Forensic Biology by Richard Li

1. Chapter 14: DNA Amplification by Polymerase Chain Reaction Forensic Biologyby Richard Li

2. Basic Principles • 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 Li

3. Basic Principles • Real time PCR- each cycle monitored • Amplification efficiency • DNA Quantitation, Short Tandem Repeat (STR), and Mitochondrial DNA (mtDNA) Forensic Biology by Richard Li

4. Essential PCR Components • 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 Li

5. Essential PCR Components • Other Components • Template DNA- 1-2.5 ng • Deoxynucleoside triphosphates (dNTPs) • Divalent cations • Monovalent cations • Buffer Forensic Biology by Richard Li

6. PCR Parameters • PCR cycles • Denaturation • Annealing • Extension • PCR controls • Used to monitor effectiveness • Positive control • Negative control Forensic Biology by Richard Li

7. Factors Affecting PCR • 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 Li

8. Reverse Transcriptase PCR • 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 Li