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Recombinant DNA Technology Help (page 3)

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By — McGraw-Hill Professional
Updated on Aug 23, 2011

Cloning Vectors

There are several choices for cloning vectors: (a) bacterial plasmids, (b) bacteriophage vectors, and (c) hybrid vectors.

  1. Bacterial Plasmids. Bacterial plasmids are naturally occurring small, circular, extra chromosomal DNA molecules. They contain origins of replication so they are autonomously replicating (i. e. , they can replicate separate from the chromosome). They naturally contain genes for antibiotic drug resistance, disease virulence factors, and gene transfer proteins. Molecular biologists have learned how to isolate and manipulate these plasmids to help in genetic engineering techniques.
  2. The larger the plasmid, the more inefficient it is as a cloning vector; larger plasmids are less easy to manipulate in vitro and less efficient in transformation. Transformation is the process by which foreign DNA (e. g. , a plasmid) is taken up and incorporated into a bacterial host cell. Therefore, smaller (2–4 kb), nontransmissible plasmids that contain two different antibiotic-resistance genes are normally used as vectors. One of the original vehicles of this kind is the E. coli plasmid pBR322. It consists of 4363 bp and contains resistance genes for the antibiotics tetracycline and ampicillin. There is a single restriction endonuclease site for the restriction enzyme BamHI in the entire pBR322 plasmid, and that site is within the tetracycline-resistance gene (tet-r). If both the donor DNA and the plasmid DNA are cut with BamHI, the donor fragments can be spliced into the plasmid as described previously. The insertion of a foreign piece of DNA within the tet-r gene destroys the ability of this plasmid to confer resistance to tetracycline on the recipient bacterial cell, causing it to become sensitive to tetracyline (Tet-s). Recipient cells that are sensitive to both antibiotics are transformed with the gene library plasmids, some of which contain the donor DNA insert of interest. Three types of cells are produced from this transformation (see Fig. 12-8). Those cells that were not transformed remain Amp-s and Tet-s and will not grow on media containing either antibiotic; those that were transformed are Amp-r, but there are expected to be both Tet-s and Tet-r cells within this group. Tet-s cells contain plasmids with a DNA insert and Tet-r cells do not; i. e. , their tet-r gene was not inactivated by the insertion of a foreign DNA molecule. One of the problems with plasmid vectors is that they can only accommodate smaller (< 5–8 kb) DNA inserts. Other vectors, such as bacteriophage λ derivatives and yeast artificial chromosomes, that can take larger DNA inserts (from 10 to over 50 kb) have been developed to overcome this problem.

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  3. Bacteriophage Vectors. Many bacteriophage vectors have been developed using lambda (λ) bacteriophage (for more on viruses, see Chapter 11). The central region of phage lambda (λ) contains genes involved in establishing and maintaining the lysogenic state, and hence is not essential for its lytic cycle. This region can be replaced with a foreign DNA insert, if it is of an appropriate size (up to 20 kb), and still allow the phage DNA to be packaged into phage heads. Large foreign inserts tend to be unstable in plasmids, so the two vectors complement one another. Furthermore, transduction (the transfer of genetic information to a bacterial cell via a viral intermediate) is a much more efficient process than transformation, and it avoids the problem of the vector closing up without an insert. Genetically manipulated phage DNA without such an insert will not be packaged properly to become functional (infective) virions. The restriction enzyme EcoRI cuts lambda DNA at both ends of the nonessential region. The two essential end regions can be isolated by electrophoresis and ligated in vitro with foreign DNA cut by that same enzyme (Fig. 12-9).
  4. Lambda-sensitive bacteria are grown on agar plates in high density to form a lawn of confluent growth. The artificially synthesized transducing phage are added in a concentration resulting in about 100 phage particles per plate, hence producing about 100 plaques of lysed bacterial cells per plate. Each plaque contains phage clones containing millions of identical phage genomes. Recombinant phage are produced when foreign DNA (e. g. , mammalian DNA ) is ligated to the manipulated phage (Fig. 12-9).

