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dna tech.docx

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McMaster University
Laura Parker

DNA Technology and Genomics I. DNA cloning permits production of multiple copies of a specific gene or other DNA segment. A. To study a particular gene, scientists needed to develop methods to isolate the small, well-defined portion of a chromosome containing the gene of interest. Techniques for gene cloning enable scientists to prepare multiple identical copies of gene-sized pieces of DNA. B. One basic cloning technique begins with the insertion of a foreign gene into a bacterial plasmid. 1. First, a foreign gene is inserted into a bacterial plasmid to produce a recombinant DNA molecule. 2. The plasmid is returned to a bacterial cell, producing a recombinant bacterium, which reproduces to form a clone of identical cells. Every time the bacterium reproduces, the recombinant plasmid is replicated as well. 3. The bacterial clone will make the protein encoded by the foreign gene. The potential uses of cloned genes fall into two general categories. a. To produce a protein product. For example, bacteria carrying the gene for human growth hormone can produce large quantities of the hormone. b. To prepare many copies of the gene itself so that the gene’s nucleotide sequence can be learned or to provide an organism with a new metabolic capability by transferring a gene from another organism. II. Gene cloning and genetic engineering were made possible by the discovery of restriction enzymes that cut DNA molecules at specific locations. A. In nature, bacteria use restriction enzymes to cut foreign DNA, to protect themselves against phages or other bacteria. They work by cutting up the foreign DNA, a process called restriction. B. Restriction enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific points in these sequences. Bacteria protect their own DNA by methylating the sequences recognized by these enzymes. C. Each restriction enzyme cleaves a specific sequence of bases or restriction site. These are often a symmetrical series of four to eight bases on both strands running in opposite directions. D. Because the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts. Copies of a DNA molecule will always yield the same set of restriction fragments when exposed to a specific enzyme. E. Restriction enzymes cut covalent sugar-phosphate backbones of both strands, often in a staggered way that creates single-stranded sticky ends. 1. These extensions can form hydrogen-bonded base pairs with complementary single-stranded stretches (sticky ends) on other DNA molecules cut with the same restriction enzyme. 2. These DNA fusions can be made permanent by DNA ligase, which seals the strand by catalyzing the formation of covalent bonds to close up the sugar-phosphate backbone. 3. Restriction enzymes and DNA ligase can be used to make a stable recombinant DNA molecule, with DNA that has been spliced together from two different organisms. III. Eukaryotic genes can be cloned in bacterial plasmids. A. Recombinant plasmids are produced by splicing restriction fragments from foreign DNA into plasmids. B. The original plasmid used to produce recombinant DNA is called a cloning vector, defined as a DNA molecule that can carry foreign DNA into a cell and replicate there. C. The process of cloning a human gene in a bacterial plasmid can be divided into six steps. 1. The first step is the isolation of vector and source DNA. a. The source DNA comes from human tissue cells grown in lab culture. The source of the plasmid is typicalRy E. coli. b. This plasmid carries two useful genes, amp , conferring resistance to the antibiotic ampicillin and lacZ, encoding the enzyme ß- galactosidase that catalyzes the hydrolysis of sugar. c. The plasmid has a single recognition sequence, within the lacZ gene, for the restriction enzyme used. 2. DNA is inserted into the vector. a. Both the plasmid and human DNA are digested with the same restriction enzyme. The enzyme cuts the plasmid DNA at its single restriction site within the lacZ gene. It cuts the human DNA at many sites, generating thousands of fragments. One fragment carries the human gene of interest. All the fragments, bacterial and human, have complementary sticky ends. 3. The human DNA fragments are mixed with the cut plasmids, and base- pairing takes place between complementary sticky ends. DNA ligase is added to permanently join the base-paired fragments. Some of the resulting recombinant plasmids contain human DNA fragments. 4. The recombinant plasmids are mixed with bacteria that are lacZ-, unable to hydrolyze lactose. This results in some bacteria that have taken up the desired recombinant plasmid DNA, and other bacteria that have not. 5. The transformed bacteria are plated on a solid nutrient medium containing ampicillin and a molecular mimic of lactose called X-gal. R a. Only bacteria that have the ampicillin-resistance (amp ) plasmid will grow. Each reproducing bacterium forms a colony of cells on the agar. b. The lactose mimic in the medium is used to identify plasmids that carry foreign DNA. (1) Bacteria with plasmids lacking foreign DNA stain blue when ß-galactosidase from the intact lacZ gene hydrolyzes X-gal. (2) Bacteria with plasmids containing foreign DNA inserted into the lacZ gene are white because they lack ß- galactosidase. 6. In the final step, thousands of bacterial colonies with foreign DNA must be sorted through to find those containing the gene of interest. a. One technique, nucleic acid hybridization, depends on base- pairing between the gene and a complementary sequence, a nucleic acid probe, on another nucleic acid molecule. (1) A radioactive or fluorescent tag is used to label the probe. (2) The probe will hydrogen-bond specifically to complementary single strands of the desired gene. (3) After denaturating (separating) the DNA strands in the bacterium, the probe will bind with its complementary sequence, tagging colonies with the targeted gene. IV. Cloned genes are stored in DNA libraries. A. A complete set of recombinant plasmid clones, each carrying copies of a particular segment from the initial genome, forms a genomic library. B. The library can be saved and used as a source of other genes or for gene mapping. 1. In addition to plasmids, certain bacteriophages are also common cloning vectors for making genomic libraries. 