Lecture 10: DNA Quantitation

 Purpose of DNA quantitation  Quantitation Methods  Interchelating dyes  Slot blot  qPCR ▪ Taqman (Life Technologies “Quantifiler”) ▪ Modified nucleotide (Iso-dC/Iso-dG; Promega Plexor HY) 2

3  Determines how much human DNA is present in DNA extracts  Downstream PCR to amplify human STRs requires about 1 ng (optimal); at least 100 pg  1 ng = 1 billionth of a gram!  Results  Reported in ng/ul ▪ Make 10 ul of a 0.1 ng/ul extract for profiling ▪ You will do this in lab

4 DNA extraction 2 ul USE INFORMATION FROM QUANTITATION TO SET UP PCR REACTION FOR STR TYPING 1 ng

 Three common methods for quantitating DNA  Interchelating Dye  Slot Blot Assay ▪ Used in crime labs throughout 1990s  Quantitative PCR (qPCR) ▪ Method of choice in most modern crime labs ▪ We’ll use this method 5

 Interchelating Dye Method  Oldest method  Dye intercalates into the DNA and then fluoresces when excited ▪ E.g. ethidium bromide, Sybergreen  Not specific to human DNA (binds any DNA) ▪ Useful with known reference blood samples ▪ Blood does not contain bacteria ▪ Not useful for questioned samples or buccal swabs ▪ Detection: Gels or spectrofluorometer 6

7 Detection range >250 pg

 Slot Blot Method  Targets DNA in specific genomes (e.g. human)  Genomic DNA is denatured and small volume is spotted onto a nitrocellulose membrane  For human DNA, targeted sequence detected by a 40-nucleotide “probe” at the D17Z1 locus (chromosome 17; highly conserved) ▪ Probe is single-stranded and biotinylated ▪ Detection is colorimetric using streptavidin/horseradish peroxidase/TMB system 8

9 1.Extract human DNA 2.Denature the DNA (heat or basic solution) 3.Spot it onto a +++-charged nitrocellulose membrane via vacuum (it will “stick”) in a “slot blot” system 4.Add ss DNA probe specific for human DNA 5.Incubate at just below melting temp of the probe 6.Wash away excess (unbound) probe 7.Add streptavidin-horseradish peroxidase complex (SA binds tightly to biotin 8.Lower pH with citrate buffer and add substrate (TMB)

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11 Detection range = 150 pg - 10 ng

 Quantitative PCR (qPCR or “real time PCR”)  Developed in the 1990s  Most sensitive ▪ Can detect DNA from a single cell!  Large range of detection (3.2 pg – 50 ng)  Amount of PCR product amplified during exponential phase of PCR correlates with the initial concentration of DNA in the extract  Do NOT confuse this with end-point PCR!

 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) Each colored line represents a different reaction

 Analyzes the cycle-to-cycle change in fluorescence signal resulting from amplification of a target sequence during PCR  Two methods is common use: Taqman and modified nucleotide  TaqMan ▪ For each target: two PCR 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 ▪ During PCR, dye is released and begins to fluoresce

16 Taqman detection range = 60 pg – 100 ng

 Appearance of fluorescent signal is captured throughout the reaction via laser scanning of the qPCR plate or tubes ▪ As the samples enter the exponential phase of amplification the fluorescence passes a fluorescence threshold ▪ The PCR cycle at which a reaction crosses this threshold is called the cycle threshold (C T ) ▪ The C T is used to assign a quant value (ng/ul) to the samples

 DNA standards at known concentrations are included in every run ▪ Standard curve is generated (line in log scale) ▪ R 2 of regression line should be > 0.98  The C T values of unknowns (e.g. evidence samples) are compared to the C T values of the standards ▪ The lower the C T the more human DNA is in the original extract used to seed the reaction

 Modified nucleotide system: iso-DC/iso-dG ▪ Very different mechanism from TaqMan ▪ Fluorescence decreases with every cycle of PCR

 System includes two PCR primers ▪ One has an iso-dC nucleotide on the 5’ end ▪ The other primer is not labeled  No probe ▪ Instead, quencher is attached to iso-dGTP nucleotides included in the Master Mix  After first cycle of PCR, the labeled primer has been incorporated ▪ The iso-dC cannot base-pair to G; it can only base-pair to iso-dG ▪ Iso-dG gets incorporated in the next cycle and fluorescence is quenched

The Promega Plexor HY qPCR system. Incorporation of the iso-dGTP as a complementary base-pair to the iso-dC quenches the fluorescence of the dye on the iso-dC molcule.

PCR cycle number

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 Standards of known concentration are also included in this system ▪ Necessary to assign real quant values to unknowns  PCR targets: ▪ Tandemly repeated sequence on chromosome 17 (FAM dye); amplifies all human DNA (male and female) ▪ Tandemly repeated sequence on Y chromosome (Cal Fluor® Orange 560 dye); amplifies only male DNA ▪ IPC: synthetic (non-human) DNA included in the Master Mix (Cal Fluor® Red 610 dye) ▪ “Internal positive control ▪ Should amplify in all samples, even those without human DNA

 At the end of the reaction the amplification products are denatured (“melted”)  The temperature at which they melt should be the same ▪ All amplicons for the same dye lane should have the same sequence and melt at the same temperature ▪ This allows analysts to verify the specificity of the reaction

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