1. Developing multiplex PCR assays for human identity testing - is there overlap with pathogen screening?
Dr. Peter M Vallone
Human Identification Project
U.S. National Institute of Standards and Technology
SoGAT XX
Warsaw, Poland
12-13 June 2007

2. Our publications and presentations are made available at:
Disclaimers
Funding: Interagency Agreement 2003-IJ-R-029 between the National Institute of Justice and NIST Office of Law Enforcement Standards
Points of view are those of the authors and do not necessarily represent the official position or policies of the US Department of Justice.
Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible.  In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.
Our publications and presentations are made available at:

3. Human Identity Project at NIST
Genotype samples with commercial assays (nucleic acid based)
Produce Standard Reference Materials
Training and Interlaboratory Studies
Develop novel multiplex assays for genotyping

4. Goals Interest in further understanding of PCR
Learn more about the SoGAT mission
Building multiplex PCR assays
Upper limits of multiplexing?
Robust performance
Speed up assay development
Can this be of use to your community?
Further my knowledge of assay design outside of forensic applications

5. Applications of Human Identity Testing
Forensic cases -- matching suspect with evidence
Paternity testing -- identifying father
Missing persons investigations
Military DNA “dog tag”
Convicted felon DNA databases
Mass disasters -- putting pieces back together
Historical investigations and genetic genealogy
Involves generation of DNA profiles usually with the same genetic markers and then MATCHING TO REFERENCE SAMPLE

6. Steps in DNA Analysis Biology Genetics Technology Steps Involved
Usually 1-2 day process (a minimum of ~5 hours)
Collection
Sample Collection & Storage
Buccal swab
Blood Stain
DNA Extraction
DNA Quantitation
qPCR
Specimen Storage
Extraction
Quantitation
Biology
Multiplex PCR
Multiplex PCR Amplification
STR Typing
If a match occurs, comparison of DNA profile to population allele frequencies to generate a case report with probability of a random match to an unrelated individual
Interpretation of Results
Genetics
Work on this one…
STR Typing
DNA separation and sizing
Usage:
Human ID
Paternity Test
Missing Person
Mass Disaster
DNA Database Search
Technology
Interpretation of Results

7. What Type of Genetic Variation?
Length Variation
short tandem repeats (STRs)
Sequence Variation
single nucleotide polymorphisms (SNPs)
insertions/deletions
CTAGTCGT(GATA)(GATA)(GATA)GCGATCGT
Tetra nucleotide repeat
GCTAGTCGATGCTC(G/A)GCGTATGCTGTAGC

8. Example Profile of a Short Tandem Repeat Assay
Information is tied together with multiplex PCR and data analysis
D8S1179
D21S11
D7S820
CSF1PO
D13S317
D16S539
D2S1338
D18S51
TPOX
VWA
FGA
D5S818
AMEL
D19S433
TH01
D3S1358
Total cost per sample
= $3.87 (Fall 2002)
16 STR loci amplified in a single reaction
0.5 ng of human genomic DNA
Identifiler STR Kit from Applied Biosystems

9. PCR Amplified DNA Template
SNP Genotyping
Allele-Specific Primer Extension G C A T
Fluorescently labeled ddNTPs polymerase
ABI PRISM® SNaPshot™
Multiplex System
SNP Primer is extended by one base unit
Oligonucleotide primer bases 5’ 3’
“tail” used to vary electrophoretic mobility
PCR Amplified DNA Template G
Animate this slide!!!!
Multiplex PCR followed by a clean up step (Exo-SAP)
Multiplex primer extension (with SNaPshot reagent mix)
Fragments separated and detected on a gel or capillary electrophoresis platform

10. Autosomal 12-plex SNP Assay
250 pg
125 pg
63 pg
31 pg

11. Forensic Assays Limited sample (0.5 – 1 ng of genomic DNA)
Multiplex (10 or more loci/amplicons in a single reaction)
Robust amplification (results must hold up in court)
Issues with degradation, inhibition and efficient sample extraction
Standardized testing throughout the forensic community

