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Fundamentals of Forensic DNA Typing
Slides prepared by John M. Butler
June 2009
Chapter 14Forensic Challenges
Chapter 14 – Forensic Challenges
Chapter SummaryDNA can be damaged or destroyed as it is exposed to environmental elements present at crime or disaster scenes. Degraded DNA often results in partial STR profiles or none at all. The use of reduced size PCR products, also known as “miniSTRs”, can in some cases enable recovery of information from degraded DNA or samples containing PCR inhibitors. Mixtures of DNA from two or more sources occur in some crime scenes and resolving the components of a mixture can be challenging in many situations. Depending on the type of mixture obtained including the ratio of contributors, the peak heights of STR alleles observed in a mixture can be used to group alleles to decipher the mixture component genotypes. Low-level DNA analysis, sometimes referred to as “low-copy number” or LCN testing, involves attempting to detect a DNA profile from only a few cells often through boosting the number of PCR cycles to enhance sensitivity. LCN testing is prone to problems, such as allelic drop-out (due to stochastic amplification effects) and allelic drop-in (due to sporadic contamination), and rigorous rules are generally applied including generating composite profiles of replicated alleles from multiple amplifications of the same DNA extract.
Degraded DNA sample
D5S818D13S317
D7S820D16S539
CSF1PO Penta D
Agarose yield gel results
Smear of degraded DNA fragments
High relative molecular mass DNA in a tight band
(a)
(b)
Good quality DNA
Degraded DNA
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14
.1
Full Profile (Good Quality)
Partial Profile (Poor Quality)
(a)
(b)
DNA size (bp) relative to an internal size standard (not shown)R
ela
tiv
e fl
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resc
enc
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nit
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RF
Us)
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.2
Comparison of Results with Good and Poor Quality DNA
STR repeat regionminiSTR primer
miniSTR primer
Conventional PCR primer
Conventional PCR primer
(a)
(b)
Conventional STR test (COfiler kit)
MiniSTR assay (using Butler et al. 2003 primers)
150 bp smaller
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.3
miniSTRs (Reduced size amplicon
Compromised Sample Improvements
• Better DNA extraction/recovery• Continued use of miniSTRs
– to improve success rates for recovery of information from compromised DNA evidence
• Replicate results for reproducibility – to improve reliability with low-template DNA testing
(a) Single SourceD3S1358 TH01 D13S317 D16S539 D2S1338
16,16 9,9.3 8,12 9,9 17,19
(b) Mixed Source
D3S1358 TH01 D13S317 D16S539 D2S1338
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 14.4
Type A Type B Type C
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4.1
Different Types of DNA Mixtures According to the German Stain Commission Classification
>2 alleles at a
locus, except tri-allelics?
Single Source DNA Sample
NO
Mixed DNA Sample
YES
Differentiate a
Major/Minor Component?
Determine STR profile and compute RMP
YES
Is the sample a mixture?
TYPE B
NO
YES
Stochastic Effects ? Possible
Low Level DNA) ?
YES
Assume # Contributor
s?
TYPE C
TYPE ANO
A biostatistical analysis must be performed
Probability of Exclusion [CPE]
“RMNE”
Likelihood Ratio [LR]
YES
NO
Are # of contributors
defined?
