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Tamarin: Principles of Genetics, Seventh Edition
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III. Molecular Genetics
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13. Genomics, Biotechnology, and Recombinant DNA
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The McGraw Hill Companies, 2001
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chain termination occurs at the adenine in the piece of DNA; that is, a DNA segment was synthesized that is six bases long (see the following gure). In the reaction mixture with ddATP, chain termination occurs opposite each of the thymines, producing DNA segments of ve, seven, and eight nucleotides. In the reaction mixture with ddGTP, chain termination occurs opposite the cytosines in positions three and nine. And, in the reaction mixture with ddCTP, chain termination occurs after synthesis of segments one, two, and four bases long. Note that the gel gives us the sequence of the complement strand of the original piece of single-stranded DNA.
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PROBLEM 4: A linear DNA molecule 1,000 base pairs long is digested with the following restriction enzymes, producing the following results:
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EcoRI Bgl II EcoRI 400 bp, 600 bp 250 bp, 750 bp 250, 350, 400 bp
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Determine the restriction map. Answer: Each enzyme alone produces two fragments, so the molecule has one site for each enzyme. Since we get different-sized fragments with each enzyme, the sites must be located asymmetrically along the DNA. Draw these sites: EcoRI 400 Z 600 BglII and 250 Z 750
The EcoRI fragment that lacks a BglII site should appear in the double digest. If BglII cuts within the 400 base-pair fragment, we would expect to see 150, 250, and 600 base-pair fragments. We don t see this, so the BglII site is not within the 400 base-pair EcoRI fragment. Thus, the map looks like this: EcoRI BglII 400 Z 350 Z 250
E X E R C I S E S
GENOMIC TOOLS
A N D
P R O B L E M S *
1. What speci c properties of type II endonucleases make them useful in gene cloning 2. The following is a double helix of DNA. What, if any, are potential restriction enzyme recognition sequences 5 -TAGAATTCGACGGATCCGGGGCATGCAGATCA-3 3 -ATCTTAAGCTGCCTAGGCCCCGTACGTCTAGT-5 3. Assuming a random arrangement of nucleotides on a piece of DNA, what is the probability that a restriction endonuclease whose recognition site consists of four bases (a four-cutter) will cut the DNA What is the probability for a six-cutter an eight-cutter 4. Under what circumstances is a restriction endonuclease unsuitable for cloning a piece of foreign DNA
5. What methods exist to create sticky ends or create ends for joining two incompatible pieces of DNA When is each method favored 6. Diagram a possible heteroduplex between two phage vectors, one with and one without a cloned insert, created by DNA-DNA hybridization. 7. What are the differences among plasmid, cosmid, expression vector, and YAC Under what circumstances is each useful 8. What are the steps by which messenger RNA can be converted into cDNA How would we obtain radioactive cDNA radioactive messenger RNA 9. What is chromosome walking When is it used 10. How would we isolate a human alanine transfer RNA gene for cloning How would we locate a clone with a human alanine transfer RNA gene in a genomic library 11. What are the differences among Southern, western, northern, and dot blotting
*Answers selected exercises and problems are on page A-14.
Tamarin: Principles of Genetics, Seventh Edition
III. Molecular Genetics
13. Genomics, Biotechnology, and Recombinant DNA
The McGraw Hill Companies, 2001
Exercises and Problems
12. How would you develop a probe for a gene whose messenger RNA could not be isolated How could an expression vector be used to isolate a cloned gene 13. How are E. coli plasmids manipulated to survive in yeast How can virus genomes, such as SV40 and phage , survive as functioning vectors when parts of their genomes are replaced by cloned DNA 14. What methods are used to get foreign DNA into eukaryotic cells What is transfection What is a transgenic mouse 15. Exonuclease III is an enzyme that sequentially removes bases from the 3 end of double-stranded DNA. The following two molecules, each 100 bp long, are digested with exonuclease III. Molecule 1 is completely digested; molecule 2 is only partially digested. Explain these results.
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