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Goals of today’s lecture 1) Explain how the polymerase chain reaction (PCR) works 2) Explain the basics of DNA sequencing 3) Explain how one can map human disease genes 4) Describe some strategies for genetic engineering in humans and plants Figure 19-6 PCR primers must be located on either side of the target sequence, on opposite strands. 5 3 Primer 3 5 Primer Region of DNA to be amplified by PCR When target DNA is single stranded, primers bind and allow DNA polymerase to work. 5 3 3 5 3 Primer Primer 5 3 5 Figure 19-7-1 THE POLYMERASE CHAIN REACTION IS A WAY TO PRODUCE MANY IDENTICAL COPIES OF A SPECIFIC GENE Primers 5 3 5 3 3 5 3 5 dNTPs 1. Start with a solution 2. Denaturation containing template DNA, synthesized primers, and an abundant supply of the four dNTPs. Heating leads to denaturation of the double-stranded DNA. Figure 19-7-2 THE POLYMERASE CHAIN REACTION IS A WAY TO PRODUCE MANY IDENTICAL COPIES OF A SPECIFIC GENE 5 3 5 5 3 5 5 3 3 5 5 3 3 5 3. Primer annealing 4. Extension At cooler temperatures, the primers bind to the template DNA by complementary base pairing. During incubation, Taq polymerase uses dNTPs to synthesize complementary DNA strand, starting at the primer. Figure 19-7-3 THE POLYMERASE CHAIN REACTION IS A WAY TO PRODUCE MANY IDENTICAL COPIES OF A SPECIFIC GENE 5. Repeat cycle 6. Repeat cycle again, of three steps (2–4) again, doubling the copies of DNA. up to 20–30 times, to produce millions of copies of template DNA. Figure 19-10-1 FLUORESCENT MARKERS IMPROVE SEQUENCING EFFICIENCY. DNA polymerase 1. Do one sequencing reaction instead of four. Reaction mix contains ddATP, ddTTP, ddGTP, ddCTP with distinct fluorescent markers. (With radioactive labels, four reactions are needed—one labeled ddNTP at a time.) Figure 19-10-2 FLUORESCENT MARKERS IMPROVE SEQUENCING EFFICIENCY. Template DNA 2. Fragments of newly synthesized DNA that result have distinctive labels. Figure 19-10-3 FLUORESCENT MARKERS IMPROVE SEQUENCING EFFICIENCY. Long fragments Short fragments Capillary tube Output 3. Separate fragments via electrophoresis in massproduced, gel-filled capillary tubes. Automated sequencing machine reads output. How Was the Huntington’s Disease Gene Found? • Huntington's disease is a rare but devastating genetic illness. • An analysis of pedigrees from families affected by the disease suggested that the trait results from a single, autosomal dominant allele. • A genetic map (or linkage map) was used to localize the Huntington's gene relative to other genetic markers (a gene that has been mapped previously). • Restriction fragment length polymorphisms (RFLPs) are differences between chromosomes that are measured as the presence or absence of a restriction endonuclease recognition site. Figure 19.10 illustrates RFLP analysis. SOUTHERN BLOTTING Location of restriction endonuclease cuts Sample 1 Double-stranded DNA 1. Restriction endonucleases cut DNA sample into fragments of various lengths. Each type of restriction endonuclease cuts a specific sequence of DNA. 2. A sample consists of all the DNA fragments of various lengths. The sample is loaded into a gel for electrophoresis. Figure 19-7 part 1 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc. Samples from four individuals Sample 1 1 2 3 4 Double stranded DNA Power supply 2. A sample consists of all the DNA fragments of various lengths. The sample is loaded into a gel for electrophoresis. 3. Electrophoresis. Use voltage difference to separate DNA fragments by size. Small fragments run faster. Figure 19-7 part 2 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc. Samples from four individuals 1 2 3 4 1 2 3 4 Single stranded DNA Double stranded DNA Power supply 3. Electrophoresis. Use voltage difference to separate DNA fragments by size. Small fragments run faster. 4. The DNA fragments are treated to make them single stranded. Figure 19-7 part 3 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc. 1 2 3 4 Single stranded DNA Stack of blotting paper Filter Gel Sponge in alkaline solution 4. The DNA fragments are treated to make them single stranded. 5. Blotting. An alkaline solution wicks up into blotting paper, carrying DNA from gel onto nylon filter, where it is then permanently bound. Figure 19-7 part 4 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc. Labeled DNA probe in solution in plastic bag Stack of blotting paper Filter Gel Sponge in alkaline solution 5. Blotting. An alkaline solution wicks up into blotting paper, carrying DNA from gel onto nylon filter, where it is then permanently bound. 6. Hybridization with radioactive probe. Incubate the nylon filter with a solution containing labeled probe DNA. The radioactive probe binds to the fragments containing complementary sequences. Figure 19-7 part 5 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc. Labeled DNA probe in solution in plastic bag X-ray film 6. Hybridization with radioactive probe. Incubate the nylon filter with a solution containing labeled probe DNA. The radioactive probe binds to the fragments containing complementary sequences. 7. Autoradiography. Place filter against X-ray film. Radioactive DNA fragments expose film, forming black bands that indicate location of target DNA. Figure 19-7 part 6 Biological Science 2/e ©2005 Pearson Prentice Hall, Inc. • The Huntington's disease gene was localized to chromosome 4 by RFLP analysis. • One gene within the isolated chromosomal region that was abnormal in people with Huntington's disease had an unusual number of CAG codons at the 5' end of the coding region. Healthy individuals have about 11–25 of these repeats, whereas affected individuals have 40 or more. • A genetic test for Huntington's disease uses PCR to determine the number of CAG repeats. Figure 19-13-1 USING ENGINEERED VIRUSES TO INTRODUCE ALLELES INTO HUMAN CELLS Viral RNA Human RNA Reverse transcriptase 1. Engineered retrovirus 2. Recombinant contains recombinant RNA, which has both viral sequences and human sequences. genes enter host cell. Figure 19-13-2 USING ENGINEERED VIRUSES TO INTRODUCE ALLELES INTO HUMAN CELLS Double-stranded DNA version of Human cell introduced genes DNA complementary to introduced RNA Reverse transcriptase 3. Viral enzymes make double-stranded DNA version of introduced genes. Host chromosome 4. Recombinant genes are inserted into host chromosome and transcribed. • Gene therapy has been used to treat a type of severe combined immunodeficiency (SCID), a fatal genetic disease whose sufferers have a profoundly weakened immune system. • The type of SCID treated is designated SCID-X1, because it is caused by mutations in a gene on the X chromosome. • Within four months after treatment, nine of the ten boys had normal levels of functioning T cells; but 30 months later, two had developed a type of cancer characterized by unchecked growth of T cells. • Although gene therapy holds great promise for the treatment of a wide variety of inherited diseases, fulfilling that promise is almost certain to require many years of additional research and testing, as well as the refinement of legal and ethical guidelines. There are currently no plans for using gene therapy to treat Patients with Huntington’s disease. Why do you think that is? A) Because the neurons of the brain are difficult to transfect with transgenes. B) Because Huntington’s disease is autosomal dominant. C) Because Huntington’s disease is caused by accumulation of a mutant protein that aggregates in the neurons. D) Because there aren’t enough Huntington’s patients to make the effort profitable. Chapter 19 Opener Biological Science 2/e ©2005 Pearson Prentice Hall, Inc. The Agrobacterium Transformation System • Agrobacterium tumefaciens is often used for genetic transformation of plants through transfer of its Ti (tumor-inducing) plasmid (Figure 19.16).