A biostatistical analysis should not be performed
Determine component profile(s) and compute RMP for
major
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4.1
Mixture Classification Flowchart
Identify the Presence of a Mixture
Consider All Possible Genotype Combinations
Estimate the Relative Ratio of the Individuals Contributing to the Mixture
Identify the Number of Potential Contributors
Designate Allele Peaks
Compare Reference Samples
Step #1
Step #2
Step #3
Step #4
Step #5
Step #6
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.5Steps in the Interpretation of Mixtures
Defined by Clayton et al. 1998
100 pg
50 pg
10 pg
Allele dropout
Severe imbalance
Good heterozygote peak balance
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.6
Heterozygote Sample Result at a Single STR Locus at Different DNA Amounts
Tri-allelic pattern
TPOX
Variant allele
D7S820D3S1358
Stutter products
6.0% 7.8%
TH01
Variant alleleIncomplete adenylation
D8S1179
-A
+A
-A
+A
Some Example Biological Artifacts with STR Markers
From Butler, J.M. (2004) Short tandem repeat analysis for human identity testing. Current Protocols in Human Genetics, John Wiley & Sons, Hoboken, NJ, Unit 14.8, (Supplement 41), pp. 14.8.1-14.8.22
DNA Degradation
Intact sample
300 base pair PCR product can be produced
Target region for PCR
300 base pair PCR product can not be produced or only in limited quantities
Degraded sampleTarget region for PCR is fragmented
Degraded DNA
Larger segments of DNA cannot be recovered when DNA molecules have fragmented into small pieces (caused by heat, water, or bacteria)
D19
AMEL
D3
D8 VWA
TH01
D21FGA D16 D18
D2
“Degraded DNA” (falls apart with high temperatures)
“Decay curve” of degraded DNA
DNA Degradation Means Less Loci Work
Control (high quality DNA)
Degraded
4000 rfu
600 rfu Much lower peak heights and loss of alleles
With degraded DNA samples, information is simply lost at
the larger sized STR loci
Full Profile (Good Quality)
Partial Profile (Poor Quality)
4000RFUs
600RFUs
Smaller sized DNA works
With degraded DNA samples, information is simply lost at the
larger sized STR loci
Typing “1 ng” degraded DNA
Same DNA with Different Quality
Signal Strength is Lower
Impact of Degraded DNA Samples
• Comparison to a phone number (string of 13 numbers)
001-301-975-4049
• If you only had “4049”…this information would be of limited value since it is not as specific (and could match other phone numbers from different area codes)
• DNA profiles are essentially a string of numbers – if the DNA is damaged, then the string of numbers is shorter and less informative…
------------4049 ----301-9-------or
STR repeat regionminiSTR primer
miniSTR primer
Conventional PCR primer
Conventional PCR primer
(A)
(B)
Conventional STR test (COfiler™ kit)
MiniSTR assay (using Butler et al. 2003 primers)
Smaller PCR products work better with low copy number or fragmented DNA templates
miniSTRs: new tool for degraded DNA
150 bp smaller
<15%<15%Stutter region
>70%>70%
100%
Heterozygous peak region
85%
MIXTURE REGIONMIXTURE REGION
9%
Higher than typical stutter product (>15%)
100%
<15%<15%
>70%>70%60%
10%
25%
Wrong side of allele to be typical stutter product
Smaller peak area than normally seen with heterozygote partner alleles(<70%)
(a)
(b)
Mixture Basics
• Mixtures arise when two or more individuals contribute to the sample being tested.
• Mixtures can be challenging to detect and interpret without extensive experience and careful training.
• Differential extraction can help distinguish male and female components of many sexual assault mixtures.
From J.M. Butler (2005) Forensic DNA Typing, 2nd Edition, p. 154
Even more challenging with poor quality data when degraded DNA is present…
Y-chromosome markers can help here in some cases…
More on Mixtures...
Some mixture interpretation strategies involve using victim (or other reference) alleles to help isolate obligate alleles coming from the unknown portion of the mixture
Most mixtures encountered in casework are 2-component mixtures arising from a combination of victim and perpetrator DNA profiles
major
minor
Ratios of the various mixture components stay fairly constant between multiple loci enabling deduction of the profiles for the major and minor components
Torres et al. (2003) Forensic Sci. Int. 134:180-186 examined 1,547 cases from 1997-2000 containing 2,424 typed samples of which 163 (6.7%) contained a mixed profile with only 8 (0.3%) coming from more than two contributors
95.1% (155/163) were 2-component mixtures
Ann Gross will discuss some recent collected casework
summaries
Amelogenin D8S1179 D21S11 D18S51
Example Mixture Data (MIX05 Study-Profiler Plus)
Single Source Sample (Victim)
Evidence Mixture (Victim + Perpetrator)
X,Y 12,12 28,31.2 15,16True “Perpetrator” Profile
Obligate Alleles (not present in the victim reference)
Y 12 28 16
http://www.cstl.nist.gov/biotech/strbase/interlab/MIX05.htm
MIX05 Case #1; Profiler Plus green loci
Victim = majorPerpetrator = minor
Sources of DNA Mixtures• Two (or more) individuals contribute to the
biological evidence examined in a forensic case (e.g., sexual assault with victim and perpetrator or victim, consensual sexual partner, and perp)
• Contamination of a single source sample from – evidence collection staff – laboratory staff handling the sample– Low-level DNA in reagents or PCR tubes or pipet tips
Reference elimination samples are useful in deciphering both situations
due to possibility of intimate sample profile subtraction
Victim Reference and Spouse or Boyfriend Reference
Examine Staff Profiles (Elimination Database), etc.
