Evaluation of the ABI Quantifiler Human DNA Quantification Kit: Optimization of Input DNA for STR An

1
Evaluation of the ABI Quantifiler Human DNA
Quantification Kit Optimization of Input DNA for
STR Analysis by CE and Determination of a True
Zero Value

• Catherine Cupples, Kristen Lewis, Jarrod
  Champagne, Rodney Dyer, and Tracey Dawson Cruz

2
Serological Screening
DNA Extraction
DNA IQ
Organic
DNA Quantitation
DNA Quality
Real-Time PCR Blot Hybridization Liquid
Hybridization
Quantifiler
QuantiBlot
Yield gel Real-Time PCR
?
INTERNAL RE-VALIDATION
INTERNAL VALIDATION
STR Amplification
?
STR Detection
STR Profile Analysis
3
Quantitation

• Human Specific
• Sensitive
• Large dynamic range
• Precise
• Low variability
• Accurate
• Narrow range of input DNA for STR amplification
• Too much or too little DNA causes stochastic
  effects

4
Integrating Quantifiler as a Quantitation Method

• Conversion from QuantiBlot to Quantifiler
• Known differences
• QuantiBlot underestimates DNA compared to
  Quantifiler
• QuantiBlot is less sensitive than Quantifiler
• Detection Range
• QuantiBlot 0.060 2 ng/µl
• Quantifiler 0.023 50 ng/µl
• Quantifiler detects amplifiable DNA

5
QuantiBlot

• Slot blot quantitation
• D17Z1 probe
• 40bp
• Human alpha satellite locus
• Human/primate specific
• Chemiluminescent or colorimetric detection
• Detection Range
• 0.060 2ng/µl

6
Quantifiler

• Real-Time PCR
• Kit components include
• DNA Standard
• PCR Reaction Mixture
• Primer Mix
• hTERT primers
• TaqMan probe
• Internal positive control template
• ABI Prism 7000 Sequence Detection System
• 40-cycle amplification
• Measures cycle-to-cycle changes in fluorescent
  signal
• Real-time data analysis
• Detection Range
• 0.023 50ng/µl

7
5 Nuclease Activity
Fluorescence quenched
Fluorescence detectable
ABI, 2003. Quantifiler Human DNA Quantitation
Kit Users Manual.
8
Quantifiler Data Analysis
9
DNA Extractions

• Affect Quality and Quantity of DNA
• Organic Extraction
• Gold standard
• Phenol-chloroform
• High quality quantity
• DNA IQ System
• Paramagnetic resin
• High quality
• 100ng maximum yield
• QIAamp Mini DNA Kit
• Silica column
• High quality quantity

10
Project GoalsEvaluation of Quantifiler

• Internal Re-validation
• DNA Input versus CE peak heights
• Affect of DNA extraction method
• Optimal heterozygote peaks
• 1500-2000 rfu
• Intra- and inter-locus balance
• True zero value
• Lack of STR success
• No typable loci

11
Peak Height Study

• DNA extractions
• Organic
• DNA IQ
• QIAamp
• 0.063-2.500ng input into Profiler Plus amp
• Examined heterozygote peak height balance

12
Comparison of Average Peak Heights from Three
Extraction Methods (n3)
620 rfu
13
Comparison of Regression Lines from Three
Extraction Methods (n3)

14
Average Heterozygote Peak Height v Product Size
(STR Locus) (n9)
15
Summary

• Extraction Methods
• Organic and QIAamp methods are comparable
• DNA IQ yields higher peak heights (higher
  quality DNA)
• Peak Height Trends
• Increasing peak heights with increasing DNA
  inputs
• Greater peak height variability gt1.5ng
• within and between extraction methods
• between loci
• Plateau at gt2.125ng
• Peak heights lt1500 rfu at 1.500ng

16
Possible Solutions

• PCR ProductFormamide
• Increase PCR Product volume
• Decrease formamide volume
• Electrokinetic injection time

17
Comparison of a 5s and 10s Injection
18
Recommendation

• Average heterozygote peak heights of 1500-2000
  rfu
• Maintain 110 PCR ProductFormamide
• 1.2µl PCR Product
• Amplify 1.500ng
• 10sec injection

19
True Zero Value

• 96 low level case samples
• undetected 225pg/µl
• Concentrated with Microcon YM-10
• STR amplified 1.500ng (target)
• 10sec injection
• Examined number of typable STR loci and alleles

20
Locus and Allele Dropout
21
Full Profile from Undetected Sample
22
Average Number of Typable Loci
23
Number of Loci Detected (Including Partial) from
Undetected Samples
9 loci 6
8 loci 3
6 loci 3
4 loci 6
2 loci 3
1 locus 6
0 loci 73
24
Summary

• Decreasing number of typable loci with decreasing
  quantitation values
• Undetected samples
• On average, one typable locus
• One full profile
• No typable loci from 73 of undetected samples
• Use as a predictor of STR success

25
Conclusions

• Peak Height Study
• Thorough internal re-validation
• Recommendation
• Maintain 110 PCR ProductFormamide
• Amplify 1.500ng
• 10sec injection
• Zero Value Study
• Predict STR success
• Many other probative samples
• High probative value

26
Acknowledgements

• Dr. Susan Greenspoon
• Sarah Seashols
• Kelly Johnson
• Dawson Cruz Lab Members
• Michelle Bonnette
• Denise Rodier
• Lindsay Thompson
• Office of the Dean of the College of Humanities
  and Sciences and the office of the Provost at
  Virginia Commonwealth University

27
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