things that can muck up a dna profile the list is long and distinguished... by christine funk, with...
TRANSCRIPT
Things that can muck up a DNA Profile
The list is long and distinguished. . .
By Christine Funk, with a lot of help from herfriends. . .
Board of Public Defense
Trial Team
651.539.4846
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!
Let’s have a giggle, shall we?
Collection Muck Ups:Typically, DNA in criminal cases has done a little traveling. . .
Primary Transfer
Picture courtesy of Jennifer Friedman
Secondary transfer
Clipping courtesy of Bob Blaiser
Collection muck up
“ 8. . .The match to the bib occurred as a result of contamination in the laboratory and was not an adventitious match. . ."
The Leskie Decision
“ 8. . . The samples from the two cases were examined by the same scientist within a close time frame."
The Leskie Decision
Mixture Interpretation
•How does one interpret a mixture, that is, which bands or peaks are alleles and which are artifacts or errors?
D5S818 D13S317
D7S820
D8S1179 D21S11 D18S51
Amel
VWAFGAD3S1358 blue panel
green panel
yellow panel
Relative Fluorescence Units
Sample Mixture ExampleProfiler Plus data
Higher than expected stutter
Higher than expected stutter
“Stutter” on wrong side of allele
“Stutter” on wrong side of alleleImbalance in X
and Y peak ratios
Imbalance in X and Y peak ratios
4 peaks at a single locus
4 peaks at a single locus
Who’s in the Mix?
Care to take a guess?
Who’s in here?
• XY 11, 15 28, 30 12, 15
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…
• XY 11, 15 28, 30 12, 15• XY 13, 15 32.2, 32.2 17, 19• XY 11, 13 28, 32.2 12, 17
Perspective is everything
12, 1212, 1312, 1412, 1512, 1612, 1712, 1812, 19
13, 1313, 1413, 1513, 1613, 1713, 1813, 19
14, 1414, 1514, 1614, 1714, 1814, 19
15, 1515, 1615, 1715, 1815, 19
16, 1616, 1716, 1816, 19
17, 1717, 1817, 19
18, 1818, 19 19, 19
Alleles Present: 13, 17, 18
12, 1212, 1312, 1412, 1512, 1612, 1712, 1812, 19
13, 1313, 1413, 1513, 1613, 1713, 1813, 19
14, 1414, 1514, 1614, 1714, 1814, 19
15, 1515, 1615, 1715, 1815, 19
16, 1616, 1716, 1816, 19
17, 1717, 1817, 19
18, 1818, 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
But Wait!! There’s more. . .
*ladders not to scale
11, 11 11, 1211, 1311, 14 11, 15 11, 16 11, 1711, 18 11, 19 11, 20 11, 21
12, 1212, 1312, 1412, 1512, 1612, 1712, 1812, 19,12, 2012, 21
13, 1313, 1413, 1513, 1613, 1713, 1813, 1913, 2013, 21
14, 1414, 1514, 1614, 1714, 1814, 1914, 2014, 21
15, 1515, 1615, 1715, 1815, 1915, 2015, 21
16, 1616, 1716, 1816, 1916, 2016, 21
17, 1717, 1817, 1917, 2017, 21
18, 1818, 1918, 2018, 21
19, 1919, 2019, 21
20, 2020, 21 21, 21
Alleles Present: 16, 17, 18, 19
11, 11 11, 1211, 1311, 14 11, 15 11, 16 11, 1711, 18 11, 19 11, 20 11, 21
12, 1212, 1312, 1412, 1512, 1612, 1712, 1812, 19,12, 2012, 21
13, 1313, 1413, 1513, 1613, 1713, 1813, 1913, 2013, 21
14, 1414, 1514, 1614, 1714, 1814, 1914, 2014, 21
15, 1515, 1615, 1715, 1815, 1915, 2015, 21
16, 1616, 1716, 1816, 1916, 2016, 21
17, 1717, 1817, 1917, 2017, 21
18, 1818, 1918, 2018, 21
19, 1919, 2019, 21
20, 2020, 21 21, 21
Alleles Present: 16, 17, 18, 19
Possible Contributors: 16, 16; 16, 17; 16, 18; 16, 19; 17, 17; 17, 18; 17, 19
Still MORE contributors: 18, 18; 18, 19; 19, 19
• 10!!
• 10 Possible Profiles in vWA!!!
• 6 profiles from D3 • 10 profiles from vWA
60 profiles so far. We have 14 loci to go. . .
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
Amplification Muck up
STR Alleles with Stutter Products
D21S11 D18S51D8S1179
DNA Size (bp)
Stutter Product
6.3% 6.2% 5.4%
Allele
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Uni
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Stutter in Pictures – First, let’s review how
it’s supposed to happen. . .