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  5. Hybrid Vectors. Cosmids are plasmids into which have been inserted the cos sites (cohesive end sites) required for packaging lambda DNA into its capsid. Cosmids can be perpetuated in bacterial cells or purified by packaging in vitro into phages. The main advantages of using cosmids are that inserts much longer than 15 kb can thereby be cloned and the ease of selecting a recombinant plasmid is greatly improved.
EXAMPLE 12. 6 Plasmid ColE1 carries a gene for resistance to rifampicin (rif-r) and the cos sites of phage lambda, which can be recognized by the cos-site-cutting (Ter) system of E. coli. Cosmids such as this can function properly, provided that two cos sites are present and the cos sites are separated by no less than 38 kb and no more than 54 kb. Cleavage of ColE1 and foreign DNA by the restriction enzyme Hind III can be used to produce linear, recombinant molecules (Fig. 12-10). Transducing phage particles can be formed if the insert between the two cos sites is 38–54 kb in length. No particles are produced if no insert is made or if the insert is larger or smaller than that range. In vitro packaging (adding heads and tails) forms transducing particles containing cosmids with cohesive termini. Upon infection of a rifampicin-sensitive (Rif-s) cell with a transducing phage particle, the linear chimera becomes circularized and replicates using the ColE1 replication system. Plating cells on medium containing rifampicin selects for those cells containing the rif-r gene, the ColE1 region, and a foreign insert.

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Identifying The Clone of Interest

Finding a cell that contains the insert of interest among all the cells of a genomic library is a major task. The process involves screening a large number of the recombinant cells or phage obtained from the transformation or transduction to find the desired clone. The likelihood of finding the desired gene fragment in a particular gene library can be estimated by the following formula, where N equals the number of recombinants required to screen, n equals the ratio of the organism's genome size relative to the average fragment size in the gene library, P equals the probability (i. e. , p = 0. 95 means there will be a 95% probability of finding the clone), and ln equals the natural log:

  1. Screening a DNA library. The type of screening method used is dependent upon the cloning vector chosen. When using a plasmid vector, locating the desired DNA fragment can be accomplished by an in situ hybridization technique known as colony hybridization (Fig. 12-11).

     

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    Recombinant colonies growing on nutrient agar plates are transferred to a piece of nitrocellulose or nylon membrane by pressing it into the plate, thereby transferring some cells from each colony (replica plating). The pattern of the colonies remains intact on the paper or membrane. The paper is then treated with a dilute sodium hydroxide solution to lyse the cells and denature the DNA into single strands. The cell contents are released, and its DNA binds tightly to the paper. Next, the sodium hydroxide is neutralized with acid. The paper is then covered with a solution containing a labeled DNA probe (a single-stranded piece of DNA that is composed of a sequence that is complementary to a portion of the gene of interest). The label can be a radioactive or fluorescent tag as discussed earlier. The probe DNA binds to, or hybridizes, to complementary DNA sequences that exist on the paper, thereby becoming indirectly bound to the paper. The paper is washed to remove any unbound probe, and is then exposed to X-ray film for autoradiography of radioactive probes or another mechanism for detecting fluorescent labels. Detected spots correspond to colonies that contain the gene of interest. Cells from the corresponding clones can be grown in broth culture, thus producing a lot of plasmid. Plasmid DNA is easily isolated from the bacterial cells and the gene of interest can be liberated from the plasmid by digestion with the same restriction endonuclease that was used for its insertion. It can then be isolated from the larger plasmid DNA by electrophoresis. For screening phage libraries, a similar technique can be used.

