detecting degradation in dna samples keith inman forensic analytical specialties, inc dayton, ohio...
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Detecting Degradation in DNA samples
Keith Inman
Forensic Analytical Specialties, Inc
Dayton, Ohio
August 11, 2006
Intact and degraded DNA
“Wedge” effect
How To Identify Challenging Samples?
experience (analyst, intra-lab, inter-lab, literature)
unsuccessful analysis using routine methods i.e., partial or null typing results
• inefficient use of analyst time
Degradation of DNA
Random breaking of DNA molecule into numerous fragments of varying sizes
Can speak of “average fragment size”
Loss of signal at high MW loci
Potential causes Uneven
amplification
• Preferential (allele)
• Differential (locus)
Loss of signal at high MW loci
Potential causes Uneven
amplification
• Preferential (allele)
Loss of signal at high MW loci
Potential causes Uneven
amplification
• Differential (locus)
Uneven signal response
Differential dye sensitivity
Fewer intact molecules - degradation
• Exposure to environmental insult
• Time
• Heat
• Moisture
• Chemicals; microorganisms
• UV light
Loss of signal at high MW loci
Effect of Heat on DNA
Solutions Detection
• Prior to amplification
• Knowledge of sample
• Age
• Condition
• Substrate
Solutions
Adjustment of primer concentrations and amp conditions Done by mfg during developmental validation
Solves problem of uneven amplification and dye sensitivity
Solutions
Detection Prior to amplification
• Differential quantitation
• Use of two primers, one for long and one for short molecules
Nuclear nuTH01 qPCR Target
target sequence spans TH01 CODIS STR locus
(2 copies/diploid genome) FAM-labeled TaqMan detection probe target sequence length: ~170 – 190 bp
STRs
probe
Nuclear nuCSF qPCR Target
target sequence flanks the CODIS CSF STR region (2 copies/diploid genome)
VIC-labeled TaqManMGB detection probe target sequence length: 67 bp
probe
STR
Using Short and Long Nuclear Targets to Assess DNA Fragmentation
nuCSF assay – detects and quantifies DNA fragments larger than ~67bp
nuTH01 assay – detects and quantifies DNA fragments larger than ~180bp
LH 0 1 2 3 4 5 15 30 45 60 LD LH
10 kbp
1.5 kb
1 kbp 800 bp
600 bp
400 bp
200 bp
Minutes of DNase Treatment
~67 bpnuCSFar
nuTH01
qPCR Degradation Ratio = nuCSF Quantity (ng) nuTH01 Quantity (ng)
For high-molecular weight DNA, expect the Degradation Ratio to be ~ 1.
For highly-degraded DNA, expect the Degradation Ratio to be > 1.
The bigger the qPCR Degradation Ratio, the more fragmented the DNA.
LH 0 1 2 3 4 5 15 30 45 60 LD LH
10 kbp
1.5 kb
1 kbp 800 bp
600 bp
400 bp
200 bp
Minutes of DNase Treatment
nuCSFar
nuTH01
~67 bp
qPCR Degradation Ratio ~ 25:“1 ng” (nuTH01) Identifiler STR Results
Interpreting the qPCR Degradation Ratio
Degradation Ratio
STR Implications
1 – 3 none
3 – 5 “wedge” effect,
possible cross-dye pull-up
>5
(>10 artifacts
expected to be significant)
increasing “wedge” effect, pull-up,
dropped-out alleles at larger loci,
off-scale peaks, called stutter peaks,
-A shouldering
Solutions
Post amplification• Yield gel
Solutions
Post Typing
•Assessment of PHR’s between loci
•At this point, a visual assessment
Solutions Increase injection time
• Increases likelihood of saturated data
• Artifacts created
• Doesn’t really work with degraded samples
Saturated data and artifacts
Solutions Amplify more DNA
Increases likelihood of saturated data
• Frequently must combine data from two amps to get full profile
New (Non-Routine) Analysis Tools for Challenging and Compromised Samples
* miniSTRs* SNPs* mitochondrial sequencing/linear-array typing enhanced PCR conditions (e.g., extra Taq, BSA) Y-STR analysis for male/female mixtures low-volume PCR amplifications increased PCR cycle numbers
Solutions
Consideration of PHR’s between loci Use of positive
controls
• Likely undegraded
• Establishes a baseline for good samples
Strategy for post-typing diagnosis of degradation
Consider the slope between loci as indicator of drop-off of signal within colors
Calculate a single summary value from the three normalized slopes as another parameter of normal undegraded sample
For each dye color, 6 data points were used to calculate the slope Y coordinate is RFU
X coordinate is peak data collection point (as determined by Genescan)
Strategy
Calculation of slope by best fit linear regression
Intercompare slopes between dye colors using correlation coefficients (r2) and paired-T tests
Results
Distribution of slopes is approximately normally distributed
A
0
5
10
15
20
25
30
35
40
-12 -11 -10-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5
Slope (RFU/data collection point)
Count
B
0
5
10
15
20
25
30
35
40
-12 -11 -10-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5
Slope (RFU/data collection point)
Count
C
0
5
10
15
20
25
30
35
40
-12 -11 -10-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5
Slope (RFU/data collection point)
Count
All slopes are negative Due to differential dye sensitivity and
multiplex complexities summarized earlier
Slopes between the three colors are not correlated Each color shows a different pattern of drop-
off in intensity between the loci
One number for evaluation
Slopes for each samples were normalized against the max and min slopes for each dye, then added to give a single normalized sum of slopes value
mnorm = (m – mmin)/(mmax – mmin)
0
5
10
15
20
25
0
0.2 0.4 0.6 0.8
1
1.2 1.4 1.6 1.8
2
2.2 2.4 2.6 2.8
3
Normalized Sum
Count
Results The average and standard deviation of the samples
can be used to calculate thresholds of departure from normal at both the 5% and 1% levels for each color
The same statistic can be used with the normalized sum to determine departures from normal at the 5% and 1% level for a single sample
Can now determine if, post typing, a sample deviates from our expectation of a normal, undegraded sample.
Threshold levels and significance levels
Avg PH Avg PH Std Dev Slope Avg
Slope Std Dev = 0.05 = 0.01
Blue 1320 515 -3.82 2.26 -7.54 -9.08 Green 1790 682 -1.00 1.67 -3.75 -4.88 Yellow 1570 592 -4.53 1.98 -7.79 -9.14 Normalized Sum 1.49 0.42 0.80 0.51
Threshold levels and significance levels
Avg PH Avg PH Std Dev Slope Avg
Slope Std Dev = 0.05 = 0.01
Blue 1320 515 -3.82 2.26 -7.54 -9.08 Green 1790 682 -1.00 1.67 -3.75 -4.88 Yellow 1570 592 -4.53 1.98 -7.79 -9.14 Normalized Sum 1.49 0.42 0.80 0.51
Avg PH Avg PH Std Dev Slope Avg
Slope Std Dev = 0.05 = 0.01
Blue 1320 515 -3.82 2.26 -7.54 -9.08 Green 1790 682 -1.00 1.67 -3.75 -4.88 Yellow 1570 592 -4.53 1.98 -7.79 -9.14 Normalized Sum 1.49 0.42 0.80 0.51
Next step Prepare degraded samples and apply the same analysis
Artificially degrade samples with DNAse
Monitor level of degradation via a yield gel
• Gives information about average base pair size when compared to a standard ladder
Next Step
Amplify and type the samples Amplify normal
amounts (1.5 – 2 ng)
Amplify larger amounts to bring up larger, more degraded loci
Acknowledgements
Dan Krane Jason Gilder Cristian Orrego Zach Gaskin