Forensic Biology by Richard Li

1. Chapter 13: DNA Quantitation Forensic Biologyby Richard Li

2. Basic Principles • Quantitation determines the amount of human DNA present in an extract • A narrow concentration range is required to “seed” the Identifiler PCR reaction • Too much or too little DNA gives rise to artifacts (false positive or false negative alleles) • Must use human-specific DNA quantitation • Bacterial DNA may be present (e.g. saliva) • Test should measure quality as well as quantity of DNA

3. Quantitation Methods • Three common methods • Slot Blot Assay • Interchelating Dye • Quantitative PCR • Method of choice in most modern crime labs • We’ll use this method in lab

4. Quantitation Methods • Slot Blot Method • Detects primate DNA • Genomic DNA is denatured (made single-stranded) and small volume is spotted onto a nitrocellulose membrane • Targeted sequence revealed by a 40-nucleotide probe at the D17Z1 locus • Probe is single-stranded and biotinylated • Detection is colorimetric using streptavidin/horseradish peroxidase/TMB system

6. Detection range = 150 pg - 10 ng

7. Quantitation Methods • Interchelating Dye Method • Fluorescent dye used • Quant-iT PicoGreen dsDNA reagent • Not specific to human DNA • Useful with known reference blood samples • Not useful for questioned samples or buccal swab samples • Fluorescence measure by spectrofluorometer

8. Detection range >250 pg

9. Quantitation Methods • Quantitative PCR (qPCR or “real time PCR”) • 1990s • More sensitive • Large range of detection • Amount of PCR product amplified during exponential phase of PCR correlates with the initial concentration • Real-time PCR most common method in forensic lab

10. Quantitation Methods • Exponential phase • 100% efficiency (plenty of primers and dNTPs) • High degree of precision in accumulation of PCR products with time: doubling per cycle • Linear phase • One or more components fall below critical concentration; amplification efficiency drops • Precision in accumulation of PCR products drops • Plateau (“end point”) • Reaction slows to a halt; components consumed

11. Plateau phase Linear phase Exponential phase Threshold (Ct)

12. Quantitation Methods • Analyzes the cycle-to-cycle change in fluorescence signal resulting from amplification of a target sequence during PCR • TaqMan Method is most popular • Two primers and one probe • Probe has a fluroescent dye on 5’ end and a quencher molecule on 3’ end • As long as probe in intact, fluorescence is quenched • IPC to detect inhibitors • May also detect total DNA: male DNA ratio • Important for intimate sexual assault samples

13. Detection range = 30 pg – 100 ng