1. Things that can muck up a DNA Profile
The list is long and distinguished. . .
By Christine Funk,
with a lot of help from her
friends. . .
Board of Public Defense
Trial Team

2. 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!

3. Let’s have a giggle, shall we?

5. Collection Muck Ups: Typically, DNA in criminal cases has done a little traveling. . .

6. Primary Transfer
Picture courtesy of Jennifer Friedman

7. Secondary transfer
Clipping courtesy of Bob Blaiser

8. Collection muck up

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

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

11. Mixture Interpretation
How does one interpret a mixture, that is, which bands or peaks are alleles and which are artifacts or errors?

12. Sample Mixture Example Profiler Plus data
4 peaks at a single locus
D5S818
D13S317
D7S820
D8S1179
D21S11
D18S51
Amel
VWA
FGA
D3S1358
blue panel
green panel
yellow panel
Relative Fluorescence Units
Higher than expected stutter
Imbalance in X and Y peak ratios
“Stutter” on wrong side of allele

13. Who’s in the Mix?
Care to take a guess?

14. Who’s in here?
XY 11, , , 15

15. But wait! What if…
XY 11, , , 15
XY 13, , , 19

16. Whoops! I meant to say… XY 11, 15 28, 30 12, 15

17. Perspective is everything

18. Alleles Present: 13, 17, 18
12, 12
12, 13
12, 14
12, 15
12, 16
12, 17
12, 18
12, 19
13, 13
13, 14
13, 15
13, 16
13, 17
13, 18
13, 19
14, 14
14, 15
14, 16
14, 17
14, 18
14, 19
15, 15
15, 16
15, 17
15, 18
15, 19
16, 16
16, 17
16, 18
16, 19
17, 17
17, 18
17, 19
18, 18
18, 19
19, 19

19. 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
12, 12
12, 13
12, 14
12, 15
12, 16
12, 17
12, 18
12, 19
13, 13
13, 14
13, 15
13, 16
13, 17
13, 18
13, 19
14, 14
14, 15
14, 16
14, 17
14, 18
14, 19
15, 15
15, 16
15, 17
15, 18
15, 19
16, 16
16, 17
16, 18
16, 19
17, 17
17, 18
17, 19
18, 18
18, 19
19, 19

20. But Wait!! There’s more. . .
*ladders not to scale

21. Alleles Present: 16, 17, 18, 19
11, 11
11, 12
11, 13
11, 14
11, 15
11, 16
11, 17
11, 18
11, 19
11, 20
11, 21
12, 12
12, 13
12, 14
12, 15
12, 16
12, 17
12, 18
12, 19,
12, 20
12, 21
13, 13
13, 14
13, 15
13, 16
13, 17
13, 18
13, 19
13, 20
13, 21
14, 14
14, 15
14, 16
14, 17
14, 18
14, 19
14, 20
14, 21
15, 15
15, 16
15, 17
15, 18
15, 19
15, 20
15, 21
16, 16
16, 17
16, 18
16, 19
16, 20
16, 21
17, 17
17, 18
17, 19
17, 20
17, 21
18, 18
18, 19
18, 20
18, 21
19, 19
19, 20
19, 21
20, 20
20, 21
21, 21

22. Still MORE contributors: 18, 18; 18, 19; 19, 19
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
11, 11
11, 12
11, 13
11, 14
11, 15
11, 16
11, 17
11, 18
11, 19
11, 20
11, 21
12, 12
12, 13
12, 14
12, 15
12, 16
12, 17
12, 18
12, 19,
12, 20
12, 21
13, 13
13, 14
13, 15
13, 16
13, 17
13, 18
13, 19
13, 20
13, 21
14, 14
14, 15
14, 16
14, 17
14, 18
14, 19
14, 20
14, 21
15, 15
15, 16
15, 17
15, 18
15, 19
15, 20
15, 21
16, 16
16, 17
16, 18
16, 19
16, 20
16, 21
17, 17
17, 18
17, 19
17, 20
17, 21
18, 18
18, 19
18, 20
18, 21
19, 19
19, 20
19, 21
20, 20
20, 21
21, 21

23. 10!!
10 Possible Profiles in vWA!!!

24. 60 profiles so far. We have 14 loci to go. . .
10 profiles from vWA
6 profiles from D3
60 profiles so far. We have 14 loci to go. . .

