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The list is long and distinguished. . . . Things that can muck up a DNA Profile. By Christine Funk, with a lot of help from her friends. . . Board of Public Defense Trial Team 651.539.4846. There are many, many ways to muck up a DNA profile.

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things that can muck up a dna profile
The list is long and distinguished. . . Things that can muck up a DNA Profile

By Christine Funk,

with a lot of help from her

friends. . .

Board of Public Defense

Trial Team

651.539.4846

there are many many ways to muck up a dna profile
There are many, many ways to muck up a DNA profile
  • Collection Muck Ups -Contamination during collection
  • Interpretation Muck Ups – Mixed Samples
  • Amplification Muck Ups
  • Degradation
  • Primer Binding Muck Ups - Peak Height Imbalance
  • Capillary Muck Ups - Blobs and Noise
  • Dye Muck Ups - Pull Up
  • Spikes
  • DNA Mutation Muck Ups - Tri-allelic Patterns; Off Ladder Alleles
  • Machine Muck Ups - Signal Data
  • Low Level DNA - Allelic Imbalance
  • Interpretation Muck ups – Examiner Bias
  • Math Muck Ups – Fun with Numbers!
primary transfer
Primary Transfer

Picture courtesy of Jennifer Friedman

secondary transfer
Secondary transfer

Clipping courtesy of Bob Blaiser

slide9

The Leskie Decision

“ 8. . .The match to the bib occurred as a result of contamination in the laboratory and was not an adventitious match. . ."

slide10

The Leskie Decision

“ 8. . . The samples from the two cases were examined by the same scientist within a close time frame."

mixture interpretation
Mixture Interpretation
  • How does one interpret a mixture, that is, which bands or peaks are alleles and which are artifacts or errors?
sample mixture example profiler plus data
Sample Mixture ExampleProfiler Plus data

4 peaks at a single locus

Higher than expected stutter

FGA

D3S1358

VWA

blue panel

Amel

green panel

Relative Fluorescence Units

D8S1179

D21S11

D18S51

“Stutter” on wrong side of allele

Imbalance in X and Y peak ratios

D13S317

yellow panel

D5S818

D7S820

who s in the mix
Who’s in the Mix?

Care to take a guess?

who s in here
Who’s in here?
  • XY 11, 15 28, 30 12, 15
but wait what if
But wait! What if…
  • XY 11, 15 28, 30 12, 15
  • XY 13, 15 32.2, 32.2 17, 19
whoops i meant to say
Whoops! I meant to say…
  • XY 11, 15 28, 30 12, 15
  • XY 13, 15 32.2, 32.2 17, 19
  • XY 11, 13 28, 32.2 12, 17
slide19

Alleles Present: 13, 17, 18

Possible Contributors to D3:

13, 13; 13, 17; 13,18;17, 17; 17,18; 18,18

SIX possible profiles

in D3

slide20

But Wait!! There’s more. . .

*ladders not to scale

slide22

Alleles Present: 16, 17, 18, 19

Still MORE contributors: 18, 18; 18, 19; 19, 19

Possible Contributors: 16, 16; 16, 17; 16, 18; 16, 19; 17, 17; 17, 18; 17, 19

slide23
10!!
  • 10 Possible Profiles in vWA!!!
slide24
6 profiles from D3

10 profiles from vWA

60 profiles so far. We have 14 loci to go. . .

why this is hard
Why this is hard:
  • You don’t know how many people are in the mix
  • You don’t know when and where their alleles overlap
  • You don’t know if you have alleles that have dropped out due to low copy numbers
  • You don’t know if you have a triallelic pattern or stutter or primer binding site mutation
str alleles with stutter products

6.3%

6.2%

5.4%

STR Alleles with Stutter Products

DNA Size (bp)

D8S1179

D21S11

D18S51

Allele

Relative Fluorescence Units

Stutter Product

stutter in pictures first let s review how it s supposed to happen
Stutter in Pictures – First, let’s review how it’s supposed to happen. . .

