Ashley Manning Hart
University at Albany
In partial fulfillment of the
Master of Science in Forensic Biology

 Extraction
 Quantitation
 Amplification
 STR DNA Analysis & Interpretation
 Report writing
 Review
 Testimony

 STR DNA analysis is very sensitive
 Inhibition of samples by either substrate or
sample itself
 Wide range of amounts and quality of DNA at
crime scene
 Best profiles from input DNA in concentration
range 0.5 – 2ng/uL
 If too much DNA is added to amplification:
stutter products, pull-up, -A peaks can be
observed
 If too little DNA is added to amplification: allelic
drop-out, allelic imbalance can be observed

 Crime scene samples can be “dirty” and contaminated
with non-human DNA
 Microbial, plant, and/or animal DNA co-extract with
human DNA recovered from the evidence
 FBI Quality Assurance Standard 9.3 requires
laboratories to quantitate the amount of human DNA in
crime scene evidence prior to subsequent DNA analysis
 Human STR DNA typing is optimized over a fairly narrow
range of human DNA
4

 Ease of interpretation – especially with DNA
mixtures
 Comparison purposes of evidence samples to
reference samples
 Ability to upload to CODIS, NDIS
 Ability to be statistically meaningful
 Ability to stand up in court

 Known samples
 Reference samples
 Offender samples
 Questioned samples
 Single source
 Mixtures: male/male, male/female,
female/female

 Quantifiler Y Human Male DNA Quantification Kit
detects male DNA
 Most valuable when male:female DNA ratio low:
 Body cavity swabs of female victim: oral, vaginal,
anal
 Bite marks, fingernail scrapings
 When male cells cannot be separated from female
cels
 Let’s explore the history of quantification first…

 UV Spectrometry
 Gel-based Methods
 Real-time PCR Methods

 Based on stacking of nitrogenous bases
 Stacking limits the amount of UV light
absorbed
 Disadvantages:
 Cannot distinguish dsDNA from ssDNA
 Not human specific
 RNA, protein and other contaminants also absorb
these UV wavelengths
 Not very sensitive
 Relatively large amount of sample needed
 Unacceptable method for crime scene evidence

 Gelsor blotting membranes utilized
 DNA specific dyes as reporter molecules
 Species non-specific
 Advantages:
 Broader quantitation range with lower limits
than UV spectrometry
 Possible to distinguish between dsDNA and ssDNA
 Disadvantages:
 Interpretation can be subjective
 Reporter molecules can be sensitive to chemicals
in the solvent
 Time-consuming – mostly hands-on

 Applied Biosystems method – chemiluminescence
or chromogenic
 Slot blot on a nylon membrane
 HUMAN AND HIGHER PRIMATE
 Probe complimentary to alpha satellite DNA on
chromosome 17 - D17S1
 Disadvantages:
 Very labor-intensive
 Interpretation can be subjective
 Low dynamic range – somewhat accurate over
low concentration range

 Commonly used: Alu probes – more areas
targeted as compared to slot blot methods
 Fluorescent probes
 Very sensitive, particularly for low copy
number DNA
 Compatible with high-throughput DNA
analysis systems
 Disadvantages:
 “Home brew” kits must be quality assurance
tested
 Can be time-consuming

 Advantages
 Less time-consuming than other methods
 Less hands-on time  ability to be automated
 Disadvantages
 Expensive kits
 Expensive platform

 Targetshaploid 64-base length segment,
Yp11.3 – the non-translated portion of the
sex-determining region of the Y-chromosome
(SRY) gene
 Contrast to Quantifiler Human Kit:
 Targets diploid 62-base length segment, 5p15.33
– an intron of the human telomerase reverse
transcriptase (hTERT) gene
 Bothkits utilize a TaqMan probe and a 5’
nuclease activity

-Two TaqMan probes target: untranslated region of SRY gene and
IPC
- Reporter dye (FAM for DNA of interest, VIC for IPC) on 5’ end of
probe
- MGB = minor groove binder – increases Tm  allows for shorter
probe
- NRQ = non-flourescent quencher – proximity to reporter dye
stops it from fluorescing until probe broken down
-P = AmpliTaq Gold DNA polymerase

-PCR temperature increased to 72ºC  P moves 5’ – 3’ and extends
forward and reverse primers
- Nucleotides (from Reaction Mix of kit) are added
- Hybridized probe is broken up  reporter dye released and able to
be detected by camera in qPCR instrument

 PRIMER MIX:
 Human Target Specific Assay
• FAM labeled probe (blue reporter dye) – targets
Yp11.3
 Internal Positive Control (IPC)
• VIC labeled probe (green reporter dye)
• 10,000 copies/rxn. of synthetic, non-human
template
• Inhibitor Detection
 TaqMan 2X Universal PCR Master Mix:
 PCR Reagents
 Passive Reference Dye
• ROX (red reporter dye) – equalizes fluorescence

