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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)

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detecting degradation in dna samples

Detecting Degradation in DNA samples

Keith Inman

Forensic Analytical Specialties, Inc

Dayton, Ohio

August 11, 2006

how to identify challenging samples
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
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
Loss of signal at high MW loci
  • Potential causes
    • Uneven amplification
      • Preferential (allele)
      • Differential (locus)
loss of signal at high mw loci7
Loss of signal at high MW loci
  • Potential causes
    • Uneven amplification
      • Preferential (allele)
loss of signal at high mw loci8
Loss of signal at high MW loci
  • Potential causes
    • Uneven amplification
      • Differential (locus)
uneven signal response
Uneven signal response

Differential dye sensitivity

loss of signal at high mw loci10
Loss of signal at high MW loci
  • Fewer intact molecules - degradation
    • Exposure to environmental insult
      • Time
      • Heat
      • Moisture
      • Chemicals; microorganisms
      • UV light
solutions
Solutions
  • Detection
    • Prior to amplification
      • Knowledge of sample
        • Age
        • Condition
        • Substrate
solutions13
Solutions
  • Adjustment of primer concentrations and amp conditions
    • Done by mfg during developmental validation
    • Solves problem of uneven amplification and dye sensitivity
solutions14
Solutions
  • Detection
    • Prior to amplification
      • Differential quantitation
        • Use of two primers, one for long and one for short molecules
nuclear nuth01 qpcr target

probe

STRs

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
nuclear nucsf qpcr target

probe

STR

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
using short and long nuclear targets to assess dna fragmentation

nuTH01

nuCSFar

Using Short and Long Nuclear Targets to Assess DNA Fragmentation

Minutes of DNase Treatment

LH 0 1 2 3 4 5 15 30 45 60 LD LH

  • 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

slide18

Minutes of DNase Treatment

LH 0 1 2 3 4 5 15 30 45 60 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

~67 bp

nuCSFar

solutions21
Solutions
  • Post amplification
      • Yield gel
solutions22
Solutions
  • Post Typing
    • Assessment of PHR’s between loci
    • At this point, a visual assessment
solutions23
Solutions
  • Increase injection time
    • Increases likelihood of saturated data
      • Artifacts created
      • Doesn’t really work with degraded samples
solutions25
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
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
solutions27
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
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
slide29
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
Strategy
  • Calculation of slope by best fit linear regression
  • Intercompare slopes between dye colors using correlation coefficients (r2) and paired-T tests
results
Results
  • Distribution of slopes is approximately normally distributed
slide32
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
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)

results36
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.
next step
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 step41
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
Acknowledgements
  • Dan Krane
  • Jason Gilder
  • Cristian Orrego
  • Zach Gaskin