Chapter 14: DNA Amplification by Polymerase Chain Reaction.

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Presentation transcript:

Chapter 14: DNA Amplification by Polymerase Chain Reaction

 Polymerase Chain Reaction (PCR)  Copies (“amplifies”)DNA in a test tube using the same type of chemistry that cells use to copy DNA  Exponential amplification of specific, short (usually 2,000 bp or less) sequences of DNA  Products are called amplicons  Highly sensitive  Can amplify small quantities  Rapid and robust 2

 Reaction ingredients:  PCR Primers ▪ Short, single-stranded DNA polynucleotides that are complementary to the sequences which flank the target region  dNTPs (in abundance)  Template DNA  DNA Polymerase ▪ Thermostable (e.g. Taq polymerase)  MgCl2 and buffer 3

 PCR steps:  Denaturation (94 deg C)  Annealing (typically deg C) ▪ Set just below melting temperature of primers ▪ rule  Extension (72 deg C) ▪ Optimum temp for taq polymerase  Cycling (denaturation, annealing, extension) ▪ Typically times 4

5 1 copy 2 copies Repeat 30 times = 2 30 = 1 billion copies

6

 PCR controls  Used to monitor effectiveness  Positive control ▪ Indicates that all reaction ingredients are working ▪ Known sample  Negative control ▪ Indicates that there is no contaminating DNA in any of the reaction reagents ▪ Includes all reaction ingredients except template DNA 7

 Template DNA degradation  Low copy number of template  Stochastic Effect  PCR inhibitors  Heme, Indigo dyes, Melanin from hairs  Contamination  Pre-PCR and post-PCR should be in separate areas  Supplies and reagents also separated 8

9 ActivityCellular ReplicationPCR Denaturation Primers Extension Number of copies produced Size of region copied Ingredients Purpose