http://www.cstl.nist.gov/biotech/strbase/interlab/MIX05.htmMIX05 Case #1; Identifiler green loci
Mixtures: Issues and Challenges
• The probability that a mixture will be detected improves with the use of more loci and genetic markers that have a high incidence of heterozygotes.
• The detectability of multiple DNA sources in a single sample relates to the ratio of DNA present from each source, the specific combinations of genotypes, and the total amount of DNA amplified.
• Some mixtures will not be as easily detectable as other mixtures.
From J.M. Butler (2005) Forensic DNA Typing, 2nd Edition, p. 155
MixtureMixtureMixture?
Mixture Mixture?
D5S818 D13S317D7S820
D8S1179 D21S11 D18S51
Amel
VWA FGAD3S1358 blue panel
green panel
yellow panel
DNA Size (bp)
Relative Fluorescence Units
amelogenin X-Y peak imbalance
A
BC
B
AC
D DCB
A
3 peaks at D8S1179
4 peaks at D21S11
4 peaks at D18S51
X
Y
DNA Size (bp)
RFUs
AA BC
AB BC
BC AC
AB AC
CC AB
BB AC
2 = major component1 = major component
1 2
1 2
1 2
1 2
1 2
1 2
AB C
Mixture Interpretation – A Major Challenge…Not a clear cut answer because DNA result is from multiple contributors
Single Source Sample
Single Source vs. Mixture Samples
Different possible combinations could have given rise to the particular mixture observed
One or two peaks observed at each locus (tested DNA region)
Locus 1 Locus 2 Locus 3 Locus 4 Locus 5
16,16 9,9.3 8,12 9,9 17,19
Mixture Sample
More than two peaks observed at more than two loci (tested DNA regions)
Locus 1 Locus 2 Locus 3 Locus 4 Locus 5
With Some Mixtures, Multiple Genotype Combinations Are Possible
A B C D
ACBDABCDBCAD
Peak Height Ratios (PHR)Minimum Peak Height (mPH)Proportion (p) or mixture proportion (Mx)
Depends on PHR and proportion of mixture components from the various contributors
Common Casework Challenges
D3S1358 TH01 D13S317 D16S539 D2S1338
MIXTURES
DEGRADED DNA
D5S818D13S317
D7S820
D16S539 CSF1PO Penta D
From Butler, J.M. (2004) Short tandem repeat analysis for human identity testing. Current Protocols in Human Genetics, John Wiley & Sons, Hoboken, NJ, Unit 14.8, (Supplement 41), pp. 14.8.1-14.8.22
Loss of signal at larger size loci
More than two alleles at multiple loci
DNA Testing Has Become Extremely Sensitive…
• What does it mean to obtain a DNA match between a suspect and material from a crime scene?
• Is the fact that a DNA profile obtained mean that this information is probative?
• More complicated samples (mixtures) and more items per case being submitted to labs
Time Line Showing the Potential for DNA Deposition/Transfer
Time
Crime Event
Opportunity for DNA Transfer from Perpetrator
Opportunity for Adventitious Transfer
Adapted from Gill, P. (2002) BioTechniques 32(2): 366-385, Figure 5
Potential to “Contaminate”
Discovery
Investigators arrive, detect, and recover evidentiary material
Laboratory analysis
Analysis completed
Higher sensitivity techniques are most likely to pick up
previously deposited (background) DNA
Chapter 14 – Points for Discussion
• Discuss advantages and disadvantages of miniSTR assays.
• Name at least two ways that the presence of a mixture can be detected.
• What are the causes of allele drop-in and allele drop-out in the context of amplifying low amounts of DNA template?