane 2: Restriction digest showing the 500 base fragment cut from a 4.5 kb plasmid vector.

Gel Electrophoresis

Gel electrophoresis is an analytical technique used to separate DNA fragments by size and charge. Notice in Figure 5 that the ”gels” are rectangular in shape. The gels are made of a gelatin-like material of either agarose or polyacrylamide. An electric field, with a positive charge applied at one end of the gel, and a negative charge at the other end, forces the fragments to migrate through the gel. DNA molecules migrate from negative to positive charges due to the net negative charge of the phosphate groups in the DNA backbone. Longer molecules migrate more slowly through the gel matrix. After the separation is completed, DNA fragments of different lengths can be visualized using a fluorescent dye specific for DNA, such as ethidium bromide. The resulting stained gel shows bands correspond to DNA molecules of different lengths, which also correspond to different molecular weights. Band size is usually determined by comparison to DNA ladders containing DNA fragments of known length. Gel electrophoresis can also be used to separate RNA molecules and proteins.

The Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) is used to amplify specific regions of a DNA strand millions of times. A region may be a number of loci, a single gene, a part of a gene, or a non-coding sequence. This technique produces a useful quantity of DNA for analysis, be it medical, forensic or some other form of analysis. Amplification of DNA from as little as a single cell is possible. Whole genome amplification is also possible.

PCR utilizes a heat stable DNA polymerase, Taq polymerase, named after the thermophilic bacterium Thermus aquaticus, from which it was originally isolated. T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase is able to withstand the high temperatures required to denature DNA during PCR. Taq polymerase’s optimum temperature for activity is between 75°C and 80°C. Recently other DNA polymerases have also been used for PCR.

A basic PCR involves a series of repeating cycles involving three main steps (see Figure 6):

  1. denaturation of the double stranded DNA
  2. annealing of specific oligonucleotide primers
  3. extension of the primers to amplify the region of DNA of interest

These steps will be discussed in additional detail below.

The oligonucleotide primers are single stranded pieces of DNA that correspond to the 5’ and 3’ ends of the DNA region to be amplified. These primers will anneal to the corresponding segment of denatured DNA. Taq Polymerase, in the presence of free deoxynucleotide triphosphates (dNTPs), will extend the primers to create double stranded DNA. After many cycles of denaturation, annealing and extension, the region between the two primers will be amplified.

The PCR is commonly carried out in a thermal cycler, a machine that automatically allows heating and cooling of the reactions to control the temperature required at each reaction step (see below). The PCR usually consists of a series of about 30 to 35 cycles. Most commonly, PCR is carried out in three repeating steps, with some modifications for the first and last step.

PCR is usually performed in small tubes or wells in a tray, each often beginning with the complete genome of the species being studied. As only a specific sequence from that genome is of interest, the sequence specific primers are targeted to that sequence. PCR is done with all the building blocks necessary to create DNA: template DNA, primers, dNTPs, and a polymerase.

Figure 6: PCR: A repeating cycle of denaturation (1), annealing (2), and extension (3). Notice that initially there is a double strand of DNA, and after denaturation, the DNA is single stranded. In the annealing step (2), single stranded primers bind. These primers are extended by Taq Polymerase, represented by the green ball (3).

The three basic steps of PCR (Figure 6) are:

• Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94 - 98°C for 30 to 60 seconds. It disrupts the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

• Annealing step: The reaction temperature is lowered to 50-65°C for 30 to 60 seconds, allowing annealing of the primers to the single-stranded DNA template. Stable hydrogen bonds form between the DNA strand (the template) and the primers when the primer sequence very closely matches the complementary template sequence. Primers are usually 17 - 22 nucleotides long and are carefully designed to bind to only one site in the genome. The polymerase binds to the primer-template hybrid and begins DNAsynthesis.

• Extension step: A temperature of around 72°C is used for this step, which is close to the optimum temperature of Taq polymerase. At this step the Taq polymerase extends the primer by adding dNTPs, using one DNA strand as a template to create a the other (new) DNA strand. The extension time depends on the length of the DNA fragment to be amplified. As a standard, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute.

Utilizing the PCR, DNA can be amplified millions of times to generate quantities of DNA that can be used for a number of purposes. These include the use of DNA for prenatal or genetic testing, such as testing for a specific mutation. PCR has revolutionized the fields of biotechnology, human genetics, and a number of other sciences. PCR was developed in 1983 by Kary Mullis. Due to the importance of this process and the significance it has had on scientific research, Dr. Mullis was awarded the Nobel Prize in Chemistry in 1993, just 10 years after his discovery.

To say that PCR, molecular cloning and the Human Genome Project has revolutionized biology and medicine would be an understatement. These efforts have led to numerous accolades, including Nobel prizes, and more may follow.


RBSE Class 12 Biology Chapter 15 Important Questions

RBSE Biology Chapter 15: MCQ Type Questions

Q.1. Restriction Endonuclease.enzyme is found naturally in ______.

Sol: (a) Bacteria.

Q.2. Which of the following enzymes are used in cutting the DNA at a specific site?

(c) Restriction Endonuclease.

Sol: (c) Restriction Endonuclease.

Q.3.DNA vector is a ______.

Sol: (a) Plasmid.

Q.4. M13 is an example of ______.

Sol: (b) Bacteriophage.

Q.5. Which of the following is a source of EcoRI?

Sol: (d) All of the above.

Q.6.Which of the following blotting techniques is used in the identification of DNA segments?

Sol: (c) Southern.

Q.7. Which enzymes join free DNA ends?

(a) Restriction endonucleases.

Sol: (b) Ligases.

Q.8.Jumping genes are called ______.

Sol: (d) Transposons.

Q.9.Which of the following techniques was discovered by Mullis in the year 1989?

(b) Polymerase chain reaction-PCR.

(c) Southern Blotting technique.

(d) Western Blotting technique.

Sol:(b) Polymerase chain reaction-PCR.

Q.10. C-DNA is used in the formation of ______.

RBSE Biology Chapter 15:Short Answer Type Questions.

Q.1. What is recombinant DNA technology? Who is credited for developing recombinant DNA technology?

Sol: The various effective measures required to incorporate changes in the DNA makeup of any organism is called recombinant DNA technology. Stanley Cohen and Herbert Boyer et al, American geneticists were credited for developing recombinant DNA technology in the year 1973.

Q.2.What are cloning vectors?

Sol: In recombinant DNA technology, in order to introduce the desired gene in the targeted plant or animal a carrier is required which can carry the desired gene and can enter the targeted plant or animal and replicates its DNA. This carrier is called the vector. Plasmid, bacteriophages and Cosmids are used as a vector in recombinant DNA technology.

Q.3. What are marker genes? Give examples of marker genes.

Sol: When the desired genes are incorporated with the vector, several unwanted products are also obtained. In order to eliminate these unwanted products and to identify the recombinant DNA in the host cell, a special type of gene is used. This produces special features in the modified or transformed cells. This gene, which is incorporated in the vector DNA, is called the marker genes. Kanamycin resistant genes are the best example of marker genes.

Q.4. What is meant by molecular probes?

Sol: The segment of DNA or RNA with the help of which C-DNA or RNA segments of some organisms can be identified are called molecular probes. The molecular probes are of the following types. DNA probes and RNA probes.

Q.5. What are marker genes? Give examples of marker genes.

Sol: When the desired gene is incorporated with the vector, several unwanted products are also obtained. In order to eliminate these unwanted products and to identify the recombinant DNA in the host cell, a special type of gene is used. This produces special features in the modified or transformed cells. This gene, which is incorporated in the vector DNA, is called the marker genes. Kanamycin resistant genes are the best example of marker genes.

Q.6. What is the genomic library?

Sol: The collection of the cloned segments of the entire genome of any organism is called the genomic library. A genomic library is formed by taking out the complete DNA content of the haploid set of chromosomes of an organism.

Q.7. What are cosmids?

Sol: Cosmids are a hybrid of plasmids and 2 (ƛ) lambda phages. Such plasmids in which the DNA sequences of Cos site of (ƛ) lambda phages are inserted are known as cosmids.

Q.8. Define the restriction endonuclease enzymes.

Sol: The enzymes which cut the DNA molecules at a specific site are called restriction endonuclease These enzymes function just like molecular scissors, which cut DNA molecules into the segments at a specific site.

Q.9. Name the gels used in gel electrophoresis technique.

Sol: There are two different types of gel, which are used in the process of gel electrophoresis technique. They are – Agarose gel and Polyacrylamide gel.

Q.10. What is a reporter gene? Give examples of the reporter gene.

Sol: There are certain genes, which produce or present some specific features in the host cell. These genes are called the reporter gene. The reporter gene produces a special effect on an account of which the cells containing these genes look different from other cells.

The LUC gene found in fire-fly or Jugnoo and produces bioluminescence is the best example for reporter genes.

Q.11.Define RFLP.

Sol: RFLP stands for Restriction Fragment Length Polymorphism. It is a molecular technique that exploits variations in homologous DNA sequences, in order to distinguish individuals, populations, or species or to pinpoint the locations of genes within a sequence.

Q.12.List out the important achievements of recombinant DNA technology.

