1. Fundamentals of Forensic DNA Typing
Chapter 6
DNA Quantitation
Fundamentals of Forensic DNA Typing
Slides prepared by John M. Butler
June 2009

2. Chapter 6 – DNA Quantitation
Chapter Summary
DNA quantitation enables an evaluation of the amount of DNA present in a sample. Real-time quantitative polymerase chain reaction (qPCR) methods also permit an assessment of the quality of a sample in terms of its ability to amplify a particular sized DNA target. A number of qPCR assays have been developed in recent years to aid evaluation of DNA quantity and quality. Quantitation can serve as a useful decision point in the overall process of DNA testing provided that the quantitation method is at least if not more sensitive than the DNA testing method.

3. Purpose of Human-Specific DNA Quantitation
All sources of DNA are extracted when biological evidence from a crime scene is processed to isolate the DNA present.
Thus, non-human DNA such as bacterial, fungal, plant, or animal material may also be present in the total DNA recovered from the sample along with the relevant human DNA of interest.
For this reason, the DNA Advisory Board (DAB) Standard 9.3 requires human-specific DNA quantitation so that appropriate levels of human DNA can be included in the subsequent PCR amplification.
Multiplex STR typing works best with a fairly narrow range of human DNA – typically 0.5 to 2.0 ng of input DNA works best with commercial STR kits.
Higher quality data saves time and money

4. Impact of DNA Amount into PCR
Reason that DNA Quantitation is Important Prior to Multiplex Amplification
Too much DNA
Off-scale peaks
Split peaks (+/-A)
Locus-to-locus imbalance
Too little DNA
Heterozygote peak imbalance
Allele drop-out
Locus-to-locus imbalance
D3S1358
10 ng template (overloaded)
2 ng template (suggested level)
DNA Size (bp)
Relative Fluorescence (RFUs)
100 pg template
5 pg template
DNA Size (bp)
Stochastic effect when amplifying low levels of DNA produces allele dropout

5. Why Do We Care About Quantitating DNA?
If we can confidently determine the amount of DNA in an extract we can then ask questions:
Will mitochondrial sequencing be required (skip STR analysis)
Should we use a miniSTR assay?
Should we use low copy number LCN methods for STRs?
Re-extract the sample?
If problems occur in the STR typing process we can have confidence that the DNA template is not the source (CE, cycler, kit)

6. Too little DNA amplified
(b)
Too little DNA amplified
(c)
Within optimal range
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 6.1
Too much DNA amplified
Figure 6.1 Illustration of STR typing results at a single heterozygous locus for a single source sample with (a) too much DNA template showing off-scale, split peaks, (b) too little DNA template where the arrow points to allele dropout due to stochastic effects, or (c) just the right amount so that two allele peaks are balanced and on-scale.

7. Slot Blot DNA Quantitation Result
20 ng
10 ng
5 ng
2.5 ng
1.25 ng
0.63 ng
Calibration standards
Unknown Samples
≈2.5 ng
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 6.2
Figure 6.2 Illustration of a human DNA quantitation result with the slot blot procedure. A serial dilution of a human DNA standard is run on either side of the slot blot membrane for comparison purposes. The quantity of each of the unknown samples is estimated by visual comparison to the calibration standards. For example, the sample indicated by the arrow is closest in appearance to the 2.5 ng standard.

8. TaqMan (5’ Nuclease) AssayR Q
Forward primer
Reverse primer 3’ 5’
TaqMan probe
Polymerization and Strand Displacement
Forward primer
Reverse primer 3’ 5’ Q R
Probe Cleavage
(release of reporter dye)
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 6.3
Fluorescence occurs when reporter dye and quencher dye are no longer in close proximity
Forward primer
Reverse primer 3’ 5’ Q R
Figure 6.3 Schematic of TaqMan (5’ nuclease) assay.
Completion of Polymerization

9. DNA Quantitation
DNA quantitation is important to determine how much human DNA (as opposed to bacterial DNA) is present in a sample
A commonly used DNA quantitation kit is called Quantifiler (sold by Applied Biosystems)
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 6.4
ABI 7500: an instrument used to perform “real-time quantitative PCR”