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Stutter in Pictures – how amplification is
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Stutter in Pictures:
amplification as it should
be. . .
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Stutter in Pictures – Amplification gone horribly awry. . .
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Stutter in PicturesG
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Stutter in PicturesG
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Stutter in PicturesG
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Stutter in PicturesG
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Stutter in PicturesG
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Stutter in PicturesG
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St… St… Stutter!!
Stutter Happens
So does teeny tiny contribution
Pop Quiz
•Why do we care about stutter?
A funny thing happened on the way to the forum. . .
Degradation
Ahem.
DNA degradation.
Degradation
Things that are insulting to DNA
Degradation: I know it when I see it!
Bonus points:•How could increasingly smaller DNA sample size be a problem in a criminal case?
When degradation isn’t degradation
Hint: Monty Pyton wouldn’t know. . .
What causes inhibition?
Size matters
Welterweight champion of the world, 1901 – 1904.
•To paraphrase Joe Walcott, “the bigger they are, the faster they fall.”
Behavior patterns of degraded DNA
Behavior patterns of degraded DNA
AmpFlSTR® Identifiler™ (Applied Biosystems)
Behavior patterns of degraded DNA
AmpFlSTR® Identifiler™ (Applied Biosystems)
AmpFlSTR® Identifiler™ (Applied Biosystems)
Behavior patterns of degraded DNA
AmpFlSTR® Identifiler™ (Applied Biosystems)
AmpFlSTR® Identifiler™ (Applied Biosystems)
AMEL
D3S1358TH01
TPOX
D2S1338
D19S433
FGA
D21S11
D18S51
CSF1PO
D16S539
D7S820
D13S317
D5S818
VWA
D8S1179
What does this have to do with a ‘ski slope?’
AmpFlSTR® Identifiler™ (Applied Biosystems)
Slide courtesy of John Butler
The Bigger stuff is over here!!
The smaller stuff is over here
Option 1
Option 2
Slide courtesy of Carll Ladd
New Topic:Peak Height Imbalance
Heterozygous peak height balance as a functionof signal intensity in RFU units.
PHB 50-150
150-300 300-1000
>
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.
Percent of Samples with PHR Less Than 70% as a Function of the Mean RFU of the Sample in the Illinois State Police Crime Lab Validation
Studies
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<500 500<Y<1000 1-2000 2-4000 >4000
Mean RFU Values per Sample
Perc
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f PH
R <
70%
So what?
Primer Binding Site Mutation
Capillary Muck Ups: Meet my friends ‘Noise’ and
‘Blob’
Noise Happens
What’s the problem with noise?
You tell me. . .
Blobs
So what’s the problem?
•Bonus points if you can tell me how a blob could muck up a DNA profile.
Pull Up
Questioned Sample
New Topic: Spikes
Spikes
Can you tell me how a spike could impact a DNA profile?
More mutations that can mess things up. . .
Triallelic Pattern
Positive control sample
•Known blood sample: 3 band profile at TPOX
•Known blood sample: D3 profile 17,>19.
Sneaky off ladder alleles:D3 Variant Allele
Machine Muck Ups
Good EPT Data
Bad EPT Data
Good Raw Data
Bad Raw Data
Good Rox
Bad Rox
Allelic Imbalance
Impress your friends:Learn the term “stochastic effects”
What happens when you have a small amount of DNA?
Allelic ImbalanceAmplifications from same dilution tube (~60 pg). (2-012802 12.59 PM)
Allelic imbalance present.“Different DNA profiles” @ THO1 and CSF1P0.
Allelic Dropout
Evidence sampleEvidence sample
Reference sampleReference 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.
D3 vWA FGA
Blue Loci*
Defendant
17 15, 17 25
Does he match?
*Note the RFU values. This lab uses a ’50 RFU cutoff.’
Defendant 17 15, 17 25Is 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.
Defendant 12, 17 15, 17 20, 25Is he excluded?
Defendant 12, 17 15, 17 20, 25Is he excluded?--No, because stochastic effect or preferential
amplification explains peak height disparity at D3; spike (pull-up) at FGA hides 20 allele.
Special ThanksBob Blaiser, AttorneyJohn Butler, National Institute of Science and TechnologySimon Ford, Lexigen, Inc.Jennifer Friedman, LA Public Defender’s OfficeJason Gilder, Forensic Bioinformatics ServicesTed Kessis, Applied DNA ResourcesDave Knudsen, Minnesota State Public Defender’s OfficeDan Krane, Forensic BioinformaticsCarll Ladd, Connecticut State Crime LabCarrie Roland, Forensic Bioinformatics ServicesMike Sganga, Senior Scientist, Attorney, Law Office of
Michael BurtBill Thompson, UC Irvine