专业英语chapter5PCR.pptVIP

Chapter 5 PCR Lectures by Gong 2009 11 Overview : Introduction The central scientific fact that makes PCR so useful is this: The genetic material of each living organism-plant or animal, bacterium or virus-possesses sequences of its nucleotide building blocks (usually DNA, sometimes RNA) that are uniquely and specifically present only in its own species. Indeed, complex organisms such as human beings possess DNA sequences that are uniquely and specifically present only in particular individuals. These unique variations make it possible to trace genetic material back to its origin, identifying with precision at least what species of organism it came from, and often which particular member of that species. Concept 1:The Mechanism of PCR The polymerase chain reaction is a test tube system for DNA replication that allows a target DNA sequence to be selectively amplified, or enriched, several million-fold in just a few hours. Within a dividing cell, DNA replication involves a series of enzyme-mediated reactions, whose end result is a faithful copy of the entire genome. Within a test tube, PCR uses just one indispensable enzyme - DNA polymerase - to amplify a specific fraction of the genome. During cellular DNA replication, enzymes first unwind and denature the DNA double helix into single strands. Then, RNA polymerase synthesizes a short stretch of RNA complementary to one of the DNA strands at the start site of replication. This DNA/RNA heteroduplex acts as a priming site for the attachment of the DNA polymerase, which then produces the complementary DNA strand. During PCR, high temperature is used to separate the DNA molecules into single strands, and synthetic sequences of single-stranded DNA (20-30 nucleotides) serve as primers. Two different primer sequences are used to bracket the target region to be amplified. One primer is complementary to one DNA strand at the beginning of the target region; a second primer is complementary to a sequence on the op