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stutter in pictures how amplification is supposed to happen
Stutter in Pictures – how amplification is supposed to happen

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stutter in pictures amplification as it should be
Stutter in Pictures: amplification as it should be. . .

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stutter in pictures
Stutter in Pictures

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Stutter in Pictures

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Stutter in Pictures

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Stutter in Pictures

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Stutter in Pictures

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Stutter in Pictures

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slide39

Stutter Happens

So does teeny tiny contribution

pop quiz
Pop Quiz
  • Why do we care about stutter?
degradation
Degradation

Ahem.

DNA degradation.

bonus points
Bonus points:
  • How could increasingly smaller DNA sample size be a problem in a criminal case?
slide50

What causes inhibition?

Hint: Monty Pyton wouldn’t know. . .

slide52

Behavior patterns of degraded DNA

  • To paraphrase Joe Walcott, “the bigger they are, the faster they fall.”

Welterweight champion of the world, 1901 – 1904.

slide56

AmpFlSTR® Identifiler™ (Applied Biosystems)

D8S1179

D21S11

D7S820

CSF1PO

TH01

D13S317

D3S1358

D16S539

D2S1338

VWA

TPOX

D19S433

D18S51

D5S818

AMEL

FGA

What does this have to do with a ‘ski slope?’

The smaller stuff is over here

The Bigger stuff is over here!!

Slide courtesy of John Butler

slide59

Option 2

Slide courtesy of Carll Ladd

slide62

Heterozygous peak height balance as a function

of signal intensity in RFU units.

Data from Holt, et al., J. Forensic Sci. 2002; 47(1):66-96.

so what s the problem
So what’s the problem?
  • Bonus points if you can tell me how a blob could muck up a DNA profile.
triallelic pattern
Triallelic Pattern
  • Known blood sample: 3 band profile at TPOX

Positive control sample

slide78

Sneaky off ladder alleles:

D3 Variant Allele

  • Known blood sample: D3 profile 17,>19.
allelic imbalance
Allelic Imbalance

Impress your friends:

Learn the term “stochastic effects”

What happens when you have a small amount of DNA?

slide87

Allelic Imbalance

Amplifications from same dilution tube (~60 pg). (2-012802 12.59 PM)

Allelic imbalance present.

“Different DNA profiles” @ THO1 and CSF1P0.

slide88

Allelic Dropout

Reference sample

Evidence sample

biased interpretation of results
It is human nature to want to ‘solve’ the crime.

The use of reference samples ‘assists’ the scientist in this process.

Biased Interpretation of results
slide92

Blue Loci*

D3 vWA FGA

Defendant

17 15, 17 25

Does he match?

*Note the RFU values. This lab uses a ’50 RFU cutoff.’

slide93

Defendant

17 15, 17 25

Is he excluded?

-- No, because additional alleles at D3 and FGA are “spurious”. The “12” is below the threshold and the FGA alleles are off ladder.

slide94

Defendant

12, 17 15, 17 20, 25

Is he excluded?

slide95

Defendant

12, 17 15, 17 20, 25

Is he excluded?

--No, because stochastic effect or preferential amplification explains peak height disparity at D3; spike (pull-up) at FGA hides 20 allele.

special thanks
Special Thanks

Bob Blaiser, Attorney

John Butler, National Institute of Science and Technology

Simon Ford, Lexigen, Inc.

Jennifer Friedman, LA Public Defender’s Office

Jason Gilder, Forensic Bioinformatics Services

Ted Kessis, Applied DNA Resources

Dave Knudsen, Minnesota State Public Defender’s Office

Dan Krane, Forensic Bioinformatics

Carll Ladd, Connecticut State Crime Lab

Carrie Roland, Forensic Bioinformatics Services

Mike Sganga, Senior Scientist, Attorney, Law Office of Michael Burt

Bill Thompson, UC Irvine