Sol: The most important achievements of recombinant DNA technology are:

  1. Human Genome Project.
  2. Cloning of the gene of haemophilia.
  3. Cloning of the hepatitis B virus.
  4. Cloning of nitrogen fixation (Nif) gene.
  5. Cloning of the human growth hormone and insulin gene.
  6. Cloning of penicillin G acylase gene for the production of penicillin

Q.13. What is DNA fingerprinting?

Sol: The method of DNA fingerprinting was first discovered by Alec Jeffreys and his coworkers in the year 1985. DNA fingerprinting is a technique that shows the genetic makeup of living things. It is a method of finding the difference between the satellite DNA regions in the genome.

Q.14.Define PCR – Polymerase Chain Reaction.

Sol: Polymerase Chain Reaction (PCR) is a technique used for creating several copies of a certain DNA segment. This technique was developed in 1983 by Kary Mullis, an American biochemist. PCR has made it possible to generate millions of copies of a small segment of DNA. This tool is commonly used in the molecular biology and biotechnology labs.

Q.15. How is a genomic library formed?

Sol: A genomic library is formed by the isolation of complete DNA of a cell. The different steps involved in the formation of the genomic library are:

The complete genome of a Donor cell Restriction endonuclease enzyme. DNA fragments Vector DNA ↓ DNA ligase Circular Recombinant DNAInsertion into the bacterial cell.Polymerisation (as a colony).Identification by DNA Probe.Formation of Genomic Library.

RBSE Biology Class 12: Long Answer Type Questions

Q.1. What are cloning vectors? Explain how cloning vectors are selected during the process of genetic engineering.

Sol: After the isolation of the desired genes, a vector is required, which can incorporate this gene and along with it, it enters into the host cell and replicates its DNA. This vector is called the cloning vector. Plasmid, bacteriophages, cosmids are the main cloning vectors used in the process of recombinant technology.

The procedure for selecting cloning vectors are as follows:

  1. The vectors should have the ability to replicate autonomously within the host cell.
  2. The vectors should be easily introduced into the host cell and should be isolated again.
  3. The vectors should contain specific restriction sites, which can be broken easily by the restriction endonuclease enzyme. The foreign DNA can be inserted easily at the restriction site.
  4. The vector should have a marker site that allows easy detection of transformed cells.
  5. The transformation should be easy and perfect.
  6. For the expression of desired foreign DNA, the vector should have some regulatory elements like a promoter, operator, etc.

Q.2. Comment on PBR 322 plasmid.

Sol: PBR 322 plasmid is the most commonly used plasmid vector. In this plasmid the two marker sites – TetR (Tetracycline resistant) and AmpR (Ampicillin resistant) are found. It contains the recognition sites for 12 different restriction enzymes. The desired DNA is inserted in between the TetR and AmpR gene with the help of the restriction enzymes.

The below diagram explains the structure of PBR 322 plasmid.

Q.3. Write a brief account of various cloning vectors used in the process of recombinant rDNA technology.

Sol: The different cloning vectors used in the process of recombinant rDNA technology are:

  • These are extrachromosomal components in the bacterial cell.
  • The DNA is a circular and double-stranded molecule.
  • They contain an origin of the replication site and can replicate independently of a bacterial chromosome.
  • They have specific restriction sites where the desired gene can be incorporated.
  • They have a marker site.
  • The plasmid may contain three to one thousands of genes in it.

Bacteriophage

  • The viruses which infect bacteria and cause lysis of bacterial cells are called bacteriophage.
  • Example: ((ƛ) ) Lambda phage and M13 phage.
  • A bacteriophage is a better vector as compared to plasmids.
  • Large DNA segments (24 Kbp) can be cloned in the bacteriophage.
  • Each bacteriophage produces a plaque in the culture. Hence, their identification is easy.
  • This is a hybrid of plasmid and ((ƛ) ) Lambda phage.
  • These cosmids can replicate within the host cells just like a plasmid.
  • Due to the presence of Cos site, these cosmids are packed like phage particles.
  • Cosmids can be used to clone the DNA segments of up to 45kbp.

Q.4. Write short notes on Southern blotting technique and DNA fingerprinting.

Southern blotting technique

This technique is used for the analysis of the DNA segments. This was developed by E.M.Southern in 1975. Hence named as southern blotting. In this technique, the DNA segments are transferred on a nitrocellulose filter. These are then identified by the hybridization with the DNA probes.

DNA fingerprinting.

DNA fingerprinting is a technique that shows the genetic makeup of living things. It is a method of finding the difference between the satellite DNA regions in the genome. This technique was discovered by Alec Jeffreys and co-workers during the 1980s. In this method, the DNA of a specific person is cut into smaller segments and is separated in the form of bands by the process of electrophoresis. The identity of a person can be established by a specific sequence found in the DNA of the person. This technique is used in resolving disputed paternity of any child and in detecting genetic disease prior to the birth of a child. It is also used in the identification of criminals.

Q.5. Write short notes on Polymerase Chain Reaction and Restriction enzyme

Polymerase Chain Reaction

PCR or Polymerase Chain Reaction is a technique used in molecular biology to create several copies of a certain DNA segment. It analyzes short sequences of DNA or RNA even in samples containing minute quantities of DNA or RNA. This technique was developed in 1983 by Kary Mullis, an American biochemist. PCR has made it possible to generate millions of copies of a small segment of DNA. This tool is commonly used in the molecular biology and biotechnology labs.

Restriction enzyme

The restriction enzyme is a protein produced by bacteria that cleaves the DNA at specific sites. This site is known as the restriction site. These enzymes protect the live bacteria from bacteriophages. They recognize and cleave at the restriction sites of the bacteriophage and destroy its DNA. Restriction enzymes are important tools for genetic engineering. They can be isolated from the bacteria and used in the laboratories.

Q.6. What is Recombinant DNA Technology? Brief out the process of Recombinant DNA Technology.

Sol: The technology used for producing artificial DNA through the combination of different genetic materials (DNA) from different sources is referred to as Recombinant DNA Technology. Recombinant DNA technology is popularly known as genetic engineering.

The recombinant DNA technology emerged with the discovery of restriction enzymes in the year 1968 by Swiss microbiologist Werner Arber,

Inserting the desired gene into the genome of the host is not as easy as it sounds. It involves the selection of the desired gene for administration into the host followed by a selection of the perfect vector with which the gene has to be integrated and recombinant DNA formed.

Thus, the recombinant DNA has to be introduced into the host. And at last, it has to be maintained in the host and carried forward to the offspring.

Process of Recombinant DNA Technology

The complete process of recombinant DNA technology includes multiple steps, maintained in a specific sequence to generate the desired product.

Step-1. Isolation of Genetic Material.

The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules.

Step-2.Cutting the gene at the recognition sites.

The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome. These reactions are called restriction enzyme digestions.

Step-3. Amplifying the gene copies through Polymerase chain reaction (PCR).

It is a process to amplify a single copy of DNA into thousands to millions of copies once the proper gene of interest has been cut using the restriction enzymes.

Step-4. Ligation of DNA Molecules.

In this step of Ligation, joining of the two pieces – a cut fragment of DNA and the vector together with the help of the enzyme DNA ligase.

Step-5. Insertion of Recombinant DNA Into Host.

In this step, the recombinant DNA is introduced into a recipient host cell. This process is termed as Transformation. Once after the insertion of the recombinant DNA into the host cell, it gets multiplied and is expressed in the form of the manufactured protein under optimal conditions

Q.7. What is the importance of genetic engineering?

Sol: Scientists working in the field of biotechnology have succeeded in achieving results which have proved advantageous in the field of medical science, agriculture and industries. By using these techniques it has become possible to improve the variety of agricultural crops and domestic animals and also the quality of other industrial products. Some important achievements of genetic engineering are as follows:

  • Cloning of Nitrogen fixation(Nif) gene in cereal crops.
  • Cloning of the Haemophilic gene.
  • Cloning of Hepatitis B virus gene.
  • Cloning of Human growth hormone and insulin gene.
  • Cloning of penicillin G acylase gene for the production of penicillin

Q.8.What are the molecular probes? List out the importance of molecular probes.

Sol: The segment of DNA or RNA with the help of which C-DNA or RNA segments of some organisms can be identified are called molecular probes.

The molecular probes are of the following types. DNA probes and RNA probes.

Importance of molecular probes.

  1. The probes are used to identify the specific DNA segments used in the research of genetic engineering.
  2. Pollutants in food can be detected with the help of molecular probes.
  3. The molecular probes are used in the field of forensic science, in resolving disputed paternity issues and establishing family relationships.
  4. These molecular probes can also be used to identify the improved variety of crops and hybridized seed of crops.

Q.9. What are the different enzymes used in the process of genetic engineering?

Sol: The different enzymes used in the process of genetic engineering are

RNA dependent DNA polymerase.

These enzymes function by polymerising the nucleotides of DNA strands on the RNA template.

DNA dependent DNA polymerase.

This enzyme functions by polymerising the nucleotides of complementary DNA strands on the template of the DNA.

Ligases – This enzyme functions by attaching the ends of the DNA fragment on the template.