12. In House Assay Design

13. Selection of Loci From Characterize sequence Literature Collaborators
Sequencing
Characterize sequence
Map in software (Lasergene)
NCBI accession number
~1000 bp/locus

14. Selection of Loci High quality sequence information
Standardize nomenclature
Strand orientation
Keep track of primer design

15. Primer Selection
Primer3 (
Stand alone version on Mac OSX
Visual OMP (
Standard design parameters (Tm ~60oC)
Amplicon length (minimize)
Restrict primers to flank target region
Take advantage of mis-priming libraries to screen primers

16. Further Primer Screening
AutoDimer software
Web based version
Stand alone version
BLAST
Avoid significant homology with other chromosomal locations and/or organisms
Save search results for further review
Confirm primer binding sites

17. Tools for Primer Selection
Screens oligos for primer-dimers interactions
Provides Tm, DG, etc (oligo calculator)
Design primers for ASPE SNP assays
Freely available

18. Simple Oligo Calculator

19. Primer-Dimer Screening
Open to new ideas and additional functionalities

20. Assay Development UV quantitation of primers (ensure reproducibility)
Run PCR in singleplex reactions
Samples for assay development
Well characterized quality – pristine
Contain a sampling of sequence variants
Sufficient quantities for testing
Well characterized template concentration (qPCR) – define sensitivity limits

21. Determination of DNA Oligomer Concentrations
Expected
100 mM
Concentrations were estimated by UV Spec using extinction coefficients determined from nearest-neighbor values
Lot-to-lot variation of oligo concentration makes it difficult to reproduce a multiplex assay

22. General PCR Conditions
Attempt to keep conditions a constant
1 x PCR buffer
1 Unit of polymerase (TaqGold)
2 mM Mg++
250 mM dNTPs
0.16 mg/mL BSA
0.2 mM of each PCR primer
0.5 – 1 ng of template DNA ( copies of target)

23. Assay Development
Use singleplex data to evaluate multiplex performance
Optimization is empirical
Balance PCR primer concentration
Replace inefficient primers
Identify and replace artifact causing primers
Integrate the lessons learned back into the informatics/strategy pipeline

