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

Joh

n M

. B

utle

r (2

00

9)

Fu

nd

am

en

tals

of

Fo

ren

sic

DN

A T

ypin

g,

Fig

ure

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

uo

resc

enc

e u

nit

s (

RF

Us)

Joh

n M

. B

utle

r (2

00

9)

Fu

nd

am

en

tals

of

Fo

ren

sic

DN

A T

ypin

g,

Fig

ure

14

.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

Joh

n M

. B

utle

r (2

00

9)

Fu

nd

am

en

tals

of

Fo

ren

sic

DN

A T

ypin

g,

Fig

ure

14

.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

Joh

n M

. B

utle

r (2

00

9)

Fu

nd

am

en

tals

of

Fo

ren

sic

DN

A T

ypin

g,

D.N

.A.

Bo

x 1

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

Joh

n M

. B

utle

r (2

00

9)

Fu

nd

am

en

tals

of

Fo

ren

sic

DN

A T

ypin

g,

D.N

.A.

Bo

x 1

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

Joh

n M

. B

utle

r (2

00

9)

Fu

nd

am

en

tals

of

Fo

ren

sic

DN

A T

ypin

g,

Fig

ure

14

.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

Joh

n M

. B

utle

r (2

00

9)

Fu

nd

am

en

tals

of

Fo

ren

sic

DN

A T

ypin

g,

Fig

ure

14

.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?