 Human male DNA, 200ng/ L – supplied with kit
 Standard curve: 50ng/ L to 23pg/ L
 Comparison of Q sample fluorescence to
standards’ fluorescence
 Quality of standard curve is critical for
estimations of quantities of human DNA

 96-well optical plate
 7500 Real-Time PCR instrument with
Sequence Detection Software
 3 phases of PCR: geometric (exponential),
linear & plateau
 qPCR measures fluorescence in real-time at
geometric phase

 Fluorescenceemitted by release of reporter
dye in sample  detected by camera
 Fluorescencethreshold set by Applied
Biosystems’ software at 0.2
 Amplificationcurve of sample crosses this
threshold after a certain number of PCR
cycles
 Thisintersection point is the Cycle Threshold
(CT) for the sample

 Varies depending on amount of DNA in
sample: well with more DNA will have a
lower CT because it takes fewer cycles for
that sample to reach the fluorescence
threshold
 CT values are most accurate and reliable
measurements of quantity of DNA in a sample

 Validation: to recognize, establish, or
illustrate the worthiness or legitimacy of
 Validation of new techniques is essential and
legally required in forensic labs
 Before Quantifiler Y Kit is implemented in a
lab, tests must be completed to ensure its
validity in the forensic community
 Validated by manufacturer, but manufacturer
not held to same rigorous standards
Definition from Merriam Webster Dictionary

 FBI Quality Assurance Standard 8.1.3
 8.1.3.1 – Testing of known and non-probative
evidence samples & Documentation of
reproducibility and precision
 8.1.3.3 – Qualifying tests administered to
analysts prior to use
 SWGDAM Revised Validation Guidelines
Standard 3
 3.1 – Known and non-probative evidence samples
 3.2 - Reproducibility and precision
 3.4 - Sensitivity studies
 3.5 - Mixture studies

 DNA extracts from simulated evidence
 Quantities and CT values compared
 Comparisons to the Amelogenin locus results
obtained from STR DNA analysis
Sample Number Sample Source
1 JRK T1 Blood card
2 JRK T1 Blood card
2 JRK T2 Buccal swab
3 JRK T2 Buccal swab
JRK 1.1 Blood
JRK 1.2 Buccal swab
JRK 1.3 Blood
JRK 2.1 Bloodstain
JRK 2.2 Gum
JRK 2.3 Buccal swab
JRK 4.2 Buccal swab
JRK 4.3 Bloodstain
JRK 4.4 Buccal swab
LMM 4.6 Buccal swab on cotton
AMH 1 Buccal swab

 QuantiBlot performed by other analysts
 Quantifiler and Quantifiler Y combined on one assay

Sample Percentage Male DNA
Electropherogram Quantifiler Y
JRK 5.5 NSF 5.57 7.16
JRK 5.6 NSF 8.84 14.88
JRK 4.8 NSF 1.34 0.716

CT Sample CT IPC
CT Sample CT IPC
Sample Type Quantifiler Quantifiler
Quantifiler Quantifiler
Y Y
Male 23.47
23.98 29.53 35.05
Standard 26.946
Female 23.02
N/A 28.83 33.74
Standard 27.018
Mixture 22.80
25.28 28.67 33.32
Standard 26.899

 Using Quantifiler Y Kit, many slopes fell
below AB’s recommended range (-3.0 to -3.6)
 Erratic slope patterns using lot number
0704042

-Log of concentration plotted against CT values
- Slope outside of acceptable, AB-validated range  overestimate of
DNA concentration to the left of the y-axis OR underestimate of DNA
concentration to the right of the y-axis
- But, CT values are independent of concentration estimates made by
the software
- Therefore, CT values were preferred for interpretation

 Issues not resolved so emphasis on
interpretation was placed on CT values rather
than quantities
 AB standards in quantification kits have been
known to vary in concentration as much as
±50 – 100% with latest quality control tests
allow for ±33% variation
 So… their QC standards may be more lax than
would be preferred in the forensic
community
 Kits with new lot numbers may yield
different results

 Analyses using Lot 0704042:
 Simulated samples & mixtures
 Reproducibility
 Reduced reaction volume
 Sensitivity
 Inhibition
 Many of the same samples analyzed again
using Lot 0706043

 Dr. Allison Eastman, Dr. Donald Orokos and
Dr. Sho Ya Wang
 NYS Police Forensic Investigation Center
 Kim and Melissa – the Queens of Quality
 Jeanette Kovari & Lisa McNab
 Amanda Brinton
 Jay Caponera
 Danielle Brownell