10. Exponential product growth
Real-Time qPCR Output
Cycle Number
Normalized Fluorescence
threshold CT
Exponential product growth
Linear
product growth
Plateau
ΔRn
Negative control a b c d e
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 6.5
Standard curve CT
Log[DNA] a b c d e
Figure 6.5 Real-time PCR output and example standard curve used to determine quantity of input DNA.
Nc = No (1 + E)c
If efficiency is close to 100% (E = 1), then the product copy number (Nc) doubles the target copy number (No) with each cycle (c).

11. (A)
1 ng sample
(B)
(A) Range of DNA concentrations reported for a 1 ng DNA sample supplied to 74 laboratories in an interlaboratory study (Kline et al. 2003). Overall the median value was very close to the expected 1 ng level with 50% falling in the boxed region. However, laboratories returned values ranging from 0.1 ng to 3 ng. (B) A target plot examining concordance and apparent precision for the various laboratory methods used. Legend: A=ACES kit; q = Quantiblot with unreported visualization method; E = Quantiblot with chemiluminescent detection; T = Quantiblot with colorimetric detection; 1, 2, 3, 4, and 5 represent methods used by only one lab.
John M. Butler (2005) Forensic DNA Typing, 2nd edition, Figure 3.4

12. Impact of DNA Amount into Multiplex PCR Reaction
We generally aim for ng
DNA amount
(log scale)
High levels of DNA create interpretation challenges (more artifacts to review)
100 ng -A
Too much DNA
Off-scale peaks
Split peaks (+/-A)
Locus-to-locus imbalance +A
10 ng
2.0 ng
Well-balanced STR multiplex
1 ng
STR Kits Work Best in This Range
0.5 ng
100 pg template
0.1 ng
Too little DNA
Heterozygote peak imbalance
Allele drop-out
Locus-to-locus imbalance
5 pg template
0.01 ng
Stochastic effects when amplifying low levels of DNA can produce allele dropout

13. Calculation of the Quantity of DNA in a Cell
1. Molecular Weight of a DNA Base Pair = 618 g/mol
A = 313 g/mol; T = 304 g/mol; A-T base pairs = 617 g/mol
G = 329 g/mol; C = 289 g/mol; G-C base pairs = 618 g/mol
2. Molecular Weight of DNA = 1.98 x1012 g/mol
There are 3.2 billion base pairs in a haploid cell ~3.2 x 109 bp
(~3.2 x 109 bp) x (618 g/mol/bp) = 1.98 x 1012 g/mol
3. Quantity of DNA in a Haploid Cell = 3 picograms
1 mole = 6.02 x 1023 molecules
(1.98 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules)
= 3.3 x g = 3.3 picograms (pg)  
A diploid human cell contains ~6.6 pg genomic DNA
4. One ng of human DNA comes from ~152 diploid cells
 1 ng genomic DNA (1000 pg)/6.6pg/cell = ~303 copies of each locus
(2 per 152 diploid genomes)
Adapted from D.N.A. Box 3.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition (Elsevier Academic Press), p. 56

14. qPCR Workshop Materials

15. qPCR qPCR is a recently developed technique First paper on qPCR:
Developed by Higuchi in 1993
Used a modified thermal cycler with a UV detector and a CCD camera
Ethidium bromide was used as intercalating reporter As [dsDNA] increased fluorescence increased
First paper on qPCR:
Higuchi, R.; Fockler, C.; Dollinger, G.; Watson, R. “Kinetic PCR analysis: real-time monitoring of DNA amplification reactions” Biotechnology (N Y) Sep;11(9):

16. PCR/qPCR What is the Difference?
In the PCR the products are analyzed after the cycling is completed (static)
gel, CE, UV, fluorimeter
End point assay
qPCR the products are monitored as the PCR is occurring (dynamic)
Once per thermal cycle
Fluorescence is measured
Kinetics of the system

