1. Chapter 13: DNA Quantitation

2.  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 2

3.  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 3

4.  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 4

5. 5

6. 6 Detection range = 150 pg - 10 ng

7.  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 7

8. 8 Detection range >250 pg

9.  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.  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. Exponential phase Linear phase Plateau phase Threshold (Ct)

12.  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. 13 Detection range = 30 pg – 100 ng