Lysozymes – This enzyme functions by dissolving the cell wall of bacteria so that the DNA of bacteria can be isolated.

Alkaline phosphates – This enzyme functions by cutting the phosphate at 5′ or 5 prime ends of the circular DNA and helps in keeping the DNA linear, so that the foreign DNA can be inserted on it. This enzyme also prevents the circular nature of DNA from forming again.

Q.10. What is PCR – Polymerase Chain Reaction? List out the applications of PCR.

Sol: Sol: Polymerase Chain Reaction (PCR) is a technique used for creating several copies of a certain DNA segment. This technique was developed in 1983 by Kary Mullis, an American biochemist. PCR has made it possible to generate millions of copies of a small segment of DNA. This tool is commonly used in the molecular biology and biotechnology labs.

The following are the applications of PCR :

In Medicine

  • Testing of genetic disease mutations.
  • Monitoring the gene in gene therapy.
  • Detecting disease-causing genes in the parents.

In Forensic Science

  • Paternity tests.
  • Used as a tool in genetic fingerprinting.
  • Identifying the criminal from millions of people.

In Research and Genetics

  • Gene Mapping.
  • Analysis of gene expression.
  • Compare the genome of two organisms in genomic studies.
  • In the phylogenetic analysis of DNA from any source such as fossils.

Q.11. Explain the process of nomenclature of restriction enzymes.

Sol: Restriction enzymes are like molecular scissors which function by cutting the DNA molecules at a specific site. These enzymes are naturally found in E.Coli, Bacillus, Streptococcus, etc.

The nomenclature of restriction enzymes are as follows:

  • The first letter of an enzyme represents the genus from which it has been isolated. This is written in capital letters.
  • The two letters after this represent the species of the genus. These are written in a small letter. These three letters are written in italics.

For example- Eco – E, Coli – from Escherichia coli

  • The fourth letter represents the strain of a genus and is written from which it has been isolated.

For example- Eco R – from R strain of E.coli

  • If more than one restriction enzymes are obtained from one organism, these are represented by a roman number.

For example- Eco-R I Eco-RII, etc.

Q.12.Explain in detail about the plasmid as a cloning vector.

Sol: Plasmids are a small, circular piece of DNA that is not the same as chromosomal DNA. Its ability to replicate is independent of chromosomal DNA. They are usually found in bacteria, but they are also present in multicellular organisms. The word Plasmid was first coined by Joshua Lederberg in 195.

Functions of Plasmids.

Plasmids have various functions, as they:

  • Facilitate the process of replication.
  • Increase the survival of the organism.
  • Carry the helpful genes to their host organisms.
  • Plasmids are frequently used as a cloning vector in the DNA recombinant technology.

Features of Plasmids.

Following are the important features of Plasmids

  • These plasmids have extrachromosomal components.
  • They have marker genes or marker sites within the plasmids.
  • The plasmid may contain three to one thousands of genes in it.
  • These plasmids are not necessary for the growth and survival of bacteria.
  • They contain specific restriction sites where the desired gene can be inserted.
  • These plasmids are circular in shape and consist of double-stranded DNA molecules.
  • They contain an origin of replication. Therefore, it is able to replicate independently within the cell.

Q.13. Do All Bacteria Have Plasmids? Draw the structure of Plasmids in bacterial cells.

Sol: Yes, Plasmids naturally exist in all bacterial cells. It functions by:

  1. Helps in their survival by producing toxins,
  2. Facilitate the process of replication in bacteria.
  3. Few plasmids contain genes that help in food digestion.
  4. The R plasmids help a bacterial cell by defending against environmental factors such as antibiotics, poison, etc.

The structure of Plasmids in bacterial cells.

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Split genes and introns

Precursors to mRNA longer than mRNA

Initial indications of a complex structure to eukaryotic genes came from analysis of nuclear RNAs during the 1970&rsquos. The precursors to messenger RNA, or pre-mRNAs, were found to be surprisingly long, considerably larger than the average mRNA size (Figure (PageIndex<1>)).

Figure (PageIndex<1>)

Denaturing sucrose gradients (with high concentration of formamide, e.g. >50%) separate RNAs on the basis of size. Analysis of nuclear RNA showed that the average size was much larger than the average size of cytoplasmic RNA. Labeled RNA could be "chased" from the nucleus to the cytoplasm ‑ i.e. nuclear RNA was a precursor to mRNA and other cytoplasmic RNAs. Was the extra RNA at the ends? or in the middle of the pre‑mRNA? More precisely, one could examine specific RNAs by hybridizing fractions from the denaturing sucrose gradients to labeled copies of, e.g. globin mRNA. The hybridizing RNA from the nucleus was about 11S (as well as mature 8S message), whereas cytoplasmic RNA of about 8S hybridized. Thus the nuclear RNA encoding globin is larger than the cytoplasmic mRNA.

Visualization of mRNA-DNA heteroduplexes revealed extra sequences internal to the mRNA-coding segments

R-loops are hybrids between RNA and DNA that can be visualized in the EM, under conditions where DNA‑RNA duplexes are favored over DNA‑DNA duplexes (Figure (PageIndex<2>)). For a simple gene structure, one sees a continuous RNA‑DNA duplex (smooth, slowly curving) and a displaced single strand of DNA (thinner, many more turns and curves &ndash single stranded DNA is not a rigid as double stranded nucleic acid, either duplex DNA or RNA-DNA).

Figure (PageIndex<2>)

EM pictures of duplexes between purified adenovirus mRNAs and the genomic DNA showed extensions at both the 3' (poly A) and 5' ends, which are encoded elsewhere on the genome. All late mRNAs have the same sequence at the 5' end this is dervied from from the tripartite leader. R‑loops between late mRNAs and adenovirus DNA fragments including the major late promoter showed duplexes with the leader segments, separated by loops of duplex DNA (Figure 3.23, bottom panel). The RNA-DNA hybrids identify regions of DNA that encode RNA. The surprising result is that RNA-coding portions of a gene are separated by loops of duplex DNA in the R-loop analysis. Examples of R-loops in genes with introns are shown in Figure (PageIndex<3>).

These data showed that the adenovirus RNAs are encoded in different segments of the viral genome i.e. the genes are split. The portion of a gene that encodes mRNA was termed an exon. The part of gene does not code for sequences in the mature mRNA is called an intron. These observations led to the Nobel Prize for Phil Sharp and Rich Roberts. Louise Chow and Sue Berget were also key players in the discovery of introns.

Figure (PageIndex<3>): R-loops between clones of rabbit beta-like globin genes (now called HBEand HBG) and mRNA from rabbit embryonic erythroid cells. A photograph from the electron microscope is shown at the the top of each panel, and an interpretive drawing is included below it. The displaced nontemplate strand of DNA forms partial or complete duplexes with the template strand in the large intron. A small intron is also visible in panel C. Panel G shows the two genes together on one large clone.

Interruptions in cellular genes were discovered subsequently, in the late 1970's, in globin genes, immunoglobulin genes and others. We now realize that mostgenes in complex eukaryotes are split by multiple introns.

Exons are more conserved than introns (in most cases), since alterations in protein-coding regions that alter or decrease function are selected against, whereas many sequences in introns can be altered without affecting the function of the gene product. Important sequences in introns (such as splice junctions, the branch point, and occassionally enhancers) are covered in some detail in Part Three.

Differences in restiction maps between cDNA and genomic clones reveal introns

Restriction maps based on copies of the mRNA (cDNA) were different from those in genomic DNA ‑ the genes were cleaved by some restriction endonucleases that the cDNAs were not, and some restriction sites were further apart in the genomic DNA. These observations were explained by the presence of intervening sequences or introns (Figure (PageIndex<4>)).

Figure (PageIndex<4>)

The experimental procedures to do this involve making a restriction map of the clones of genomic DNA, and then identifying the regions that encode mRNA by hybridization of labeled cDNA probes to the restriction digests. Cloned genomic DNA digested with appropriate restriction endonucleases, separated by size on an agarose gel, and then transferred onto a nylon or nitrocellulose solid support. This Southern blot is then hybridized with a labeled probe specific to the cDNA (composed only of exons). The pattern of labeled fragments on the resulting autoradiogram shows the fragments that contain exons. Alignment of these with the restriction map of the gene gives an approximation of the position of the exons.

The blot-hybridization approach can be combined with a PCR (polymerase chain reaction) analysis for higher resolution. Primers are synthesized that will anneal to adjacent exons. The difference in size of the PCR amplification product between genomic DNA and cDNA is the size of the intron. The PCR product can be cloned and sequenced for more detailed information, e.g. to precisely define the exon/intron junctions.

Subsequently, the nucleotide sequence of exonic regions and preferably the entire gene is determined. The presence of introns were confirmed and their locations defined precisely in DNA sequences of isolated clones of the genes.

Types of Exons

Eukaryotic genes are a combination of introns and exons. However, not all exons do the same thing (Figure (PageIndex<5>)). In particular, the protein-coding regions or genes are a subset of the sequences in exons. Exons include both the untranslated regions and the protein-coding, translated regions. Introns are the segments of genes that are present in the primary transcript (or precursor RNA) but are removed by splicing in the production of mature RNA. Methods used to detect coding regions will not find all exons.