  2. Screening an Expression Library. The most common procedures for detecting protein-secreting clones usually involve antibodies in an immunoassay. Antibodies are protein molecules produced by immune cells that are capable of binding to particular sites on other proteins called antigens. Antibodies are produced in response to a foreign protein antigen introduced into vertebrate animals. Antibodies against one protein antigen are usually highly specific and they can be produced and then purified for use in immunoassays. A label or tag can be attached to antibodies to enable their detection once they have bound to a corresponding antigen. The most sensitive labels are radioactive isotopes used for radioimmunoassay (RIA) or enzyme labels used in enzyme linked immunosorbent assay (ELISA). The latter are often preferred because of the handling and disposal problems associated with radioactive materials.
EXAMPLE 12. 7 Agar containing lysozyme and antibodies to a specific protein of interest is poured over bacterial colonies on a Petri plate and allowed to harden. Colonies lysed by lysozyme release their proteins. If the protein of interest is present, the antibodies will react with it and form a ring of precipitate around the colony.

Site-Directed Mutagenesis and Protein Engineering

It is possible to introduce one or more nucleotide alterations of known composition and location into specific genes or regulatory sequences. For example (Fig. 12-12), a plasmid carrying a gene of interest can be nicked at one position with an endonuclease.

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The plasmid DNA is then denatured and intact single-stranded circles are isolated. Short (13–30 bases) oligonucleotides of known complementary structure (usually synthesized de novo) can be made to have a mutant base at a desired site. This oligonucleotide is renatured with the intact single stranded circles to serve as a primer for in vitro replication of a new DNA strand that is not completely complementary to that of the plasmid strand. The replicated circles are sealed with DNA ligase. Those covalently closed circles are isolated and used to transform bacteria. During in vivo replication, each strand of the plasmid serves as a template for producing a progeny strand. Thus, some plasmids are produced with wild-type gene sequences and some with a single base-pair mutation at a known site. This example is just one of the many different ways to introduce known and random mutations into DNA sequences. After isolation, the mutants can be identified and then evaluated for their effects on the functioning of the gene or regulatory sequence. Biochemical and structural characteristics of proteins can be altered by making directed changes in the amino acid sequence. For example, protein stability, activity, temperature resistance, and pH optimum are just some of the qualities that can be changed using this technique. This is referred to as protein engineering.

Polymorphisms

A polymorphism is the existence of two or more alleles at a locus in a population. Conventionally, a polymorphic element or locus is one at which the frequency of the most common allele is less than 0. 99. Polymorphisms may exist minimally at three levels: (1) chromosome, (2) gene, or (3) restriction fragment length. DNA sequence polymorphisms can be as simple as a single nucleotide difference (known as SNP for single nucleotide polymorphism) or an insertion or deletion of a number of nucleotides (indel). Either of these types of polymorphisms can lead to differences in the abilities of restriction enzymes to recognize and cut a specific site. This type of polymorphic measure is called a restriction fragment length polymorphism (RFLP). Chromosomal polymorphisms that are large enough to be detected under the light microscope may involve euploidy, aneuploidy, translocations, inversions, duplications, or deficiencies.

EXAMPLE 12. 8 If the EcoRI restriction enzyme site, GAATTC, exists in three particular nucleotide positions in a gene sequence (allele 1) and mutation results in an alteration of the middle site to CAATTC (allele 2). The enzyme will no longer be able to recognize and cut the DNA strands at this position, resulting in a single 500-bp EcoRI fragment for allele 2 when compared with the two fragments (150 and 350 bp) predicted by cutting of allele 1. New restriction sites can also be created by mutation where there previously were none or different ones (see Example 12. 9).

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One technique used to analyze RFLPs is Southern blotting, named after E. M. Southern, who first developed it. A restriction enzyme digest of an individual's DNA is electrophoresed (i. e. , separated from other DNA sequences) on an agarose gel and then denatured to single strands. The single-stranded fragments are then transferred from the gel to nitrocellulose paper in the following manner. The gel is placed on normal filter paper that has been soaked in concentrated salt solution. The nitrocellulose paper is placed on top of the gel, with dry blotting paper and a weight on top of that. The salt solution moves through the gel, carrying the DNA fragments with it onto the nitrocellulose paper where they become trapped. The fragment pattern on the gel is thereby faithfully transferred onto the nitrocellulose. The fragment(s) of interest can then be located on the nitrocellulose by in situ hybridization with a radioactive DNA probe, followed by autoradiography.

