Degenerate Oligonucleotide PrimedPCR: Thermalcycling Modifications for Forensic DNA Analysis

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Degenerate Oligonucleotide Primed-PCR
Thermalcycling Modifications for Forensic DNA
Analysis

• Denise N. Rodier
• rodierdn_at_vcu.edu

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Low Copy Number DNACSI does it, why cant you?

• Multiplex STR kits are optimized for 1ng-2.5ng of
  pristine DNA
• Low copy number samples (high quality, low
  quantity or low quality, high quantity) are
  typically lt0.16ng
• Avoid using entire sample
• Original sample should be able to be retested

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Our Solution WGA

• Whole Genome Amplification
• Sequence independent preamplification of entire
  genome from limited starting material for
  subsequent molecular analysis
• DOP-PCR, iPEP, MDA
• Characterized in
• Tumor diagnosis
• Prenatal or pre-implantation diagnostics
• Forensic science?

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Current Research WGA

• Experimental Plan
• Test methods/modifications using genomic DNA
  followed by STR analysis
• (WGA products quantified and compared)
• Most efficient method optimized and validated
  for use with forensic multiplex PCR kits and
  platforms
• Test method on casework samples and samples with
  multiple contributors

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DOP-PCR

• A partially-degenerate primer used in conjunction
  with low annealing temperatures (30C) during
  first five amplification cycles
• 5-CCGACTCGAGNNNNNNATGTGG-3
• Primers randomly anneal to the genome
• Coverage is genome wide, theoretically amplifying
  through all STR loci used in forensic DNA analysis

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Published DOP-PCR thermalcycling parameters

• Step 1 5 minutes, 95C
• Step 2 (5 cycles) 1 minute, 94C
• 1.5 minutes, 30C
• 3 minutes, 30?72C
• 3 minutes, 72C
• Step 3 (35 cycles) 1 minute, 94C
• 1 minute, 62C
• 2 minutes, 72C
• (14 seconds at each cycle)
• Step 4 7 minutes, 72C
• Step 5 Hold at 7C

Non-specific annealing
Specific annealing
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What is DOP-PCR?
DOP Primers
DNA
DOP Products
STR Genotyping??
Microsatellites
CGH or FISH
SNP Genotyping
LOH, Linkage Studies
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Semen Stain
After DOP
Before DOP
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The problem

• While there is some success in finding loci,
  there are other issues
• peak imbalance
• drop out
• allele shifting
• preferential amplification
• Drop-in?

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DOP-PCR Optimization

• Increase ramp/elongation times for non-specific
  amplification steps
• Increase cycle numbers for non-specific
  amplification steps
• Modify the primer
• Add proofreading enzymes
• Alter buffering conditions

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Increasing the elongation time

• Step 2 (5 cycles) 1 minute, 94C
• 1.5 minutes, 30C
• 3 minutes, 30?72C
• 3 minutes, 72C
• Modify to 1 minute, 2 minutes, 5 minutes, 8
  minutes, 10 minutes, 12 minutes (n 4 for each
  time point)
• Positive control (K562, 1ng), negative control,
  0.125ng, 0.062ng

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Increasing elongation time -1
? 0.8912 for 0.125ng input 0.8833 for 0.062ng
input
40kb
5kb
Average fragment size increases with increased
elongation time
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Increasing elongation time -2
Average fold increase in total DNA ranged from
145 to 218 fold for 0.125ng inputs 100 to 363
fold for 0.062ng for all time points tested No
correlation between ramp/elongation time and yield
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Increasing elongation time -3



Three minute samples had highest success 65
for 0.125ng inputs and 45 for 0.062ng inputs
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Cycle number titration

• Step 2 (5 cycles) 1 minute, 94C
• 1.5 minutes, 30C
• 3 minutes, 30?72C
• 3 minutes, 72C
• Modify to 3 cycles, 4 cycles, 7 cycles, 9 cycles,
  12 cycles, 15 cycles (n 4 for each cycle set)
• Positive control (K562, 1ng), negative control,
  0.125ng, 0.062ng

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Increasing cycle number -1
Nearly 1000 fold increase
? 0.9432 for 0.125ng input 0.9799 for 0.062ng
input
Strong correlation between cycle number total
yield for sample
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Primer modification

• Standard DOP-PCR primer
• 5-CCGACTCGAGNNNNNNATGTGG-3
• Modify 5 end of primer to increase degeneracy (n
  4)
• 5-CTCGAGNNNNNNNNNNATGTGG-3 (10N primer)
• Use standard thermalcycling parameters
• 5-NNNNNNNNNNNNNNNNATGTGG-3 (16N primer)
• 40 cycles of non-specific amplification
• no specific amplification step

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Modified Primer - 1
10N primer shows large increase in STR
success gt70 of loci success
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DOP-PCR implications future considerations

• Modifications to standard DOP-PCR reaction may
  improve STR success
• Cycle number and/or primer
• Analyze lower yield samples increase sample
  number test alternate sources
• Test proofreading enzymes, altered buffering
  conditions, and/or additives
• Test optimized DOP-PCR reaction with
  non-probative case samples, degraded samples

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Dawson Cruz Lab

• Dr. Tracey Dawson Cruz
• Lindsay Thompson
• Jarrod Champagne
• Cathey Cupples
• Michelle Bonnette

Funding NIJ Forensic DNA RD
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Questions??