17. Why Real Time qPCR? Advantages
The availability of commercial qPCR kits (labs have almost entirely switched to this method for DNA quantitation)
Higher throughput and reduced user intervention
Automated set up
Simple data analysis
Experimental data rapidly analyzed in software; interpolating into the calibration curve
qPCR will be sensitive to the same inhibitors as faced in a traditional STR test (both PCR based)

18. Why Real Time qPCR? Advantages
No post PCR manipulation (reduced contamination issues)
High sensitivity (down to a single copy number ?)
Large dynamic range: ~30 pg to ~30 ng
Assays are target specific (autosomal, mito, Y) and can be multiplexed – to a degree…
Still are subject to stochastic effects with low amount of DNA

19. Why Real Time qPCR? Challenges qPCR is subject to inhibition
internal PCR controls (IPC) can help
qPCR quantitation precision suffers at low copy numbers (below 30 pg by a factor of 2)
When working below 100 pg qPCR is still subject to variability and uncertainty
The exponential relationship between CT and quantity means that small changes in CT lead to large variations in quantity

20. Why Real Time qPCR? Challenges
qPCR quantitates specific target sequences, it does not quantify “DNA”
In highly degraded samples, assays that amplify short target sequences will detect and measure more DNA than assays that amplify long target sequences (relevant to STR typing)
Accurate qPCR quantitation assumes that each unknown sample is amplified at the same efficiency as the Calibrant sample in the dilution series
Results are relative to the Calibrant (these can vary)

21. Efficiency is dropping < 100%
PCR Amplification
4 phases of PCR amplification
Lag (doubling, but not detected)
Exponential (doubling)
Linear (less than doubling)
Plateau (little change)
The exponential phase is where we make our qPCR measurements
Efficiency is dropping < 100%

22. PCR Efficiency Taking our previous relationship 2N
The efficiency of the PCR can be represented as:
XN = X0 (1 + E)N
XN predicted copies
X0 starting copy number
E efficiency (0 to 1)
N number of cycles

23. PCR Efficiency Starting with 100 copies and 100% and 28 cycles 90% 80%
XN = 100(1 + 1)28
2.68 x 1010 copies
90%
XN = 100( )28
6.38 x 109 copies
80%
XN = 100( )28
1.40 x 109 copies

24. Summary
Quantitation is an important step in the overall process of DNA typing
PCR is an exponential process; 2N
Of the 4 phases of qPCR the exponential is where qPCR measurements are made
We can determine E from a plot of cycles versus amplified copies of target DNA

25. Importance of the Calibrant!
Things to keep in mind about Calibrants
The Calibrant is usually a pristine well-characterized DNA sample
Not extracted
Not subjected to the same environment as your unknown(s)
Will not contain inhibitors, Ca++ etc
May be from a cell line or mixed source sample
May exhibit lot-to-lot variation (monitor this)

26. Overview of SRM 2372 Values and Use
See
NIST
Attenuance (λ260)
Informational Values
DNA Concentration (ng/µL)
1 OD
= 50 ng/µL
Certified Values
Alu qPCR
Other assays
Interlab Study Confirms Assay Relative Bias
SRM 2372 Components
Quantifiler A B C
Different Assays
Different Calibrants
Forensic Labs
Adjust calibrant values for each lot
“Calibrated” NIST-Traceable Calibrant for Use in Daily Work
Measure Unknown
DNA Samples

27. Difference in DNA Quantitation Capability vs. STR Typing Sensitivity
Nuclear DNA quantities
1 ng
This gap has kept labs proceeding with “no result” slot blot samples
Quantiblot Limit of Detection (LOD)
STR typing (28 cycles) LOD
100 pg
Low Copy
Number Realm
LCN STR typing (34 cycles) LOD
Real-time qPCR LOD
1 pg (less than a single cell)
mtDNA possible due to higher copy #

28. Chapter 6 – Points for Discussion
What problems might exist with having quantitation assays that are less sensitive than downstream DNA testing methods?
How can reliable DNA quantitation aid decisions in terms of what route to proceed with?
What is the optimal quantity of DNA for most commercial STR kits? What is the effect of too much or too little DNA being amplified?