Chapter 13: DNA Quantitation

 Determining the amount of DNA is a sample is essential  A narrow concentration range is required for PCR  Must use human-specific DNA quantitation methods  Quality and Quantity of DNA Forensic Biology by Richard Li2

 Slot Blot Assay  Interchelating Dye  Quantitative PCR Forensic Biology by Richard Li3

 Slot Blot Method  Detect primate DNA  Genomic DNA is denatured and small volume is spotted onto a nitrocellulose membrane  Single-stranded DNA is immobilized onto a nylon membrane  Targeted sequence revealed by a 40-nucleotide probe at the D17Z1 locus Forensic Biology by Richard Li4

 Interchelating Dye Method  Fluorescent dye used  Quant-iT PicoGreen dsDNA reagent  Limit 250pg  Not specific to human DNA  Useful with known reference samples  Fluorescence measure by spectrofluorometer Forensic Biology by Richard Li5

 Quantitative PCR methods  Detects PCR inhibitors  Most sensitive  Amount of PCR product amplified during PCR correlates with the initial concentration  End-point PCR  Real time PCR Forensic Biology by Richard Li6

 Real Time PCR  1990’s  Analyzes the cycle-to-cycle change in fluorescence signal resulting from amplification of a target sequence during PCR  TaqMan Method Forensic Biology by Richard Li7