2. Fragments of foreign DNA can be spliced into a phage genome using a restriction enzyme and DNA ligase. An advantage of using phage as vectors is that phage can carry larger DNA inserts than plasmids can. 3. The recombinant phage DNA is packaged in a capsid in vitro and allowed to infect a bacterial cell. 4. Infected bacteria produce new phage particles, each with the foreign DNA. C. A more limited kind of gene library can be developed by starting with mRNA extracted from cells. 1. The enzyme reverse transcriptase is used to make single-stranded DNA transcripts of the mRNA molecules. 2. The mRNA is enzymatically digested, and a second DNA strand complementary to the first is synthesized by DNA polymerase. 3. This double-stranded DNA, called complementary DNA (cDNA), is modified by the addition of restriction sites at each end. 4. Finally, the cDNA is inserted into vector DNA. 5. A cDNA library represents that part of a cell’s genome that was transcribed in the starting cells. 6. This is an advantage if a researcher wants to study the genes responsible for specialized functions of a particular kind of cell. 7. By making cDNA libraries from cells of the same type at different times in the life of an organism, one can trace changes in the patterns of gene expression. V. Eukaryote genes can be expressed in prokaryotic host cells. A. A cloned eukaryotic gene can be made to function in a prokaryotic host by inserting an expression vector, a cloning vector containing a highly active prokaryotic promoter. The prokaryotic host will then recognize the promoter and proceed to express the foreign gene that has been linked to it. B. The presence of long noncoding introns in eukaryotic genes may prevent correct expression of these genes in prokaryotes, which lack RNA-splicing machinery. C. This problem can be avoided by using a cDNA form of the gene. D. Molecular biologists can avoid incompatibility problems by using eukaryotic cells as hosts for cloning and expressing eukaryotic genes. 1. Yeast cells are as easy to grow as bacteria and, unlike most eukaryotes, have plasmids. 2. Scientists have constructed yeast artificial chromosomes (YACs) that combine the essentials of a eukaryotic chromosome (an origin site for replication, a centromere, and two telomeres) with foreign DNA. 3. These chromosome-like vectors behave normally in mitosis and can carry more DNA than a plasmid. 4. Another advantage of eukaryotic hosts is that they are capable of providing the posttranslational modifications that many proteins require. VI. The polymerase chain reaction (PCR) amplifies DNA in vitro. A. DNA cloning is the best method for preparing large quantities of a particular gene or other DNA sequence. B. When the source of DNA is small or impure, the polymerase chain reaction (PCR) is quicker and more selective. C. This technique can quickly amplify any piece of DNA without using cells. 1. The DNA is incubated in a test tube with special DNA polymerase, a supply of nucleotides, and short pieces of single-stranded DNA as a primer. 2. PCR can make billions of copies of a targeted DNA segment in a few hours. This is faster than cloning via recombinant bacteria. 3. In PCR, a three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules. a. The reaction mixture is heated to denature the DNA strands. b. The mixture is cooled to allow hydrogen-bonding of short, single- stranded DNA primers complementary to sequences on opposite sides at each end of the target sequence. c. A heat-stable DNA polymerase extends the primers in the 5’  3’ direction. The DNA polymerase used was isolated from prokaryotes living in hot springs. 4. By being complementary to sequences bracketing the targeted sequence, the primers determine the DNA sequence that is amplified. 5. PCR is so specific and powerful that only minute amounts of partially degraded DNA need be present in the starting material. 6. Occasional errors during PCR replication impose limits to the number of good copies that can be made when large amounts of a gene are needed. 7. Increasingly, PCR is used to make enough of a specific DNA fragment to clone it merely by inserting it into a vector. VII. One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis. A. Gel electrophoresis separates nucleic acids or proteins on the basis of their rate of movement through a gel in an electrical field. Rate of movement depends mostly on size electrical charge of the macromolecules. B. Restriction fragment analysis detects DNA differences that affect restriction sites. In restriction fragment analysis, the DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis. 1. When the mixture of restriction fragments from a particular DNA molecule undergoes electrophoresis, it yields a band pattern characteristic of the starting molecule and the restriction enzyme used. 2. The separated fragments can be recovered undamaged from gels, providing pure samples of individual fragments. C. We can use restriction fragment analysis to compare two different DNA molecules representing, for example, different alleles of a gene. 1. Because the two alleles differ slightly in DNA sequence, they may differ in one or more restriction sites. 2. If they do differ in restriction sites, each will produce different-sized fragments when digested by the same restriction enzyme. 3. In gel electrophoresis, the restriction fragments from the two alleles will produce different band patterns, allowing us to distinguish the two alleles. 4. Restriction fragment analysis is sensitive enough to distinguish between two alleles of a gene that differ by only one base pair in a restriction site. D. A technique called Southern blotting combines gel electrophoresis with nucleic acid hybridization. 1. Although electrophoresis will yield too many bands to distinguish individually, we can use nucleic acid hybridization with a specific probe to label discrete bands that derive from our gene of interest. 2. The probe is a radioactive single-stranded DNA molecule that is complementary to the gene of interest. 3. Southern blotting reveals not only whether a particular sequence is present in the sample of DNA, but also the size of the restriction fragments that contain the sequence. 4. One of its many applications is to identify heterozygous carriers of mutant alleles associated with genetic disease. 5. In the example below, we compare genomic DNA samples from three individuals: an individual who is homozygous for the normal ß-globin allele, a homozygote for sickle-cell allele, and a heterozygote. a. We combine several molecular techniques to compare DNA samples from three individuals. b. We start by adding the same restriction enzyme to each of
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