EXAMPLE 12. 9 The normal gene for the b-globin chain of human hemoglobin has a GAG codon for glutamic acid as the sixth amino acid from the N terminus. Individuals with sickle-cell anemia have a mutant, GTG for valine, at that same position. Since fetal hemoglobin does not contain β-globin chains, it is impossible to obtain fetal hemoglobin for prenatal analysis of this genetic disease. However, fibroblasts (which normally do not make hemoglobin) contain the gene for b-chain of hemoglobin, and these cells can be retrieved by amniocentesis (see Human Cytogenetics section in Chapter 7, p. 193). The total DNA from fibroblast cells is digested with the restriction endonuclease MstII and the fragments are separated by electrophoresis on an agarose gel. The DNA is then transferred onto a nitrocellulose membrane by Southern blotting, denatured to single strands, incubated with a radioactive b-globin gene probe, and autoradiographed. Only one DNA band of 1300 bp appears on the autoradiograph for normal hemoglobin (HbA), whereas two bands of lengths 200 and 1100 bp appear for sickle cell hemoglobin (HbS). Hence, the GAG codon in the b-chain gene of HbA is not part of a recognition site for MstII, but the mutation to GTG in HbS creates a new MstII site.

A similar technique, referred to as northern blotting, is used to identify RNA molecules that are similar to a probe sequence. Transfer of a protein electrophoresis pattern from a gel to a paper is called western blotting. In this case, the probe is usually a labeled antibody against the protein of interest.

Analysis of polymorphisms is useful in medical and forensic genetics.

  1. Medical Genetics. Medical genetics is the application of genetics to health promotion and disease diagnosis. The analysis of SNPs and other polymorphisms has been heralded as an important aspect of finding the genetic causes of complex diseases, such as heart disease. These types of diseases are most likely caused by the interactions of multiple genetic factors with environmental influences, such as diet. The International HapMap Project (http://www. hapmap. org) is cataloging the common haplotypes, or regions of linked SNP variants (Fig. 12-13) within the human population in different countries across the globe.

     

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    There are estimated to be up to 10 million common SNPs in the human genome, so identifying clusters of linked SNPs eases the job of identification. Haplotypes can be used as markers associated with various complex diseases. These markers may lead geneticists to specific genes that influence the disease phenotype. Haplotype analysis has been used to identify a mutation in a gene, CFH1 (complement factor H), that increases the risk of developing age-related macular degeneration (AMD). This disease is the leading cause of blindness among people over age 50 and affects more than 10 million Americans. Haplotype mapping is also useful for tracing ancestral lineages because they are tightly linked so recombination does not occur very often between them.

Forensic genetics can be used to determine the identity or nonidentity of DNA from cells (e. g. , blood, hair, semen) left at the scene of a crime with the DNA of cells of any suspect. It can also be used in cases of disputed parentage or for identifying the parentage of missing children. This branch of genetics utilizes a technique known as DNA fingerprinting to distinguish the DNA of a human from that of any other person. It depends on the fact that there are different numbers of tandem repetitive DNA sequences scattered throughout different human genomes. Any DNA sequence that exists inmultiple copies strung together in various tandem lengths is referred to as a minisatellite or a variable number of tandem repeats locus (VNTR locus). The number, pattern, and length of these repeats are unique for each individual. Regardless of length, each repeat contains a common (usually < 20 bp) core sequence that can be recognized by an appropriate radioactive probe. DNA is extracted from a convenient sample of cells (e. g. , white blood cells) and subjected to cleavage by one or more restriction endonucleases. The fragments are separated on a gel, denatured to single strands, transferred to a filter by Southern blotting, exposed to a radioactively labeled probe, and then autoradiographed. The number shown of bands and sizes on the autoradiograph are unique for each individual.

Practice problems for these concepts can be found at:

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