2015 National Conference
Orlando, Florida
Thurs, April 30- Sat, May 2, 2015
Advanced DNA Analysis Methods Session
2015 Innocence Network National Conference
Advanced DNA Analysis Session
Demystifying Touch DNA
Alan Keel
Forensic Analytical Sciences, Inc.
May 2, 2015
Orlando, Florida
Demystifying “Touch DNA”
• Where does DNA come from?
– Cells: Forensic Interest
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sperm
white blood cells
epithelial cells (body cavity fluids)
skin cells (corneocytes)
hair roots
adipocytes (fat cells)
osteocytes (bone cells)
Demystifying “Touch DNA”
• Where does DNA come from?
– Cells: Forensic Interest
• Sperm, white blood cells, epithelial cells, skin cells
(corneocytes), hair roots, adipocytes, osteocytes
– Wet Cells: Body fluid
• Body orifice swabs, stains
– Sperm, white blood cells, epithelial cells
Wet cells: from body fluid
Typical pretrial vaginal swab cell debris
Typical post-conviction vag swab cell debris
Post-conviction vaginal swab cell debris
Demystifying “Touch DNA”
• Where does DNA come from?
– Cells: Forensic Interest
• Sperm, white blood cells, epithelial cells, skin cells
(corneocytes), hair roots, adipocytes, osteocytes
– Wet cells
• Body orifice swabs, stains
– Dry cells
• Skin cells
• Accumulation via habitual wear/use over time
Dry cells: skin cells (corneocytes)
Hat band
Dry cells: dead skin cells
Sandal strap
Demystifying “Touch DNA”
• Where does DNA come from?
– Cells: Forensic Interest
• Sperm, white blood cells, epithelial cells, skin cells
(corneocytes), hair roots, adipocytes, osteocytes
– Wet cells
• Body orifice swabs, stains
– Dry cells
• Accumulation via habitual wear/use over time
– Touch DNA cells
• Transfer from momentary/incidental
touch/handling – usually low numbers/amount of
DNA
Where does Touch DNA come from?
• Transfer of cells from momentary/incidental
touch
– Need moisture/friction to effect the transfer
• From dead cornified skin cells of fingers?
• From sperm cells?
• From white blood cells?
•From epithelial cells!
Where do the epithelial cells come from?
• Body fluid = saliva/nasal mucous
• Mouth, nose, lips, eyes, face in general
• Wipe mouth, lick fingers, cough/sneeze
into our hands, wipe nose, pick nose, pick
teeth
• Touch our faces 20X/hour
• Additional sources of moisture: hundreds
of pores on each fingertip/rub eyes
Touch DNA Biology
• Cells do not originate from the hands, but
from the face and are being transferred by
the hands to the things we touch, while still
moist and via sweat.
• Challenge is to locate this biology, recover
it, identify it (microscopy), analyze it.
2015 Innocence Network National Conference
The Difference between
the Analysis of Low Levels of DNA
and
Low Copy Number [LCN] DNA Analysis
And Why it Matters to You
Alan Keel
Forensic Analytical Sciences, Inc.
May 2, 2015
Orlando, Florida
Normal v. LCN DNA Testing
Normal
• Can attempt with any
amount of DNA
• Standard/usual cycle
number
• Evaluate result on its
own merits on a locus
by locus basis and
holistically:
– State minimum
number of contributors
LCN
• Only indicated with tiny
amounts of DNA
• Increased cycle number
• Two or more
amplifications
• Nested amplifications
• Consensus results
• Reduced reaction volume,
post-amp cleanup, increased
injection times, increased
PCR product load
Why is this distinction important?
• No legal hurdle to get your (often low-level) data
accepted
• LCN sensitivity makes it difficult to establish the
relevance of biological evidence.
– Victim/accused occupy the same environment
– Argue inadvertent contamination
• Very few labs offer Low Copy Number analysis
– Conscientious effort to “get a result” via Standard PCR
even when little to no DNA detected
– Many labs have LCN guidelines based on 2013 NDIS
STD 4.2.1.10, but not true LCN procedure
• What is NDIS STD 4.2.1.10? (Jan 31, 2013)
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NDIS STD 4.2.1.10: “Based upon a lab’s internal
validation, any DNA typing results generated from
limited quantity and/or quality DNA template using
conditions that have demonstrated increased
stochastic effects are defined as Low Template or
Low Copy DNA analyses”.
Stochastic Effects include:
– Allele drop out/in
– Increased stutter
– Peak height imbalance
• LCN Conditions:
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Additional cycles
Post PCR purification
Reduced reaction volume
Injection enhancement: time and/or voltage X
Nested PCR
NDIS STD 4.2.1.10 Today
(Effective January 1, 2015)
• SWGDAM Guidelines for STR Enhanced
Detection Methods (October 2014)
– Attempt to Distinguish between Standard,
Enhanced Detection, and LCN Methodologies
• NDIS adopts SWGDAM distinctions:
– “DNA records developed with Enhanced
Detection Methods validated in accordance
with the QAS and SWGDAM Guidelines for
STR Enhanced Detection Methods… may be
submitted to NDIS:
STD 4.2 NDIS Acceptance of Records
• Must adhere to FBI QAS STDs 9.5/17
• 4.2.1.1 Record shall be interpretable. Any
data used to make an exclusion can be
included in the DNA record submitted to
NDIS.
