Chapter 14: DNA Amplification by Polymerase Chain Reaction

 Polymerase Chain Reaction (PCR)  Exponential amplification of specific sequences of DNA to yield sufficient amplified product: Amplicons  Highly sensitive  Can amplify small quantities  Rapid and robust Forensic Biology by Richard Li2

 Real time PCR- each cycle monitored  Amplification efficiency  DNA Quantitation, Short Tandem Repeat (STR), and Mitochondrial DNA (mtDNA) Forensic Biology by Richard Li3

 Thermostable DNA Polymerases  AmpliTaq Gold  Hot start PCR approach  PCR Primers  Oliogonucleotides that are complementary to the sequences which flank the target region  Multiplex PCR- more than one region Forensic Biology by Richard Li4

 Other Components  Template DNA ng  Deoxynucleoside triphosphates (dNTPs)  Divalent cations  Monovalent cations  Buffer Forensic Biology by Richard Li5

 PCR cycles  Denaturation  Annealing  Extension  PCR controls  Used to monitor effectiveness  Positive control  Negative control Forensic Biology by Richard Li6

 Template 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 Forensic Biology by Richard Li7

 Ribonucleic Acid (RNA)  Linear molecule containing four types or nucleotides linked together by phosphodiester bonds  mRNA  The Central Dogma  Francis Crick  Reverse Transcriptase-PCR (RT-PCR)  Amplify complementary DNA (cDNA) copies of RNA Forensic Biology by Richard Li8