lecture 10: dna quantitation. purpose of dna quantitation quantitation methods interchelating...
TRANSCRIPT
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)
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Purpose of Quantitation
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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
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2. DNA extract
1. Evidence
3. DNA quantitati
on
4. STR typing
DNA extraction
2 ul
USE INFORMATION FROM QUANTITATION
TO SET UP PCR REACTION FOR STR
TYPING
1 ng
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
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Quantitation Methods
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
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Detection range >250 pg
Quantitation Methods
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
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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)
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Detection range = 150 pg - 10 ng
Quantitation Methods
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!
Quantitation Methods
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
Quantitation Methods
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
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Taqman detection range = 60 pg – 100 ng
Quantitation Methods
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
Quantitation Methods
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
Quantitation Methods
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
Quantitation Methods
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.
Quantitation Methods
PCR cycle number
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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
Quantitation Methods
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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