DNA QUANTITATION

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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

 Results
 Reported in ng/ul

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Purpose of Quantitation

1. Evidence

DNA extraction

2. DNA extract
2 ul

4. STR 3. DNA
typing quantitation
USE INFORMATION
FROM QUANTITATION
1 ng TO SET UP PCR
REACTION FOR STR
TYPING

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 Quantitation Methods
 Methods for quantitating DNA
 Spectroscopy
 Interchelating Dye
 Picogreen
 Slot Blot Assay
▪ Used in crime labs throughout 1990s
 Quantitative PCR (qPCR)
▪ Taqman (Life Technologies “Quantifiler”) (Method of choice in
most modern crime labs)
▪ Modified nucleotide (Iso-dC/Iso-dG; Promega Plexor HY)
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 Spectrophotometry

 traditional method of DNA quantitation.

 involves measuring the absorbance of the sample
at 260nm on a spectrophotometer.
 shows little sample-to-sample variation.
 technique is not species-specific
(any bacterial or fungal DNA that has co-purified cannot be
distinguished from the human target DNA and also single-
stranded DNA and nucleotides cannot be distinguished
from double-stranded DNA)

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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 spectrofluorometer

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Detection range >250 pg

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 PICOGREEN
 involves the use of a fluorescent dye PicoGreen
dsDNA quantitation reagent.
 allow high-throughput quantitation.
 utilises the increased fluorescent intensity that is
observed when PicoGreen binds to dsDNA.
 The fluorescent intensity of the PicoGreen dye is
measured with a spectrofluorometer capable of
producing the excitation wavelength of ~480 nm and
recording at the emission wavelength of ~520 nm.

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PICOGREEN

 quantification by comparison of the sample

fluorescence to the fluorescence of a set of
standards that are included in every sample
run.
 not specific for human DNA
 detection limit of 25pg/ml.

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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 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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 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
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
 Plateau phase

Linear phase

Exponential
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
Taqman detection range = 60 pg – 100 ng

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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

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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