ISC Biotechnology Question Paper 2013 Solved for Class 12

Maximum Marks: 80
Time allowed: Three hours

  • Candidates are allowed additional 15 minutes for only reading the paper. They must NOT start writing during this time.
  • Answer Question 1 (Compulsory) from Part I and five questions from Part II, choosing two questions from Section A, two questions from Section B and one question from either Section A or Section B.
  • The intended marks for questions or parts of questions are given in brackets [ ].
  • Transactions should be recorded in the answer book.
  • All calculations should be shown clearly.
  • All working, including rough work, should be done on the same page as, and adjacent to the rest of the answer.

Part-1
(Answer all questions)

Question 1.
(a) Mention any one significant difference between each of the following : [5]
(i) Gene and Genome
(ii) Multi potent cell and Uni potent cell
(iii) Galactose and Glycine
(iv) Batch culture and Continuous culture
(v) Coding region and Non-coding region

(b) Answer the following questions : [5]
(i) Name the enzyme used in PCR. What is the source of this enzyme?
(ii) Why is Bt-cotton resistant to boll worm?
(iii) Mention any two methods of ex-situ conservation of germplasm.
(iv) What is Proteomics?
(v) Glucose and fructose have the same chemical formula (C6H1206), yet they differ in chemical properties. Why ?

(c) Write the Ml form of the following : [5]
(i) GDB
(ii) PIR
(iii) YAC
(iv) NCBI
(v) ddNTP

(d) Explain briefly : [5]
(i) Bacterial Artificial Chromosome
(ii) Vascular differentiation
(iii) Phenylketonuria
(iv) Quartemary protein
(v) Designer oils
Answer:
(a) (i) Gene: Gene is the unit of the genome, consisting of a sequence of DNA that occupies a specific position (locus) on a chromosome and determines a particular characteristic in an organism.

Genome: Genome is the total genetic information or all the genes contained in a haploid set of chromosomes in eukary otes, in a single chromosome in bacteria, or in the DNA or RNA of viruses.

(ii) Multipotent: These cells have the ability to differen-tiate into many of the various type of specialized cell types and can develop into any cell of a particular group or type. e.g., umbilical cord stem cells.

Unipotent: These cells can undergo unlimited reproductive divisions, but can only differentiate into a single type of cell or tissue, e.g., skin cells.

(iii) Galactose: It is a part of disaccharide that is made- up of two sugars. It is found in milk alongwith glucose. Galactose does not occur freely in nature. It is produced in the body during the digestion of disaccharide lactose.

Glycine: Glycine is a neutral amino acid and one of the 20 building blocks of protein. It is a non-essential amino acid, used in purine synthesis, and is a neurotrans¬mitter.

(iv) Batch culture: It is a type of culture in which nutrients are fed continously depending upon the amount consumed without removing growth products.

Continuous culture: It is a open type of culture in which nutrients are supplied from time to time alongwith removal of product in same volume.

(v) Coding region: Coding region (exon) is a part of the DNA that actually codes for a protein.

Non-coding region: Non-coding region (introns) is that part of DNA that does not code directly for a protein.

(b) (i) The enzyme used in PCR is TAQ – DNA polymerase I and the source of this enzyme is Thermus aquaticus.

(ii) Bt-co.tton is an insect-resistant Genetically Modified (GM) variety of cotton seed, which contains a cry gene from Bacillus thuringiensis to kill the bollworm.

(iii) Ex-situ conservation of germplasm refers to maintaining or conserving the germplasm of organism outside their natural seed habitats. Two methods of ex-situ germplasm conservation are seed banks, botanical gardens, zoological parks etc.

(iv) Proteomics is the study of entire complement of proteins, particularly their structures and functions on the large scale. The term “proteomics” was first coined in 1997 and used to make an analog>’ with genomics, the study of the genes. The word “proteome” is derived from “protein’ and “genome”, and this was coined by Marc Wilkins in 1994. It is constantly changing due to intracellular and extracellular factors.

(v) Both Glucose and fructose have the same chemical formula, but they are different because of the different arrangement of the atoms within the molecules. Glucose is an aldose with a -CHO group at position 1 while fructose is a ketose with a -C = O at position 2.

(ii) Protein Information Resource

(iii) Yeast Artificial Chromosome.

(iv) National Centre for Biotechnolog> Information

(v) Dideoxynucleoside triphosphate.

(d) (i) Bacterial artificial chromosome (B AC) is a cloning l ector construct, based on a fertility plasmid (or F-plasmid). which is used for transforming and cloning in bacteria, usually E.coli. An ori gene for maintainance of F factor, a selectable marker and many restriction sites for insertion of foreign DNA. The bacterial artificial chromosome’s usual insert size is 300 to 350 kbp.

(ii) Vascular tissues are complex tissues, each consisting of a number of different types of cells. Vascular differentiation refers to the process by which different types cell types arise from precursor cells and become different in structure and function from each other.

(iii) Phenylketonuria is the recessive genetic disorder caused by the absence of the enzyme phenylalanine hydroxylase which catalyzes the conversion of phenylpyruvic acid into hydroxyphcnyl pyruvic acid. It is caused due to mutation of gene.

(iv) Quartemary proteins are the multimeric proteins i.e.. proteins hav ing more than two or more polypeptide chains which are linked to form quartemary structure, e.g.. Haemoglobin.

(v) Designer Oil: “Designer oil” that reduces LDL (‘bad“) blood cholesterol levels in humans and increases energy expenditure which may prevent people from gaining weight. The oil incorporates a phytosterol-based functional food ingredient Phytrol (TM) from Forbes into oil using proprietary technology.

Part-II
(Answer any five questions)

Question 2.
(a) Give a comparative account of DNA and RNA on the basis of their following characteristics: [4]
(i) Chemical composition and structure
(ii) Location and function
(b) Mention the uses of the following in genetic engineering techniques : [4]
(i) Shuttle vectors and Expression vectors
(ii) Restriction endonucleases
(c) What is electroporation ? [2]
Answer:
(a) (i) DNA:

  • DNA has 2-Deoxyribose sugar.
  • It contains cytosine and thymine as pyrimidine.
  • It has a double stranded helix struc-ture.
  • RNA has ribose sugar.
  • It contains cytosine and uracil as pyrimidine.
  • It has a single stranded helix.
  • DNA occurs in the nucleus chloroplast and mitochondria of cell.
  • It controls transmission of hereditary characters.

(b) (i) Shuttle vectors exist and work and allow DNA to be transferred between both prokaryotes and eukaryotes. The shuttle vector has two origins of replication i.e.. on E and ori Euk allowing replication to occur in either system/host. It “shuttles” between two different species. It can be used to perform reverse genetics, e.g.. Yeast episomal plasmid (YEP). Expression vectors allow expressing certain genes directly from their recombinant DNAs A typical expression vector will have a promoter upstream of the DNA containing the sequence to be expressed.

(ii) Restriction endonucleases are enzymes that cleave DNA at specific nucleotide sequences. The sequence recognized is often four to six nucleotides long. For example, the restriction endonucleases Eco RI recognize the sequence. GAATTC.

(c) Electroporation is a mechanical method used to introduce polar molecules into a host cell through the cell membrane. In this procedure, a brief exposure to a high electric voltage pulse temporarily disturbs the phospholipid bilayer, allowing introduction of molecules like DNA to pass into the cell.

Question 3.
(a) What is gene cloning ? Mention the steps involved in this process
(b) Explain the following :
(i) Acidic and basic amino acids
(ii) Phospholipids and glycolipids
(c) State any four objectives of germplasm conservation.
Answer:
(a) The process of formation of similar copies of a desired gene is called gene cloning. Gene cloning is the technique of recombinant DNA technology’ in which a desired gene of interest having a characteristic feature is cloned. Gene cloning involves the replication of DNA fragments by the use of self-replicating vector’s genetic material for its multiplication, expression or integration into host chromosome.

Steps involved in gene cloning :

  • In cloning a gene the first step is to isolate the DNA segment from the organism that contains the gene of interest.
  • Remove the gene of interest from the DNA, by using restriction enzymes or by PCR.
  • Vector is also treated with same restriction enzyme, to cleave it. Vector come to possessing single strand at the ends called stick>’ ends.
  • Then the enzy me DNA ligase is used to insert the gene of interest to be cloned into the plasmid. Vector having sicky ends to form recombinant DNA.
  • The plasmid or vector acts as a vehicle that transports the desired gene into a host cell, the process is known as transformation.
  • Now, these recombinant plasmids are inserted into bacterial host cells, where they replicate to amplify the desired gene, the process is called gene cloning.
  • Now the cell can be plated out on an agar medium. The colony of cells containing the desired cloned gene can be identified and isolated.

(b) (i) Amino acids are the basic structural unit of all proteins. A free’ neutral amino acid (a single amino acid) always has an amino group -NH2. a carboxyl group -COOH, hydrogen -H and a chemical group or side chain -”R”.

Acidic amino acid :
Two amino acids have acidic side chains at neutral pH. These are aspartic acid or aspartate (Asp) and glutamic acid or glutamate (Glu). Their side chains have carboxylic acid groups whose pKa’s are low enough to lose protons, becoming negatively charged in the process. Such amino acids are highly polar.

