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Published byEverett Kelley Modified over 9 years ago
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Chapter 13: DNA Quantitation
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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
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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 3
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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 4
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6 Detection range = 150 pg - 10 ng
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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
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8 Detection range >250 pg
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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
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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
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Exponential phase Linear phase Plateau phase Threshold (Ct)
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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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13 Detection range = 30 pg – 100 ng
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