Forensic Biology by Richard Li

1. Chapter 13: DNA Quantitation Forensic Biologyby Richard Li

2. Basic Principles • 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 Li

3. Quantitation Methods • Slot Blot Assay • Interchelating Dye • Quantitative PCR Forensic Biology by Richard Li

4. Quantitation Methods • 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 Li

5. Quantitation Methods • 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 Li

6. Quantitation Methods • 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 Li

7. Quantitation Methods • 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 Li