Basic amino acid :
Three amino acids have three basic side chains at neutral pH. These are arginine (Arg), lysine (Lys). and histidine (His). Their side chains contain nitrogen and resemble ammonia, which is a base. Their pKa’s are high enough that they tend to bind protons, gaining a positive charge in the process.

(ii) Phospholipids are the phosphorylated triglyceride lipids in which one fatty acid is replaced by phosphate group added by phosphorylation. Glvcolipids are the glycosylated lipids in which sugar residue galactose or carbohydrate molecule is added by glycosylation. Phospholipids and glycolipids both are the derivatives of lipids. They form an essential component of cell membrane which plays a role in structure, maintenance and also help in eliciting certain immune reactions.

(c) Objectives of Germplasm Conservation :

  • Conservation of rare germplasm arising through somatic hybridization.
  • Storage of pollen for enhancing longevity.
  • Maintainance of recalcitrant seeds.
  • To develop genes for adaptations / endurance to varying, unfavorable biotic/abiotic stresses / environments.
  • To develop high yielding varieties.

Question 4.
(a) Why are enzymes temperature sensitive ? Briefly explain the mode of action of enzymes on their substrate. [4]
(b) How is the hormone insulin synthesized, using genetic engineering technique ? State two ways in which this technique is better than the techniques used earlier. [4]
(c) What is a supra-molecular assembly ? [2]
Answer:
(a) Enzymes are temperature sensitive because almost all enzymes are proteins have tertiary structure and only function in a specific range of temperature. Exposing enzymes to high temperature break bonds and can cause them to denature, which alter the shape of the enzyme. Due to change in shape the substrate no longer ‘fits’ inactive site of the enzyme and can no longer function as normal.

Mode of enzyme action : It can be explained by this models :

Lock and key mechanism : This model was proposed by Emil Fisher in 1898. It is also called the template model. According to this model the union of the substrate and the enzyme takes place at the active site, more or less in a manner in which a key fits in a lock and results in formation of an enzyme substrate complex. As the two molecules are involved, this hypothesis is also known as the concept of inter molecular fit. The ES complex is highly unstable and almost immediately this complex breaks to produce the end product of the reaction and regenerate the free enzyme. The ES complex results in the release of energy.

Examples:
Catalase : It catalyzes the decomposition of hydrogen peroxide into water and oxygen.
2H2O2 → 2H2O + O2
One molecule of catalyses can break 40 million molecules of hydrogen peroxide each second.

The first major medicinal product of genetic engineering is human insulin called Humulin. Insulin is a protein that acts as a hormone to stimulate uptake of blood sugar into tissues, such as the liver and the muscles.
Following are the steps which are involved in insulin synthesis :

  • Isolate the gene responsible for producing human insulin protein. The gene is a part of the DNA in a human chromosome.
  • Then remove a circular piece of DNA called plasmid from a bacterial cell. Special restriction enzymes are used to cut the plasmid ring open with sticky ends.
  • With the plasmid ring open, the gene for insulin is inserted into the plasmid ring and the ring is closed with ligase enzyme forming recombinant DNA. This process is called recombinant technology’.
  • The bacterial plasmid DNA now contains the human insulin gene and is inserted into a bacteria.
  • Many plasmids with the insulin gene are inserted into many bacterial cells. When the bacterial cells reproduce by dividing, the human insulin gene is also cloned in the newly cloned cells.
  • Human insulin protein molecules produced by bacteria are gathered and purified by down stream process by culturing the genetically engineered bacteria, limitless supplies of insulin may be produced.

Two ways in which genetic engineering is better than the technique used earlier:

  • Insulin produced by genetic engineering is pure and has no allergic reaction.
  • The human insulin is much cheaper when produced by r-DNA technology than was the insulin from cows, as it could be produced much more quickly in greater quantity.

(c) A supra molecular assembly or “super molecule” is a well defined complex of molecules held together by non-covalent bonds. Molecules are combined in the form of sphere or rod. The dimensions of supra molecular assemblies can range from nanometres to micrometers. The process by which a supra molecular assembly forms is called molecular self-assembly.

Question 5.
(a) What is plant tissue culture ? Discuss the organization of a tissue culture laboratory under the following headings: [4]
(i) Media preparation
(ii) Culture room.
(b) Explain any two methods used for the identification of recombinant host cells from the non-recombinant host cells. [4]
(c) Name any four in vivo techniques employed in haploid production. [2]
Answer:
(a) Plant tissue culture is the technique of in vitro maintenance and growth of plant cells, tissues and organs under aseptic conditions on a suitable artificial culture medium contained in small containers under controlled environmental conditions of temperature and light.

(i) Media Preparation Room : An area is required for preparation of media. In such space there should be provision for bench space for chemicals, labware, culture vessels, closures and miscellaneous equipment required for media preparation and dispensing. In this room provision is also made for placing hot plates or stirrers, pH meter, balance, waterbath, burners, oven, autoclave, culture vessel, refrigerator etc.

(ii) Culture Room : All types of cultured plant tissues are incubated under the conditions of well controlled temperature, humidity, illumination and air circulation. The culture room should have light and temperature control system. Generally temperature is maintained at 25±2°C and 20-98% relative humidity and uniform air ventilation. The cultures are grown in diffused light and darkness each for a period of 12 hours.

(b) The introduction of the recombinant DNA in to a suitable host cell is followed by the selection of those cells, which contain the recombinant vectors. There are various selection methods that are based on the expression or non-expression of some of the traits present in the vector or alongwith the cloned gene.

Antibiotic sensitivity : Recombinant plasmid has many traits such as ori recognition site and selectable marker gene. Some of these traits are resistant to certain antibiotics. If the antibiotic resistant gene is present alongwith the cloned gene, it is very easy to select the recombinant transformants directly on a medium supplemented with respective antibiotic.

In most of the cases there are two stages of selection. First is the selection on the basis of . transformed cells i.e., the cells that have taken a plasmid. The second one is to identify the transformed cells that have the recombinant plasmid. The presence of a desired DNA insert can be confirmed either by isolating the recombinant plasmids and digesting it with the same restriction enzyme used for making the recombinant vectors, by PCR, by southern hybridisation with DNA probes, by northern hybridisation with RNA probes and by direct DNA sequencing

lnsertional inactivation : Another method to differentiate between recombinant and non¬recombinant is on the basis of their ability to produce colour.
lnsertional inactivation: In this method, a recombinant DNA is within the coding sequence of an enzyme p-galactosidase. This results into the inactivation of enzyme which is referred to as insertional inactivation.

The bacterial colonies whose plasmids do not have insert, produce blue colour but those with an insert or the recombinant do not produce any colour and are identified as recombinant colonies.

(c) In vivo techniques employed in haploid production are gynogenesis, ovule and rogenesis, genome elimination by distant hybridisation or chemical treatment and semigamy.

Question 6.
(a) Write the principle and any two applications of each of the following biochemical techniques : [4]
(i) Ion – exchange chromatography
(ii) Gel – permeation
(b) What is a genetic code ? Enlist three important properties of genetic code. [4]
(c) What are DNA probes ? [2]
Answer:
(b) (i) Principle of Ion-exchange chromatography : It is defined as the reversible exchange of ions in solution with ions electrostatically bound to some sort of insoluble support medium. Separation is obtained since different molecules have different degree of interaction with the ion-exchanger due to difference in their charges, charge densities and distribution of charge on their surfaces. These interactions can be controlled by varying conditions such as ionic strength and pH.

An ion-exchanger consists of an insoluble matrix to which charged groups have been covalently bound. Ion exchange separations are carried out mainly in columns packed with an ion-exchanger. There are two types of ion-exchanger, namely cation and anion exchangers. Cation exchangers possess negatively charged groups and these will attract positively charged cations. Anion exchangers have positively charged groups that will attract negatively charged anions. After the ion exchange the molecules can be eluted from the matrix by selective desorption. The selective desorption can be achieved by changes in pH and /or ionic concentration or by affinity elution, in which case an ion that has greater affinity for the exchange than has the bound ion is introduced into the system.

  • Polystyrene and polyphenolic ion exchange resins are more often used to separate srhall molecule such as amino acids, small peptides, nucleotides, N-bases, cyclic nucleotides, organic acids.
  • The cellulose ion exchangers are commonly used for proteins, including enzymes, polysaccharides and nucleic acids.

Principle of Gel-permeation chromatography: Gel permeation / filtration chromatography is a separation technique which uses molecular sieves, composed of neutral cross-linked carriers e.g., polymers like agarose, dextrans of different pore sizes. Therefore, it can separate macromolecule of different sizes from one another. Molecules smaller than pore size either the carrier and are retained. They are later eluded (in order of molecular size) and collected. Other names that have been suggested for this technique are : get filtration, molecular or size exclusion chromatography or molecular sieve chromatography.

  • Separation of polysaccharide, enzymes, antibodies and other proteins.
  • Separation of non-polar species such as triglycerides in non-aqueous mobile phases.
  • Used to analyse the molecular-weight distribution of organic soluble polymer.