• 4.2.1.7 Record meets 1/Database rarity
– Still require entry at 10 loci
• 4.2.1.10 LCN records are not acceptable
STANDARD v. ED DNA Testing
Standard
Enhanced Detection
• Can attempt with any
amount of DNA
• Standard/usual cycle
number
• Evaluate result on its
own merits on a locus
by locus basis and
holistically:
• Can attempt with any
amount of DNA
• Increased cycle number
• Post-amp purification
• Reduced reaction vol.
• Increased inj. time/volt.
• Nested amplifications
– State minimum
number of contributors
• Reagent enhancement (add’l
Taq polymerase/BSA
• LCN Analysis????
So Now, What is LCN Analysis?
A Mere Subset of Enhanced
Detection
• Enhanced Detection
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Any amount of DNA
Can incr. the cycle #
Can incr. inj time/volt
Can do nested PCR
Can alter the reagents
Can do post-PCR
cleanup
• Low Copy Number
– Indicated with tiny
amounts of DNA
– Includes any/all ED
measures
– Replicate analyses
should be conducted
on each sample
– Consensus profile
• Post-PCR Enhancement Measures
– Stochastic Effects that might have occurred during a
particular amplification are not affected by post-PCR
measures: injection time/voltage, amount of PCR
product utilized, cleanup
– SWGDAM 3.1 Essentially, any peak that can’t be
eliminated as an artifact (spike/baseline/pull-up/dye
blobs) should be considered to be PCR product.
– SWGDAM 3.1 Any PCR product that can’t be
eliminated as a stutter or minus A peak should be
considered an allele.
– Alleles should be reproducible from injection to
injection.
– These measures help discriminate signal from noise
and do not alter any stochastic effect produced during
the PCR.
– No scientific reason these measures should be
grouped with those measures that alter the PCR
conditions and “increase the potential for stochastic
effects”.
Final Considerations
• Must ensure low-level DNA analyses are clearly
distinguished from LCN analyses.
– New NDIS STD 4.2.1.10 and Enhanced Detection Guidelines
muddy this distinction.
• Enhanced Detection OK, but LCN is a subset of Enhanced
Detection and not OK – self contradictory
• No Enhanced Detection classification is necessary, but we
are likely stuck with it.
– Will continue to lead to admissibility challenges for relevant biol.
– Will result in false convictions from irrelevant biology.
• The Innocence Network should consider
convening an expert panel to draft a position on
what constitutes Standard vs. Enhanced
Detection vs. LCN DNA analysis.
United States District Court, D. New Mexico.
UNITED STATES of America, Plaintiff,
v.
John Charles McCLUSKEY, Defendant.
No. CR 10–2734 JCH. June 20, 2013.
Background: Defendant, who was charged
with murder, moved for a Daubert hearing
and to exclude government's DNA and
serology test results.
US v McCluskey
Two DNA Analysis Inadmissibility Findings
× Results of low copy number (LCN) DNA testing by
New Mexico Department of Public Safety (NMDPS)
Laboratory were not sufficiently reliable to be
admissible in murder trial; LCN testing carried a
greater potential for error due to difficulties in
analysis and interpretation caused by four
stochastic effects.
× Unless government could prove at murder trial, as
a foundational matter, that the quantity of DNA
contributed to a mixed sample by an individual
exceeded 250 pg, the government's evidence with
regard to that individual would be excluded as an
unreliable low copy number (LCN) result.
What went wrong?
1. LCN testing should not be defined by any
particular amount of DNA in a sample but
by the extraordinary analytical
procedures used to obtain a result.
2. NMDPS Lab classified the testing of
samples in this case as LCN by definition
based on 2013 NDIS STD 4.2.1.10.
3. Court misled by defense expert.
What went wrong?
• LCN testing requires unusual conditions
which are not normally employed.
– Increased amplification cycles (>32 to 60)
– Replicate amplifications
– Reduced reaction volume
– Nested amplifications
– Determination of a consensus result
– Post-amplification product purification
• None of the above were employed by
NMDPS in this case.
Additional factors criticized by the Court
• Injection time: 5 sec/10 sec effects.
– 10 sec injection reproduced the initial data and
added clarity to the entire result: Signal v. noise.
• Replicate testing: faulted the lab for not
doing when it was impossible to so do.
– Limited sample, all the DNA used in one amp
– Catch 22, the replicates would have been
inadmissible due to DNA quantity.
– Demonstrates normal analysis (not LCN analysis)
The Four Stochastic Effects cited
by the Court*
1. Peak height imbalance: one: TPOX 51% v. 70%
2. Allelic Drop-out: no result at some loci
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Butler: “an extreme form of heterozygote peak
height imbalance”
Krane: Drop out at one locus implies you can’t be
sure about any other locus
Krane: other labs would be inclined to “describe the
whole sample as simply being inconclusive” when
such result was obtained. The Court relied heavily
on Krane.
3. Allelic Drop-in: not considered by the Court
4. Stutter variability: not criticized by the Court
*Butler, Fundamentals of Forensic DNA Typing, 2010
Inadmissible “LCN” Evidence
Co-Defendant
Room Temp Storage since 1980
Frozen Storage since 1980
8pg (low level DNA) sample
125pg (low copy number?) sample