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Degenerate Oligonucleotide PrimedPCR: Thermalcycling Modifications for Forensic DNA Analysis

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Title: Degenerate Oligonucleotide PrimedPCR: Thermalcycling Modifications for Forensic DNA Analysis


1
Degenerate Oligonucleotide Primed-PCR
Thermalcycling Modifications for Forensic DNA
Analysis
  • Denise N. Rodier
  • rodierdn_at_vcu.edu

2
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

3
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?

4
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

5
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

6
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
7
What is DOP-PCR?
DOP Primers
DNA
DOP Products
STR Genotyping??
Microsatellites
CGH or FISH
SNP Genotyping
LOH, Linkage Studies
8
Semen Stain
After DOP
Before DOP
9
The problem
  • While there is some success in finding loci,
    there are other issues
  • peak imbalance
  • drop out
  • allele shifting
  • preferential amplification
  • Drop-in?

10
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

11
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

12
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
13
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
14
Increasing elongation time -3



Three minute samples had highest success 65
for 0.125ng inputs and 45 for 0.062ng inputs
15
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

16
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
17
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

18
Modified Primer - 1
10N primer shows large increase in STR
success gt70 of loci success
19
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

20
Dawson Cruz Lab
  • Dr. Tracey Dawson Cruz
  • Lindsay Thompson
  • Jarrod Champagne
  • Cathey Cupples
  • Michelle Bonnette

Funding NIJ Forensic DNA RD
21
Questions??
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