The genetic code is called a triplet code, i.e sequence of three nitrogenous bases on m-RNA that specifies the recognition of a particular of a single amino acid. Thus, the information encoded in the sequence of nitrogenous bases must be read in groups of three, (UAC, GGC, UGC).

Three important properties:

  1. Triplet code : Three adjacent nitrogen bases constitute a codon which specifies the placement of one amino acid in a polypeptide.
  2. Start signal : Polypeptide synthesis is signaled by AUG or methionine codon and GUG — Valine codon. They have dual function.
  3. Stop signal: Polypeptide chain termination is signaled by three termination codons — UAA, UAG, and UGA. They do not specify any amino acid and are hence also called non-sense codon.
  4. Universal code : The genetic code is applicable universally i.e., the codon specifies the same amino acid from a virus to a tree or human being.
  5. Non-ambiguous codon : One codon specifies only one amino acid and not any other.

(c) DNA Probe : It is a solution of radioactive, single-stranded DNA or oligodeoxy nucleotides (a DNA segment of few to several nucleotides). The name probe signifies the fact that this DNA molecule is used to detect and identify the DNA fragment in the gel/membrane that has a sequence complementary to the probe. The probe hybridises with the complementary DNA on the membrane to the greater extent with a low non-specific binding on the membrane. This step is known as hybridisation reaction.

Question 7.
(a) How can the following plants be obtained, using genetic transformation techniques . [4]
(i) Drought and salinity tolerant plants
(ii) Somatic hybrids
(b) Explain the process involved in the transcription of DNA to mRNA. Also, mention any two post transcriptional changes that occur in the mRNA formed. [4]
(c) What are Okazaki fragments ? How are they joined ? [2]
Answer:
(i) Drought tolerance : Water is crucial for all living things. Plants use water as a solvent, a transport medium, an evaporative coolant, physical support, and as a major ingredient for photosynthesis. Without sufficient water, agriculture is impossible. Therefore, drought tolerance is an extremely important agricultural trait.

One way of engineering drought tolerance is by taking genes from plants that are naturally drought tolerant and introducing them to crops. The resurrection plant (Xerophyta viscosa), a native of dry regions of southernmost Africa, possesses a gene for a unique protein in its cell membrane. Experiments have shown that plants given this gene are less prone to stress from drought and excess salinity.

Some genes have been found that control the production of the thin, protective cuticle found on leaves. If crops can be grown with a thickened waxy cuticle, they could be better equipped for dealing with dryness.

Salt tolerance: Irrigation has enabled the transformation of arid regions into some of the world’s most productive agricultural areas. Excess salinity, however, is becoming a major problem for agriculture in dry parts of the world. In several cases, scientists have used biotechnology to develop plants with enhanced tolerance to salty conditions.

Researchers have noticed that plants with high tolerance to salt stress possess naturally high levels of a substance called glycine betaine. Further, plants with intermediate levels of salinity tolerance have intermediate levels, and plants with poor tolerance to salinity have little or none at all. Genetically modified tomatoes with enhanced glycinebetaine production have increased . tolerance to salty conditions.

Another approach to engineering salt tolerance uses a protein that takes excess sodium and diverts it into a cellular compartment where it does not harm the cell. In the lab, this strategy was used to create test plants that were able to flower and produce seeds under extreme salt levels. Commercially available crops with such a modification are still several years away.

(ii) Process, other than the sexual cycle has recently become available for higher plants, which can lead to genetic recombination. This non-conventional genetic procedure involving fusion between isolated somatic protoplasts under in vitro conditions and subsequent development of their product (heterokaryon) to a hybrid plant is known as somatic hybridisation.

Application of Somatic Hybridisation :

  • Somatic cell fusion appears to be the only means through w hich two different parental genomes can be recombined among plants that cannot reproduce sexually (asexual or sterile).
  • Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can be fused to produce fertile diploids and polyploids.
  • Somatic cell fusion overcomes sexual incompatibility barriers. In some cases, somatic hybrids between two incompatible plants have also found application in industry or agriculture.
  • Somatic cell fusion is useful in the study of cytoplasmic genes and their activities and this information can be applied in plant-breeding experiments.

(b) The process of transcription: Transcription is the process of creating a messenger RNA strand from DNA, performed by the enzyme RNA polymerase, Transcription always occurs in a 5′ → 3′. direction, with polymerase moving 3′ → 5′ along the DNA strand.

Transcription Initiation : There are three steps in transcription :
Initiation : RNA synthesis begins after the RNA polymerase attaches to the DNA and unwinds it. RNA synthesis will always occur on the template strand.

Elongation : RNA polymerase unwinds the DNA double helix and moves downstream and elongates the RNA transcript by adding ribonucleotides in a 5′ → 3′ direction. Each ribonucleotide is added to the growing mRNA strand using the base pairing rules (A binds with T, G binds with C). For each C encountered on the DNA strand a G is inserted in the RNA, for each Q a C and for each T, an A is inserted. Since there is no T in RNA, U is inserted whenever an A is encountered. After RNA polymerase has passed, the DNA restores its double stranded structure.

Termination: When the mRNA is complete, the mRNA is released and the RNA polymerase releases from the DNA.

Two post transcriptional changes that occur in the mRNA formed are:
RNA transcripts eukaryotes are modified or processed, before leaving the nucleus to produce functional wRNA. It is processed in two ways :
(1) 5 ‘ capping : Capping of the pre-mRNA involves the addition of 7-methylguanosine (m7G) to the 5′ end.,
(2) 3′ polyadenylation: The pre-mRNA processing at the 3′ end of the RNA molecule involves cleavage of its 3′ end and then the addition of about 200 adenine residues to form a poly (A) tail. The cleavage and adenylation reactions occur if a polyadenylation signal sequence (5′ – AAUAAA-3′) is located near the 3′ end of the pre-mRNA molecule, followed by another sequence, which is usually (5′ -CCA-3’).

(c) Okazaki fragments are short, newly synthesized DNA fragments produced discontinously in pieces during DNA replication. They are formed on the lagging template strand and are complementary to the lagging template strand. Okazaki fragments are joined together by DNA ligase enzyme.

Question 8.
(a) What is meant by the term genomics ? Write the differences between structural genomics and functional genomics. [4]
(b) Name and explain any four methods of synchronization of cells. [4]
(c) What is meant by Expressed sequence tags ? [2]
Answer:
(a) The word ‘genomics’ has taken root from the term ‘genome’ which is an organism’s total genetic constitution mapping, sequencing and analyzing the genomic information to solve a medical, industrial or biological query. Genomics studies investigate structure and function of genes and do this simultaneously for all the genes in a genome. Genomics is broadly categorized into structural and functional genomics.

Structural Genomics : The structural genomics deals with DNA sequencing, sequence assembly, sequence organisation and management. Basically it is the starting stage of genome analysis i.e,. construction of genetic, physical or sequence maps of high resolution of the organism. The complete DNA sequence of an organism is its ultimate physical map. Due to rapid advancement in DNA technology and completion of several genome sequencing projects for the last few years, the concept of structural genomics has come to a state of transition. Now it also includes systematics and determination of 3D structure of proteins found in living cells. Because proteins in every group of individuals vary and so there would also be variations in genome sequences.

Functional Genomics: It is based on the information of structural genomics the next step is to reconstruct genome sequences and to find out the function that the genes do. This information also lends support to design experiment to find out the functions that specific genome does. The strategy of functional genomics has widened the scope of biological investigations. This strategy is based on systematic study of single gene / protein to all genes/proteins.

Therefore, the large scale experimental methodologies (along with statistically analysed / computed results) characterise the functional genomics. Hence, the functional genomics provide the novel information about the genome. This eases the understanding of genes and function of proteins, and protein interactions.

(b) Cell culture synchronization : Cells in suspension cultures vary greatly in size, shape, DNA, and nuclear content. Moreover, the cell cycle time varies considerably within individual cells. Therefore, cell cultures are mostly asynchronous.

It is essential to manipulate the growth conditions of an asynchronous culture in order to achieve a higher degree of synchronization. A synchronous culture is one in which the majority of cells proceed through each cell cycle phase (G1: S, G2 and M) simultaneously.

Synchronization can be achieved by following methods :

  • Physical methods include selection by volume (size of cell aggregate.)
  • Chemical methods include starvation (depriving suspension cultures of an essential growth compound and culture supplying).
  • Chemical methods include inhibition (temporarily blocking the progression of events in the cell cycle using a biochemical inhibitor and then releasing the block).

(c) An Expressed Sequence Tag or EST is a short sub-sequence of a transcribed cDNA sequence represents a partial gene. They may be used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination, used in micro-arrays.

Question 9. .
(a) What is Human Genome Project ? Mention its objectives and significant achievements. [4]
(b) Write short notes on : [4]
(i) Locus – link
(ii) Microprocessor
(iii) EMBL
(iv) Taxonomy Browser
(c) What is site-directed mutagenesis ? [2]
Answer:
(a) The Human Genome Project (HGP) : This is an international scientific research project with a primary goal to determine the sequence of chemical base pairs which make-up DNA and to identify the approximately 25,000 genes of the human genome from both a physical and functional standpoint.

