Chapter 13: DNA Quantitation

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

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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

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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

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Detection range = 150 pg - 10 ng

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

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Detection range >250 pg

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

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

Exponential phase

Linear phase

Plateau phase

Threshold (Ct)

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

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Detection range = 30 pg – 100 ng