Lecture 10: DNA Quantitation. Purpose of DNA quantitation Quantitation Methods Interchelating...

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Forensic Biologyby Richard Li, with additions and edits by Ruth Ballard

Lecture 10: DNA Quantitation

Outline

Purpose of DNA quantitationQuantitation Methods

Interchelating dyes Slot blot qPCR

▪ Taqman (Life Technologies “Quantifiler”)▪ Modified nucleotide (Iso-dC/Iso-dG; Promega

Plexor HY)

Purpose of Quantitation

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

Purpose of Quantitation

2. DNA extract

1. Evidence

3. DNA quantitati

4. STR typing

DNA extraction

USE INFORMATION FROM QUANTITATION

TO SET UP PCR REACTION FOR STR

TYPING

Quantitation Methods

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

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 spectrofluorometer6

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

1. Extract human DNA2. Denature the DNA (heat or basic solution)3. Spot it onto a +++-charged nitrocellulose membrane via vacuum (it

will “stick”) in a “slot blot” system4. Add ss DNA probe specific for human DNA5. Incubate at just below melting temp of the probe6. Wash away excess (unbound) probe7. Add streptavidin-horseradish peroxidase complex (SA binds tightly

to biotin8. Lower pH with citrate buffer and add substrate (TMB)

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

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 (CT)

▪ The CT 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)▪ R2 of regression line should be > 0.98

The CT values of unknowns (e.g. evidence samples) are compared to the CT values of the standards▪ The lower the CT 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

Quantitation Methods 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

Quantitation Methods 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

Lecture 10: DNA Quantitation. Purpose of DNA quantitation Quantitation Methods Interchelating...

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