Benefits: The work on interpretation of genome data is still in its initial stages. It is anticipated that detailed knowledge of the human genome will provide new avenues for advances in medicine and biotechnology. A number of companies, such as Myriad Genetics started offering easy ways to administer genetic tests to a variety of illnesses, including breast cancer, disorders of homeostasis, cystic fibrosis, liver diseases and many others. Also, the etiologies for cancers, Alzheimer’s disease and other areas of clinical interest are considered likely to benefit from genome information and possibly may lead in the long term to significant advances in their management.

There are also many tangible benefits for biological scientists. For example, a researcher investigating a certain form of cancer may have narrowed down his/her search to a particular gene. By visiting the human genome database on the world wide web, this research can examine what other scientists have written about this gene, including (potentially) the three-dimensional structure of its product, its function(s), its evolutionaty relationships to other human genes, or to genes in mice or yeast or fruit flies, possible detrimental mutations, interactions with other genes, body tissues in which this gene is activated, diseases associated with this gene or other data types.

(b) Locus Link is a National Center for Biotechnology Information (NCBI) online resource. It is designed to link together related information on genetic loci and gene products from several sources.

Microprocessor : A microprocessor or processor is the heart of the computer and it performs all the computational tasks, calculations and data processing etc. inside the computer. Microprocessor is the brain of the computer. In the computers, the most popular type of the processor is the Intel Pentium chip and the Pentium IV is the latest chip by Intel Corporation. The microprocessors can be classified based on the following features.
Instruction Set: It is the set of the instructions that the Microprocessor can execute.
Bandwidth : The number of bits processed by the processor in a single instruction.
Clock Speed : Clock speed is measured in the MHz and it determines that how many instructions a processor can processed.

European Molecular Biology Laboratory (EMBL) : It was established to collect, organise and distribute data on nucleotide sequence and other information rebated to them. Nucleotide Sequence Database (also known as EMBL -Bank) constitutes Europe’s primary nucleotide sequence resource. Main sources for DNA and RNA sequences are direct submission from individual researches, genome sequencing projects and patent applications.

Taxonomy Browser: This search tool provides taxonomic information on various species. The Taxonomy database of NCBI has information (including scientific and common names) about all organisms for which some sequence information is available (over 79,000 species). The server provides genetic information and the taxonomic relationship of the species in question. Taxonomy has links with other servers of NCBI e.g., structure and PubMed.

(c) Site-directed mutagenesis is a molecular biology technique in which a mutation is created at a specific site in the DNA molecule.


Questions in Regards to Isolating Promotor Region for Recombinant DNA Techniques - Biology

Biology chapter exam prep

Gene cloning is crucial to any application involving one gene because _____. ( Concept 20.1)

A)naturally occurring DNA molecules are very long and contain many genes

B)it provides a means to produce large quantities of its protein product

C)genes occupy only a small proportion of the chromosomal DNA in eukaryotes, the rest being noncoding nucleotide sequences

D)it provides a means to produce many copies of a gene in short period of time

E)All of the listed responses are correct.

What is an advantage to using a bacterial artificial chromosome (BAC) for generating a genomic library compared to a plasmid or phage that has historically been used for this process? ( Concept 20.1)

A)BACs carry DNA fragments much larger than plasmids or phages and greatly minimize the number of clones needed to make up the genomic library.

B)The use of BACs reduces the frequency with which specific genes will be cut within the coding region by restriction enzymes and divided up among two or more clones.

C)Because of their size, BACs are much less difficult to work with in the lab than plasmids or phages.

D)The first and second responses listed are correct.

E)The first three listed responses are correct.

In which of the following would it be advantageous to create and work with a cDNA (complementary DNA) library rather than a genomic library? ( Concept 20.1)

A)a study of the role that noncoding RNA plays in regulating the expression of the coding genes of a genome

B)sequencing the enhancer region of a gene that regulates neural development of a frog

C)a study of a protein involved in eye development of a salamander and the regulation of the gene that expresses it

D)a comparison of the sequences of introns within a gene shared among different lineages of reptiles

E)A cDNA library would not be the appropriate choice for any of the listed responses.

The expression of the PAX-6 gene when vertebrate and fruit fly versions of the gene are exchanged between these animal groups illustrates _____. ( Concept 20.1)

A)that the same gene can have very different functions in different types of animals

B)that some coding genes have products other than proteins in different types of animals

C)the common ancestry in the evolution of these animal groups

D)that the mechanisms of gene expression vary among different animal groups

E)that a gene that plays a major role in the development of one type of organism often has a reduced role in another

Which of the following enzymes is key to the automation of PCR (polymerase chain reactions)? ( Concept 20.1)

Bacteria use restriction enzymes to _____. ( Concept 20.1)

An enzyme that cuts DNA at a symmetrical sequence of bases is called _____. ( Concept 20.1)

When a typical restriction enzyme cuts a DNA molecule, the cuts are staggered so that the DNA fragments have single-stranded ends. This is important in recombinant DNA work because _____. ( Concept 20.1)

A)it allows a cell to recognize fragments produced by the enzyme

B)the single-stranded ends serve as starting points for DNA replication

C)the fragments will bond to other fragments with complementary single-stranded ends

D)it enables researchers to use the fragments as introns

E)only single-stranded DNA segments can code for proteins

In genetic engineering, "sticky end" refers to _____. ( Concept 20.1)

A)a technique for finding a gene of interest within a nucleus without destroying the cell

B)the ability of plasmids to stick to a bacterial cell wall and thus be taken up into the bacterium

C)short bits of single-stranded DNA left at the end of DNA molecules cut by restriction enzymes

D)the site on mRNA that sticks to the DNA during transcription

E)None of the listed responses is correct.

Which of the following enzymes could seal a nick in one strand of a double-stranded DNA molecule by creating a sugar-phosphate bond between the adjacent, unjoined nucleotides? ( Concept 20.1)

To create recombinant DNA with long-term stability, it is necessary to have which of the following in the test tube? ( Concept 20.1)

E)heat-resistant DNA polymerase

What two enzymes are needed to produce recombinant DNA? ( Concept 20.1)

A)a restriction enzyme and a topoisomerase

B)a restriction enzyme and a ligase

C)a restriction enzyme and a polymerase

D)a polymerase and a ligase

E)a polymerase and a topoisomerase

In recombinant methods, the term "vector" refers to _____. ( Concept 20.1)

A)the enzyme that cuts DNA into restriction fragments

B)the sticky ends of a DNA fragment

D)a plasmid or other agent used to transfer DNA into a living cell

E)a DNA probe used to locate a particular gene

Which arrangement of the following four enzymes represents the order in which they would be used in a typical gene-cloning experiment resulting in the insertion of a cDNA into a bacterial plasmid? Begin with the gene's mRNA transcript. ( Concept 20.1)

A)restriction enzyme, reverse transcriptase, DNA polymerase, DNA ligase

B)restriction enzyme, DNA ligase, reverse transcriptase, DNA polymerase

C)reverse transcriptase, DNA polymerase, restriction enzyme, DNA ligase

D)reverse transcriptase, DNA ligase, DNA polymerase, restriction enzyme

E)reverse transcriptase, restriction enzyme, DNA polymerase, DNA ligase

A scientist wishing to create an organism capable of breaking down several kinds of toxic waste combines genes from several species of bacteria to create a single "superbacterium." Which of the following would be needed to do this? ( Concept 20.1)

E)All of the listed responses are correct.

A nucleic acid probe is used to _____. ( Concept 20.1)

B)produce a large amount of DNA from a tiny amount of DNA

C)make exact copies of DNA sequences

D)identify genes that have been inserted into bacterial plasmids or separated by electrophoresis

What is the source of the reverse transcriptase used in recombinant DNA technology? ( Concept 20.1)

D)cultured phage-infected mammalian cells

E)either retroviruses or cultured phage-infected mammalian cells

Because eukaryotic genes contain introns, they cannot be translated by bacteria, which lack RNA-splicing machinery. But if you want to engineer a bacterium to produce a eukaryotic protein, you can synthesize a gene without introns. A good way to do this is to _____. ( Concept 20.1)

A)alter the bacteria so that they can splice RNA

B)use a nucleic acid probe to find a gene without introns

C)work backward from mRNA to make a version of the gene without introns

D)use a phage to insert the desired gene into a bacterium

E)use a restriction enzyme to remove introns from the gene

DNA synthesized using an RNA template is called _____. ( Concept 20.1)

In the polymerase chain reaction (PCR), the sequence of bases in the primers is important because it _____. ( Concept 20.1)

A)determines which segment of the genome will be amplified

B)always matches a stop codon

C)always causes a silent mutation

D)determines how many cycles of the reaction are needed to obtain a sufficient amount of amplified DNA

E)determines the number of tandem repeats in a genome

A molecular biologist has isolated a short segment of DNA that she wants to replicate in vitro. First she heats the DNA, which separates the two strands, and then she adds _____. ( Concept 20.1)

A)nucleotides, primers, and polymerase

B)ribosomes, nucleosomes, and messenger RNA

D)transfer RNA, matching amino acids, and messenger RNA

E)ribosomes, matching amino acids, and primers

In the polymerase chain reaction (PCR) technique, a heating phase and a cooling phase alternate. An original sample of DNA would have to pass through how many total rounds of heating and cooling before a sample is increased eight times in quantity? ( Concept 20.1)

Single nucleotide polymorphisms (SNPs) _____. ( Concept 20.2)

A)are single base-pair variations in the genomes of the human population

B)are genetic markers used to study the genetic basis for disease

C)are small nucleotide differences among individuals located in coding and non-coding sequences in the genome

D)can be the molecular basis for different alleles

E)All of the listed responses are correct.