24. Literature from our Mplex Work
Butler, J.M. (2005) Constructing STR multiplex assays. Methods in Molecular Biology: Forensic DNA Typing Protocols (Carracedo, A., ed.), Humana Press: Totowa, New Jersey, 297: [preprint]
Butler, J.M., David, V.A., O’Brien, S.J., Menotti-Raymond, M. (2002) The MeowPlex: a new DNA test using tetranucleotide STR markers for the domestic cat. Profiles in DNA, Promega Corporation, Volume 5, No. 2, pp. 7–10.
Butler, J.M., Schoske, R., Vallone, P.M. Highly multiplexed assays for measuring polymorphisms on the Y-chromosome. (2003) Progress in Forensic Genetics 9 (Brinkmann, B. and Carracedo, A., eds.), Elsevier Science: Amsterdam, The Netherlands, International Congress Series 1239, pp
Butler, J.M., Shen, Y., McCord, B.R. (2003) The development of reduced size STR amplicons as tools for analysis of degraded DNA. J. Forensic Sci 48(5)
Butler, J.M., Schoske, R., Vallone, P.M., Kline, M.C., Redd, A.J., Hammer, M.F. (2002) A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers. Forensic Sci. Int. 129:
Butler, J.M., C.M. Ruitberg, Vallone, P.M. (2001) Capillary electrophoresis as a tool for optimization of multiplex PCR reactions, Fresenius J. Anal. Chem. 369:
Coble, M.D. and Butler, J.M. (2005) Characterization of new miniSTR loci to aid analysis of degraded DNA. J. Forensic Sci. 50:
Devaney, J.M. Pettit, E.L., Kaler, S.G., Vallone, P.M., Butler, J.M., Marino, M.A. (2001) Genotyping of two mutations in the HFE gene using single-base extension and high-performance liquid chromatography. Anal. Chem. 73:
Just, R.S., Irwin, J.A., O'Callaghan, J.E., Saunier, J.L., Coble, M.D., Vallone, P.M., Butler, J.M., Barritt, S.M., Parsons, T.J. (2004) Toward increased utility of mtDNA in forensic identifications. Forensic Sci. Int. 146S: S147-S149.
Menotti-Raymond, M.A., David, V.A., Wachter, L.A., Butler, J.M., O’Brien, S.J. (2005) An STR forensic typing system for genetic individualization of domestic cat (Felis catus) samples. J. Forensic Sci. 50(5):
Schoske, R., Butler, J.M., Vallone, P.M., Kline, M.C., Prinz, M., Redd, A.J., Hammer, M.F. (2001) Development of Y STR megaplex assays. Proceedings of the Twelve International Symposium on Human Identification 2001, Promega Corporation.
Schoske, R., Vallone, P.M., Ruitberg, C.M., Butler, J.M. (2003) Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci. Anal. Bioanal. Chem., 375:
Schoske, R. (2003) The design, optimization and testing of Y chromosome short tandem repeat megaplexes. Ph.D. dissertation, American University, 270 pp.
Schoske, R., Vallone, P.M., Kline, M.C., Redman, J.W., Butler, J.M. (2004) High-throughput Y‑STR typing of U.S. populations with 27 regions of the Y chromosome using two multiplex PCR assays. Forensic Sci. Int. 139:
Vallone, P.M., Just, R.S., Coble, M.D., Butler, J.M., Parsons, T.J. (2004) A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome. Int. J. Legal Med., 118:
Vallone, P.M. and Butler, J.M. (2004) Multiplexed assays for evaluation of Y-SNP markers in U.S. populations. Progress in Forensic Genetics 10, Elsevier Science: Amsterdam, The Netherlands, International Congress Series 1261,
Vallone, P.M. and Butler, J.M. (2004) Y-SNP typing of U.S. African American and Caucasian samples using allele-specific hybridization and primer extension. J. Forensic Sci. 49(4): 723‑732.
Vallone, P.M., Decker, A.E., Butler, J.M. (2005) Allele frequencies for 70 autosomal SNP loci with U.S. Caucasian, African American, and Hispanic samples. Forensic Sci. Int. 149:
Vallone, P.M., Decker, A.E., Coble, M.D., Butler, J.M. (2006) Evaluation of an autosomal SNP 12-plex assay. Progress in Forensic Genetics 11, Elsevier Science: Amsterdam, The Netherlands, International Congress Series 1288,
Vallone, P.M., Fahr, K., Kostrzewa, M. (2005) Genotyping SNPs using a UV photocleavable oligonucleotide in MALDI-TOF MS. Methods in Molecular Biology: Forensic DNA Typing Protocols (Carracedo, A., ed.), Humana Press: Totowa, New Jersey, 297:
Multiplex Assays Developed at NIST
Various Y-SNPs (6-plexes)
Y-STR 10-plex & 20-plex
Mitochondrial SNP 11-plex
Autosomal SNP 12-plexes
Coming soon:
Autosomal STR 26-plex

25. Autosomal 26-plex Under Development (19plex so far)10 11 12 13 14 15 16 17 18 5 4 3 2 7 6 8 19
6FAM
VIC
NED
PET
1 ng DNA; 30 cycles

26. Rapid PCR with Short Amplicon STRs
1 ng of genomic DNA
Initial results amplifying a
STR 3-plex in 36 minutes
Could show promise for rapid screening

27. Areas of Concern/Differences
Speed ~1 day
Extraction 1 hour
Quantitation 1 hour
PCR ~2.5 hours
Capillary/Gel separation 1 hour
Faster mutation rate – unique variants (or closely spaced sites)
We use qPCR for quantitation not detection, but multiplex assay design strategy may apply

28. Mitochondrial SNPs Closely spaced polymorphisms
Haplotypes will vary from person to person

29. Standard Reference Materials
Traceable standards to ensure accurate measurements in our U.S. crime laboratories
SRM 2391b – CODIS STRs
SRM 2392-I – mtDNA
SRM 2395 – