Recombinant DNA Technology Help (page 3)
Recombinant DNA Technology
Two primary advances helped usher in the new era of molecular genetics in the late 1970s to the early 1980s: (1) the use of recombinant DNA technology (genetic engineering) used to isolate and manipulate genes in vitro in order to endow cells with new synthetic capabilities and (2) the ability to synthesize and determine the linear order of nucleotides of DNA molecules (DNA sequencing). Recombinant DNA technology has made it possible to clone (isolate and make copies of individual genes) and transfer genes between bacterial species and strains or from eukaryotes into bacteria (or vice versa), causing the engineered cells to produce, sometimes in relatively large quantities, proteins of great economic importance such as enzymes (e. g. , amylases, proteases), hormones (e. g. , insulin, growth hormone), and interferons (lymphocyte proteins that prevent replication of many viruses). These proteins are made in such small quantities in human cells that the cost of their extraction and purification from blood or cadaver tissues has been very expensive, thus restricting their medical use in prevention and treatment of disease. Many products are now produced successfully using genetic engineering techniques; such products include blood-clotting factors (e. g. , tissue plasminogen activator, or TPA, used to activate the breakdown of blood clots and to prevent recurrence of blood clots in heart attack patients), and complement components (part of the immune system). In 1980, the United States Supreme Court decreed that new life forms created by genetic engineering could be patented, although this is not true in many other countries. This decision has contributed to the investment of large sums of money by private corporations into the development of many useful recombinant strategies. Public outcry against recombinant, or GM (genetically modified), crops in Europe had led the European Union (E. U. ) to ban or require extensive labeling requirements of foods that contain ingredients from GM crops. This has caused significant tension between the U. S. food industry and E. U. countries.
There are many hopes for the future benefit of genetic engineering technology in helping solve some of society's problems. In the agricultural arena, soybeans, corn, and cotton have been genetically engineered or modified to contain added herbicide tolerance and pest-resistance genes. These crops are also known asGMOs (genetically modified organisms). For example, one type of GM corn (Bt corn) contains a gene from the bacterium Bacillus thuringiensis that produces a protein that kills insect larvae. Larvae of the European corn borer can be costly pests for corn farmers. Having the pesticide gene in the corn eliminates the need to spray the crop with a pesticide. Other crops have been or are being engineered to contain genes that encode vaccines, added nutrients, and disease resistance, as well as resistance against cold, drought, and salinity. These few examples suffice to demonstrate the possibilities of this new technology and explain why there is such great excitement in the scientific, medical, agricultural, and pharmaceutical communities concerning its further development. While this technology is exciting and has proven useful, some have voiced concerns over potential environmental hazards, unknown risks to human health, and economic impact. These issues are still being investigated and must be resolved in order to make this technology useful and safe.
Most genetic engineering projects begin with the cloning, or isolation and amplification, of a gene. There are two major purposes for cloning a gene: (1) to obtain the sequence of its DNA (e. g. , in order to discover how an abnormal gene differs structurally from its normal allele) and (2) to produce large amounts of a gene product (i. e. , protein). Gene cloning generally involves three major steps: (1) A DNA fragment (or gene) of interest is isolated. (2) The fragment is spliced into a cloning vehicle called a cloning vector, such as a phage or a plasmid. (3) The vector is introduced into a host cell where it is replicated many times using the host cell's DNA replication machinery. A good cloning vector should have three characteristics: (1) the ability to replicate autonomously from the main chromosome to make many copies within the cell, (2) one or more unique recognition sites for restriction enzymes so it can be opened at a single position to receive a foreign DNA insert (i. e. , the gene of interest being cloned), and (3) a gene that encodes resistance to an antibiotic agent that will allow for the distinguishing of cells that contain the cloning vector as opposed to cells that do not.
Once the gene is cloned it can be analyzed (restriction mapped and its DNA sequence determined) and then transferred into a host cell or organism that will allow it to be expressed as a protein product. The host system might be a genetically engineered (transgenic) plant or a mammalian cell culture, yeast culture, or bacterial cell system that is used as a biological factory to produce large quantities of the desired product.
