Lecture 10: DNA Quantitation
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Purpose of DNA quantitation
Quantitation Methods
 Interchelating dyes
 Slot blot
 qPCR
▪ Taqman (Life Technologies “Quantifiler”)
▪ Modified nucleotide (Iso-dC/Iso-dG; Promega Plexor HY)
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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!
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Results
 Reported in ng/ul
▪ Make 10 ul of a 0.1 ng/ul extract for profiling
▪ You will do this in lab
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DNA extraction
2 ul
1 ng
USE INFORMATION
FROM QUANTITATION
TO SET UP PCR
REACTION FOR STR
TYPING
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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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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
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Detection range >250 pg
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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 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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Detection range = 150 pg - 10 ng
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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
Plateau phase
Linear phase
Exponential
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
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 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
 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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