1. Chapter 14: DNA Amplification by Polymerase Chain Reaction

2.  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

3.  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

4.  PCR steps:  Denaturation (94 deg C)  Annealing (typically 50-60 deg C) ▪ Set just below melting temperature of primers ▪ 4 + 2 rule  Extension (72 deg C) ▪ Optimum temp for taq polymerase  Cycling (denaturation, annealing, extension) ▪ Typically 20-30 times 4

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

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7.  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

8.  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. 9 ActivityCellular ReplicationPCR Denaturation Primers Extension Number of copies produced Size of region copied Ingredients Purpose