Cutting and Ligating DNA Molecules
- Enzymes that Create Cohesive Ends. DNA cut by restriction enzymes that leave complementary cohesive ends can be rejoined when mixed together in a test tube. They are brought together by their complementary sequences and then an enzyme called T4 DNA ligase seals the nicks. A common "cutting" and "pasting" scenario is shown in Fig. 12-1,where one of the cut fragments is a linear piece of genomic DNA from amammal and the other is a circular bacterial plasmid. When these two fragments come together in the presence of DNA ligase, a stable, covalently closed, circular recombinant DNA molecule is created. This recombinant molecule is a hybrid or chimera of DNA from bacteria and a mammal.
- Joining Blunt-Ended DNA Fragments. DNA fragments that contain complementary cohesive ends can be joined by the action of the enzyme DNA ligase, as discussed earlier. This enzyme, originally isolated from the bacterial phage T4, catalyzes the formation of a phosphodiester bond between adjacent nucleotides. This enzyme can also be used to join blunt-ended fragments together, but there are several methods that can be employed.
- Blunt-End Ligation. DNA ligase can be used to directly join the blunt ends of double-stranded DNA (ds DNA ) fragments. An advantage of this method is that it can join two defined sequences without introducing any additional material between them. The inability to control which pairs of blunt ends become joined is an obvious disadvantage (Fig. 12-2).
- Homopolymer Tails. An enzyme called terminal deoxynucleotidyl transferase can add any available deoxynucleotides to the 30 end of a single-stranded region of DNA without need of a template. If a string of A nucleotides are added onto the vector DNA and thymines (T) are added onto the foreign DNA , homopolymer tails are produced that should base-pair. The gap (absence of one or more nucleotides in a DNA molecule) can be filled by DNA polymerase I using the other complete strand as a template. The nick (absence of a phosphodiester bond between adjacent deoxyribose sugars) can be sealed byDNA ligase (Fig. 12-3).
- Linkers. A third method employs short DNA segments, called linkers or adapters, that contain a restriction enzyme recognition site; these can be synthesized chemically. Linkers can be added covalently to the ends of a plasmid or to an insert by blunt-end ligation (Fig. 12-4). This method imposes no restriction on the choice of sites to generate the ends, yet allows retrieval of the insert by cleavage with the appropriate restriction enzyme.
Blunt-ended DNA fragments can also be modified at their ends to become overhangs. These ends can pair with other complementary ends and be sealed by DNA ligase. There are two main methods for modification of blunt-ends.
It is also possible to convert cohesive ends into blunt ends by treatment with either an enzyme that will extend the shorter side of a 5' overhang or one that will degrade the cohesive end of a 3' overhang. Both of these methods employ the use of a DNA polymerase that either synthesizes new DNA in the case of extension or "filling in," or acts as a 3' to 5' nuclease in the case of degrading the 3' overhang. In either case, the original restriction enzyme recognition site is generally destroyed. In the following diagram, N-N represents any conventional complementary base pair.
Polymerase Chain Reaction (PCR)
Cloning DNA segments for use in a variety of purposes (i. e. , DNA sequencing) was formerly a relatively laborious in vivo process. However, in 1985, an in vitro technique, called the polymerase chain reaction (PCR), was developed for making large amounts of any DNA sequence without the need for cloning using gene banks or libraries. (see page 341). The PCR technique (Fig. 12-5) requires a pair of primers that are usually short (12–20 nucleotides long) pieces of chemically synthesized DNA (oligonucleotides), having nucleotide sequences specifically complementary to those on opposite strands flanking the target region to be copied.
