1. Detecting Degradation in DNA samples
Keith Inman
Forensic Analytical Specialties, Inc
Dayton, Ohio
August 11, 2006

2. Intact and degraded DNA

3. “Wedge” effect

4. 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

5. Degradation of DNA
Random breaking of DNA molecule into numerous fragments of varying sizes
Can speak of “average fragment size”

6. Loss of signal at high MW loci
Potential causes
Uneven amplification
Preferential (allele)
Differential (locus)

7. Loss of signal at high MW loci
Potential causes
Uneven amplification
Preferential (allele)

8. Loss of signal at high MW loci
Potential causes
Uneven amplification
Differential (locus)

9. Uneven signal response
Differential dye sensitivity

10. Loss of signal at high MW loci
Fewer intact molecules - degradation
Exposure to environmental insult
Time
Heat
Moisture
Chemicals; microorganisms
UV light

11. Effect of Heat on DNA

12. Solutions Detection Prior to amplification Knowledge of sample Age
Condition
Substrate

13. Solutions Adjustment of primer concentrations and amp conditions
Done by mfg during developmental validation
Solves problem of uneven amplification and dye sensitivity

14. Solutions Detection Prior to amplification Differential quantitation
Use of two primers, one for long and one for short molecules

15. 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

16. 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

17. Using Short and Long Nuclear Targets to Assess DNA Fragmentation
Minutes of DNase Treatment
LH LD LH
nuCSFar
nuTH01
nuCSF assay – detects and quantifies DNA fragments larger than ~67bp
nuTH01 assay – detects and quantifies DNA fragments larger than ~180bp
10 kbp
1.5 kb
1 kbp
800 bp
600 bp
400 bp
200 bp
~67 bp

18. Minutes of DNase Treatment
LH LD LH
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.
10 kbp
1.5 kb
1 kbp
800 bp
600 bp
400 bp
200 bp
nuTH01
nuCSFar
~67 bp

19. qPCR Degradation Ratio ~ 25: “1 ng” (nuTH01) Identifiler STR Results

20. Interpreting the qPCR 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

21. Solutions
Post amplification
Yield gel

22. Solutions Post Typing Assessment of PHR’s between loci
At this point, a visual assessment

23. Solutions Increase injection time
Increases likelihood of saturated data
Artifacts created
Doesn’t really work with degraded samples

24. Saturated data and artifacts

25. Solutions Amplify more DNA Increases likelihood of saturated data
Frequently must combine data from two amps to get full profile

26. 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

27. Solutions Consideration of PHR’s between loci Use of positive controls
Likely undegraded
Establishes a baseline for good samples

28. 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

29. 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)

30. Strategy Calculation of slope by best fit linear regression
Intercompare slopes between dye colors using correlation coefficients (r2) and paired-T tests

31. Results
Distribution of slopes is approximately normally distributed

32. 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

34. 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)

36. 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.

37. Threshold levels and significance levels

38. Threshold levels and significance levels

40. 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

41. Next Step Amplify and type the samples
Amplify normal amounts (1.5 – 2 ng)
Amplify larger amounts to bring up larger, more degraded loci

42. Acknowledgements
Dan Krane
Jason Gilder
Cristian Orrego
Zach Gaskin