Separating DNA fragments by gel electrophoresis is useful for which of the following? ( Concept 20.2)

A)identifying DNA fragments for RFLP analysis

B)purifying specific DNA fragments

C)distinguishing between different alleles of a gene

D)identifying a plasmid or a virus by examining its restriction fragment pattern

E)All of the listed responses are correct.

Southern blotting is _____. ( Concept 20.2)

A)a method of DNA amplification

B)a technique used to study RFLPs

C)how bacteria take up DNA from the surrounding solution

D)the insertion of DNA into a plant's chromosomes

E)used to determine the product of a particular gene

Which of the following is the first step of the Southern blotting procedure? ( Concept 20.2)

A)hybridizing the DNA with a radioactive probe

B)digesting the DNA with a restriction enzyme

C)separating the DNA fragments using gel electrophoresis

D)transferring the DNA to a blot

E)using the blot to expose photographic film

The dideoxyribonucleotide chain-termination method _____. ( Concept 20.2)

A)produces a ladder of DNA fragments, with each individual band labeled with one of four different fluorescent tags

B)can be used to sequence entire eukaryotic chromosomes in a single reaction

C)is very slow, requiring several weeks to determine a sequence of about 200 nucleotides

D)does not involve electrophoresis

E)is difficult to automate and must be performed under close human supervision

The term "RFLP" stands for _____. ( Concept 20.2)

A)restriction fragment length polymorphism

B)reverse fragment ligated polymerization

C)really fast ligation protocol

D)restriction fragment ligation procedure

E)RNA fragment length pool

RFLPs have been tremendously useful for genomic mapping studies because _____. ( Concept 20.2)

A)they are found only in the coding sequences of genes

B)they are found only in the promoter regions of genes

C)they are found only in disease-causing genes

D)they are not restricted to genes, and are abundantly scattered throughout the genome

E)they are found only in expressed genes

The efficiency of cloning, and the ability to generate healthy cloned animals, has been largely hampered by the difficulty of _____. ( Concept 20.3)

A)completely reversing epigenetic alterations in donor cell nuclei such as DNA methylation and chromatin packing

B)inducing recombination in differentiated donor cells in order to restore the full genomic complement

C)transforming donor cells with genes encoding proteins required for normal embryonic development

D)physically removing the nucleus from the egg cell that will ultimately receive the donor cell nucleus

E)implanting the clone into the surrogate mother

"Therapeutic cloning" refers to _____. ( Concept 20.3)

A)the use of cloned embryos as a source of stem cells that could be used to treat disease

B)treating patients with therapeutic proteins made using recombinant DNA technology

C)cloning animals to obtain organs that could be used for transplantation into humans

D)treating a genetic disease by obtaining cells from an individual with the disease, introducing genes into the cells in order to repair the genetic defect, and then reintroducing the cells back into the individual

E)All of the listed responses are correct.

Nuclear transplantation involves _____. ( Concept 20.3)

A)inserting a sperm cell into an egg cell in vitro

B)placing the nucleus from an egg cell into an enucleated somatic cell

C)removing the nucleus of an egg cell and replacing it with the nucleus of a somatic cell

D)the use of microarray analysis and RNA interference

E)the use of reverse transcriptase to make copies of the genes that are being expressed.

_____ can give rise to any type of cell whereas _____ can give rise to a subset of cell types. ( Concept 20.3)

A)Heterozygous cells . homozygous cells

B)Adult stem cells . embryonic stem cells

C)Embryonic stem cells . adult stem cells

D)Totipotent cells . nerve cells

E)Adult stem cells . totipotent cells

During the process of differentiation, cells _____. ( Concept 20.3)

B)exchange DNA with other cells via the process of horizontal gene transfer

C)gain and lose genes, depending on what type of cell they will become

D)express different genes in response to cell signaling

E)randomly turn on and off genes until the right combination is reached

Dolly, the sheep, was cloned from an adult cell. She had a number of health problems and died at a relatively young age. Three mules that were born in 2003 were cloned from fetal cells. If it turns out that the mules remain healthy and live normal lives, how would this outcome tie in with Gurdon's observations with tadpoles? ( Concept 20.3)

A)Gurdon found no correlation between the age of the donor cells and the ability of the transplanted nucleus to direct development.

B)Gurdon found that nuclei from older donor cells were more likely to correctly direct differentiation and give rise to healthy tadpoles.

C)Gurdon found a positive correlation between the age of the donor nuclei and the ability of the nuclei to direct differentiation.

D)Gurdon found that the ability of a transplanted nucleus to direct normal development was inversely related to the age of the donor.

E)None of the listed responses is correct.

All of the following are true regarding induced pluripotent stem (iPS) cells except _____. ( Concept 20.3)

A)iPS cell technology may provide a more morally acceptable approach to therapeutic cloning

B)iPS cells have been demonstrated to function identically to embryonic stems cells

C)the reprogramming of diseased cells in humans to form iPS cells could provide model systems for studying the origins of the disease

D)iPS cell technology could offer the potential to regenerate nonfunctional or diseased tissues and avoid the risk of transplant rejection in the diseased patient

E)iPS cells are formed by added genes to the genome of differentiated skin cells

All of the following are current applications of DNA technology in medicine except _____. ( Concept 20.3)

A)clinical use of iPS cells harvested from organ-impaired individuals for the culturing and transplantation of a functioning organ in the diseased individual

B)use of genome-wide association studies to identify SNPs (single-nucleotide polymorphisms) linked to disease

C)use of microarray assays to analyze the expression patterns of genes associated a type of cancer

D)use of retroviruses to introduce normal alleles of genes into diseased cells for disorders involving one defective gene

E)genetically engineering organisms, from bacteria to goats, into protein factories that produce vital human proteins such as insulin, anticlotting agents, and human growth hormone

A genetic marker is _____. ( Concept 20.4)

A)a place where a restriction enzyme cuts DNA

B)a chart that traces the family history of a genetic trait

C)a particular nucleotide sequence at a particular locus whose inheritance can be followed

D)a radioactive probe used to find a gene

E)an enzyme used to cut DNA

Human nerve cells differ from human muscle cells because different sets of genes are expressed in each type of cell, different genes are transcribed into mRNA and translated into protein. Which of the following techniques would be the most efficient way to identify the genes that these cells express? ( Concept 20.4)

A)gel electrophoresis of DNA fragments

C)isolating and analyzing all the proteins from each type of tissue

Gene therapy involves _____. ( Concept 20.4)

A)adding a functioning version of a defective gene to the cells of an individual

B)allowing individuals to follow the natural progression of a genetic disorder, accompanied by psychological counseling, then drug treatment when the condition becomes life-threatening

C)no serious ethical questions

D)replacing organs affected with genetic disorders by transplants

E)All of the listed responses are correct.

A molecular biologist used a retroviral vector to introduce a gene coding for a certain human enzyme into mouse cells. One cell line was isolated that was able to make the human enzyme, but it had lost the ability to express an endogenous, normally expressed gene in the process. What is the best explanation for these results? ( Concept 20.4)

A)The virus caused the mouse cells to become diseased.

B)The virus had transferred a gene from one mouse cell to another.

C)The virus inserted the gene encoding the human enzyme within the sequence of a normally expressed endogenous gene.

D)The virus was too small to carry the entire gene.

E)The enzyme acted as a nuclease enzyme, cutting up mouse DNA.

DNA fingerprints are used to determine whether Sam could be the father of Becky's baby. Sam is not the father if _____ genetic fingerprint shows some bands not present in _____ genetic fingerprint. ( Concept 20.4)

E)the baby's . Sam's or Becky's

DNA fingerprints used as evidence in a murder trial look something like supermarket bar codes. The pattern of bars in a DNA fingerprint shows _____. ( Concept 20.4)

A)the order of bases in a particular gene

B)the presence of various-sized fragments of DNA

C)the presence of dominant or recessive alleles for particular traits

D)the order of genes along particular chromosomes

E)the exact location of a specific gene in a genomic library

Which of the following would be considered a transgenic organism? ( Concept 20.4)

A)a bacterium that has been treated with a compound that affects the expression of many of its genes

B)a human treated with insulin produced by E. coli bacteria

C)a fern grown in cell culture from a single fern root cell

D)a rat with rabbit hemoglobin genes

E)All of the listed responses are correct.

Transgenic organisms can be scientifically or commercially useful only if _____. ( Concept 20.4)

A)the inserted ("foreign") gene is drawn from the human genome

B)the inserted ("foreign") gene is expressed in the host organism

C)the host organism is a microorganism

E)All of the listed responses are correct.

In genetic engineering, the highly active plasmid from Agrobacterium tumefaciens is used to _____. ( Concept 20.4)



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