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slides2spring08 - Anthony Griffiths Susan Wessler Richard...

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Introduction to Genetic Analysis Ninth Edition Chapter 20: Gene Isolation and Manipulation Copyright © 2008 by W. H. Freeman & Company Anthony Griffiths • Susan Wessler • Richard Lewontin Sean B. Carroll
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Cloning PCR What is the source of DNA here? Obtaining Specific Sequences - Two Major Methods
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Obtaining Specific Sequences - Two Major Methods Cloning PCR What is the source of DNA here? Chromosomal DNA
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PCR PCR (Polymerase chain reaction) is based upon an in vitro DNA polymerase reaction. A thermostable DNA polymerase (most commonly from Thermus aquaticus ) is used. This allows you to anneal primers to the target DNA (primers are sequences of ~20 bp that match the target), extend the primers, denature (melt) the DNA, and repeat the process a number of times (often 30 to 35 times).
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Another Source of DNA - complementary DNA Is this eukaryotic or prokaryotic RNA? Is this rRNA, tRNA, or mRNA?
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Another Source of DNA - complementary DNA Is this eukaryotic or prokaryotic RNA? Eukaryotic - note the poly(A) tail. Is this rRNA, tRNA, or mRNA? mRNA - only mRNA is polyadenylated.
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Another Source of DNA - complementary DNA Why use cDNA rather than chromosomal DNA?
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Cloning with Restriction Enzymes Regardless of the source of the DNA, cloning requires: The cloning vector .
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