These primers thus define the ends of the DNA segment that will be duplicated. The original template source of DNA does not have to be highly purified, and even a very small amount of template can serve as the initiator for the PCR. For example, cheek cells can be scraped from the inside of a cheek and placed directly into the PCR reaction. The DNA sample is heated, allowing the complementary DNA chains to separate (denaturation step). The primers are then added together with a heat-tolerant DNA -polymerizing enzyme. The primers bind to the single-stranded chains during the cooling phase (annealing step), and the polymerizing enzyme extends the primer through the rest of the fragment (extension step), creating double-stranded DNA molecules. The process is then repeated; i. e. , the mixture is reheated, and during the ensuing cooling phase the excess primers (or newly added primers) bind to template strands and become extended by the polymerizing enzyme to produce more double-stranded molecules. One cycle of denaturation, annealing, and extension can take several minutes, and about 20–30 cycles of heating plus DNA synthesis are normally run during gene amplification by the PCR (with heating and cooling times, this process can take several hours). After 20 cycles, a single DNA molecule can theoretically be amplified to about 1 million copies (called amplicons), and after 30 cycles to about 1 billion copies. With this quantity of DNA , nucleotide sequencing can be done easily.
Probes can also be used to locate genes or DNA segments of interest. Probes are commonly composed of a DNA sequence that is similar to the DNA or gene of interest. Because of the large quantity of DNA generated by the PCR, highly radioactive probes are not required, and the target segments can be detected by using nonradioactive probes or stains.
EXAMPLE 12. 4 Human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immunodeficiency syndrome (AIDS). Very few susceptible cells actually harbor the virus in an infected person. It is estimated that 1–10 copies of viral DNA per million cells can be detected through the use of the PCR and suitable probes.
There are many variations of PCR. Multiplex PCR reactions contain primers for several different DNA targets, allowing for amplification of multiple, different sized DNA regions in a single PCR reaction. Nested PCR reactions consist of two successive amplification reactions, the first containing a primer set that targets a larger region of DNA. The second reaction contains primers that result in the amplification of a DNA target region within the first amplicon. This type of PCR approach helps reduce the amplification of non-specific DNA sequences.
A library is a collection of DNA fragments from a single source, such as a particular organism or a tissue. Libraries are created for the purpose of isolating a specific DNA fragment (or gene) of interest from a particular source. There are three general methods for achieving this by using recombinant DNA technology to (1) clone all of the genes of an organism into a genomic DNA library, (2) clone individual genes or DNA sequences of interest into a cDNA library, (3) clone gene fragments into an expression library, or (4) clone DNA fragments from an environmental sample into an environmental genomic DNA library. Identification of recombinant clones in a genomic or cDNA library can be carried out using a DNA probe that contains complementary sequences to the gene fragment of interest while identification of expression library recombinants requires the use of a protein specific antibody.
Genomic DNA Library. In shotgun experiments, genomic DNA from a donor organism is cut into many pieces by the same restriction endonuclease used to cleave the plasmid vector at its single restriction endonuclease site. The two kinds of fragments—various pieces of genomic DNA and the plasmid—are mixed in vitro and allowed to randomly rejoin by their complementary (cohesive, or sticky) ends to form circles (Fig. 12-1).
If it is important that a DNA insert be connected in the proper orientation into a vector for expression of the foreign DNA , the vector and insert can be cleaved with two restriction enzymes that generate different cohesive tails at the ends of each fragment. The insertion can then occur in only one orientation (Fig. 12-6).
Each new recombinant molecule is designed to contain a different piece of the genomic DNA. Recipient bacterial cells are made permeable for transformation by the naked DNA of the plasmids by treatment with a cold calcium chloride solution. When plated on nutrient agar containing an antibiotic, each transformed cell will multiply many times to form a colony or clone, all cells of which contain multiple copies (sometimes hundreds) of the same chimeric plasmid. The transformed cells can grow on the medium containing the antibiotic since they contain a gene that confers resistance to the drug. The large set of clones that collectively contains all the donor's DNA (i. e. , its genome) is known as a genomic DNA library. It should be noted that all the DNA is cloned, not just individual genes.