25. 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

26. Amplification Muck up

27. STR Alleles with Stutter Products
DNA Size (bp)
D8S1179
D21S11
D18S51
Allele
Relative Fluorescence Units
Stutter Product
6.3%
6.2%
5.4%

28. Stutter in Pictures – First, let’s review how it’s supposed to happen. . .G T C C A G 1 2 3 4

29. Stutter in Pictures – how amplification is supposed to happenG T C C A G 1 2 3 4

30. Stutter in Pictures: amplification as it should be. . .G T C C A G G T C C A G 1 1 2 2 3 3 4 4

31. Stutter in Pictures – Amplification gone horribly awry. . .1 2 3 4

32. Stutter in Pictures G T C C A G 1 2 3 4

33. Stutter in Pictures G T C C A G 1 2 3 4

34. Stutter in Pictures G T C C A G 1 2 3 4

35. Stutter in Pictures G T C C A G 1 2 3 4

36. Stutter in Pictures G T C C A G C A G G T C 1 1 2 2 3 3 4 4

37. Stutter in Pictures G T C C A G C A G G T C 1 1 2 2 3 3 4 4

38. St… St… Stutter!!

39. Stutter Happens
So does teeny tiny contribution

41. Pop Quiz
Why do we care about stutter?

42. A funny thing happened on the way to the forum. . .

43. Degradation
Ahem.
DNA degradation.

44. Degradation

45. Things that are insulting to DNA

46. Degradation: I know it when I see it!

48. Bonus points:
How could increasingly smaller DNA sample size be a problem in a criminal case?

49. When degradation isn’t degradation

50. What causes inhibition?
Hint: Monty Pyton wouldn’t know. . .

51. Size matters

52. Behavior patterns of degraded DNA
To paraphrase Joe Walcott, “the bigger they are, the faster they fall.”
Welterweight champion of the world, 1901 – 1904.

53. Behavior patterns of degraded DNA
AmpFlSTR® Identifiler™ (Applied Biosystems)

54. Behavior patterns of degraded DNA
AmpFlSTR® Identifiler™ (Applied Biosystems)
AmpFlSTR® Identifiler™ (Applied Biosystems)

55. Behavior patterns of degraded DNA
AmpFlSTR® Identifiler™ (Applied Biosystems)
AmpFlSTR® Identifiler™ (Applied Biosystems)

56. What does this have to do with a ‘ski slope?’
AmpFlSTR® Identifiler™ (Applied Biosystems)
D8S1179
D21S11
D7S820
CSF1PO
The smaller stuff is over here
TH01
D13S317
D3S1358
D16S539
D2S1338
D19S433
VWA
TPOX
D18S51
The Bigger stuff is over here!!
D5S818
AMEL
FGA
Slide courtesy of John Butler

57. Option 1

59. Option 2
Slide courtesy of Carll Ladd

61. New Topic: Peak Height Imbalance

62. Heterozygous peak height balance as a function
of signal intensity in RFU units.
PHB
50-150
>
1000 rfu’s
Observed minimum
48%
57%
58%
78%
Theoretical minimum
Mean -3 SD
mean
40%
80%
54%
85%
63%
88%
75%
92% N
145
140
233
209
Data from Holt, et al., J. Forensic Sci. 2002; 47(1):66-96.

64. So what?

65. Primer Binding Site Mutation

66. Capillary Muck Ups: Meet my friends ‘Noise’ and ‘Blob’

67. Noise Happens

68. What’s the problem with noise?
You tell me. . .

69. Blobs

70. So what’s the problem?
Bonus points if you can tell me how a blob could muck up a DNA profile.

71. Pull Up

72. Questioned Sample

73. New Topic: Spikes

74. Spikes

75. Can you tell me how a spike could impact a DNA profile?

76. More mutations that can mess things up. . .

77. Triallelic Pattern Known blood sample: 3 band profile at TPOX
Positive control sample

78. Sneaky off ladder alleles:
D3 Variant Allele
Known blood sample: D3 profile 17,>19.

79. Machine Muck Ups

80. Good EPT Data

81. Bad EPT Data

82. Good Raw Data

83. Bad Raw Data

84. Good Rox

85. Bad Rox

86. Allelic Imbalance
Impress your friends:
Learn the term “stochastic effects”
What happens when you have a small amount of DNA?

87. Allelic Imbalance
Amplifications from same dilution tube (~60 pg). ( PM)
Allelic imbalance present.
“Different DNA THO1 and CSF1P0.

88. Allelic Dropout
Reference sample
Evidence sample

91. 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.

92. Blue Loci* Defendant 17 15, 17 25 Does he match?
D vWA FGA
Defendant ,
Does he match?
*Note the RFU values. This lab uses a ’50 RFU cutoff.’

93. Defendant ,
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.

94. Defendant
12, , , 25
Is he excluded?

95. Defendant
12, , , 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.

96. 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