Sequencing The Polymerase Chain Reaction (PCR) and Its ：测序聚合酶链反应（PCR）和.pptVIP

The Polymerase Chain Reaction Adapted from a presentation written for Principles of Gene Manipulation · November 6, 2000 Jennifer Cooper · America Madrigal · Lale?a Vellanoweth AMPLIFICATION: PCR and Its Applications Definition of PCR Requirements for PCR PCR Process A. Denaturation B. Annealing C. Extension D. Cycling (repeat A-C) PCR for HLA DQ-alpha The Polymerase Chain Reaction (PCR) is an in vitro method to amplify a specific region of DNA. PCR is extremely sensitive, with the capability of amplifying minuscule quantities of DNA. In the reaction Sample – template Primers High temperature resistant polymerase; e.g., Taq Deoxynucleotide triphosphates – dNTPs (dATP, dGTP, dCTP, dTTP) Buffer Mg++, KCl Thermocycler – instrument programmed to change samples rapidly from one set temperature to another There are three basic steps in PCR 1. Denaturation (~95oC) 2. Annealing (~55oC, but varies) 3. Extension (~72oC) Cycling repeats Steps 1-3 up to 35 times. PCR RequirementsThe Details Amount Needed very small; intact DNA from one cell to see on a gel, need 1011 final copies; need 104 starting copies Concern –competition with primers for annealing by Too much starting template Too much product from excessive cycling Remember, the association rate of two strands increases with the square root of the length of the DNA. Longer strands anneal more quickly than shorter. So . . . Templates and products are longer than primers. In high concentration, they reanneal before primers can anneal. PCR REQUIREMENTS: sample - template Even degraded DNA is OK if sample is large enough Fossils Remains Old samples from crime scenes PCR REQUIREMENTS - primers Two primers of known sequence flank region you are interested in anneal to opposite strands of template prime toward the region between them non-complementary to each other lack internal complementarity of sufficient length to anneal to unique site in the genome (~20 nt) 1/420 = 1 site of ident