- Gene or cDNA Library. This method for isolating genes is much more specific than a genomic DNA library. Instead of cloning all of the donor's DNA , only specific genes (i. e. , open reading frames) or gene fragments are cloned. If the desired protein is very small (15–20 amino acids) or a short segment of protein sequence is known, it is possible (using the genetic code) to chemically synthesize a corresponding DNA molecule. One of the earliest protein-coding sequences synthesized in this manner was that for the hormone somatostatin (14 amino acids; 42 base pairs in the DNA strand).
- Expression Library. Expression libraries are built using vectors that contain very efficient regulatory sequences that allow genes to be highly expressed in host cells. These regulatory sequences are the promoters from host cell genes that are known to be efficiently expressed. Libraries of gene fragments can be created in expression vectors using the types of cloning procedures discussed for genomic libraries in section. These libraries are screened for clones able to produce the desired protein product. Alternatively, cloned genes can be fused to the strong promoters for expression of a useful gene product, such as insulin.
- Environmental genomic DNA Library. An environmental genomic library is created in the same way as a traditional genomic DNA library except that it contains DNA fragments from a population of organisms present in a sample taken from the environment, such as soil or ocean water. Instead of containing the genome of a single organism, it contains pieces of genomes from many different organisms, such as the hundreds of different types of bacteria that might be present in a soil sample. Environmental libraries can be screened for the presence of specific genes or used for sequencing the "genomes" present in the sample. This type of analysis is sometimes referred to as metagenomics because the organismal source material is unknown and of a community nature (i. e. , not from a single cultured source). It allows for the genetic study of organisms in their natural environment that may be impossible to isolate using standard culturing techniques.
EXAMPLE 12. 5 A portion of the protein sequence for somatostatin is shown below. The genetic code was used to perform reverse genetics (from protein to DNA ) in order to figure out a hypothetical DNA sequence for this protein. Because of the degeneracy of the genetic code, the third base position in many mRNA codons may possibly contain more than one nucleotide. The potential nucleotides that can fill this position are indicated as letters in the 3rd position of the codon and below this position. This synthetic piece of DNA is known as a degenerate oligonucleotide.
Larger synthetic genes have been built using (1) PCR or (2) a combination of oligonucleotide synthesis, PCR, and restriction enzyme cutting and ligation. Most proteins, however, are too long to allow chemical synthesis of the corresponding DNA. In this case, it is possible to isolate the total population of mRNA molecules from those cells that are specialized to make the protein. For example, human insulin is produced only by the pancreas, even though the insulin gene is present in all human nucleated body cells. A purified preparation of insulin mRNA can be isolated from pancreatic cells. A synthetic oligonucleotide of thymidines (oligo-dT) is hybridized to the poly-A tail of the mRNA strand. The viral enzyme reverse transcriptase (RNA-dependent DNA polymerase) is added to make a single-stranded DNA copy (cDNA or complementary DNA ) that ends in a hairpin loop (Fig. 12-7).
The mRNA template is then destroyed with alkali or an RNase enzyme. The hairpin end of the remaining cDNA serves as a primer for extension synthesis of a complementary strand byDNA polymerase I. The loop is then removed by an enzyme called S1 nuclease to produce a double-stranded cDNA molecule. These blunt-ended molecules can now be spliced into a suitable vector and cloned as previously described. Alternatively, linkers can be added to the ends of the cDNA to facilitate cohesive end cloning. A library of cDNA fragments is called a cDNA library. The advantage to this kind of library is that when cloning eukaryotic genes, DNA sequences that represent only the coding (exon) information will be obtained since mature mRNA does not contain introns.
A specialized type of cDNA library called an EST library (for Expressed Sequence Tag) is used to make a fairly quick assessment of the variety of expressed genes in an organism or tissue. This is done by sequencing hundreds of cDNA library clones and then comparing that sequence information with known databases of gene sequences.
There are several choices for cloning vectors: (a) bacterial plasmids, (b) bacteriophage vectors, and (c) hybrid vectors.
- 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.
- 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).
- 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.
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.
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).
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.
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:
- 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).
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.
- 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.
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.
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).
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.
- 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.
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.
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