2112 Lectures 35-36 Study Guide.doc

Primer sequences are chosen so that hybridize to

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Primer sequences are chosen so that hybridize to sequences at opposite ends of the target segment, on the 3’ end of the strand. With each cycle, the total number of target segment molecules of the correct length doubles, so the total number is 2^n, where n = number of cycles. This process produces billions of copies of the target sequence. B. What is unique about the polymerase used in PCR? Taq polymerase is obtained from a bacterial species, thermos aquaticus. This species lives in hot springs and the stability of its DNA polymerase at high temp enables the enzyme to function above 95 degrees Celsius. 10) You have ligated your PCR fragment (containing the gene that encodes your protein of interest) into an expression plasmid. How will you determine that the sequence of your gene is intact, contains no mutations, and will produce the desired protein? (Pg. 414, paragraph 3, sentence 1; Pg. 411, Fig. 20.3) You can determine the complete nucleotide sequence of a DNA molecule by a process called DNA sequencing. This process sequences cut up DNA fragments. 11) In question 8 (above) you described a technique for obtaining a protein-coding gene directly from a bacterial chromosome. A. Why would this approach not work in mammalian cells? (Pg. 346, paragraph 1, sentences 6 and 8) Mammalian cells contain sequences of DNA nucleotides that are usually not continuous, as they are interrupted by introns. B. How would you obtain a DNA fragment that contains only the coding region of a mammalian gene? Reverse transcriptase polymerase chain reaction (RT-PCR): RT enzyme synthesizes DNA copies of each mRNA, called reverse transcripts. Short complementary strands of thymine deoxyribose can be added to the 3’ end of mRNA and used for primer for synthesis of a DNA strand. Once mRNA is degraded, a second DNA strand complementary to the first is synthesized by DNA polymerase. The resulting double stranded DNA is called cDNA. cDNA lacks introns and can be used for protein expression in bacteria. 12) How would you determine where a particular gene is expressed in an organism? (Pg. 421, column 2, paragraph 3 and pg. 422, Fig. 20.9; Pg. 423, 2 nd column, paragraph 2, sentences 1-2, 5-7 and Fig. 20.12; Pg. 424, Figure 20.13) mRNA can be detected by nucleic acid hybridization with molecules of a complementary sequence. The complementary molecule, a short single stranded DNA or RNA is called a nucleic acid probe. The probe can be synthesized so that it is complementary to mRNA, using cloned genes.
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  • Fall '08
  • RAMMAPORT
  • DNA

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