forensic biology by richard li
DESCRIPTION
Chapter 13: DNA Quantitation. Forensic Biology by Richard Li. 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 - PowerPoint PPT PresentationTRANSCRIPT
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
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
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
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
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
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