Forensic Biology by Richard LiBasic PrinciplesSlide 3Essential PCR ComponentsSlide 5PCR ParametersFactors Affecting PCRReverse Transcriptase PCRChapter 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 robustForensic Biology by Richard Li 2Real time PCR- each cycle monitoredAmplification efficiencyDNA Quantitation, Short Tandem Repeat (STR), and Mitochondrial DNA (mtDNA)Forensic Biology by Richard Li 3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 regionForensic Biology by Richard Li 4Other ComponentsTemplate DNA- 1-2.5 ngDeoxynucleoside triphosphates (dNTPs)Divalent cationsMonovalent cationsBufferForensic Biology by Richard Li 5PCR cyclesDenaturationAnnealingExtensionPCR controlsUsed to monitor effectivenessPositive controlNegative controlForensic Biology by Richard Li 6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 separatedForensic Biology by Richard Li 7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 RNAForensic Biology by Richard Li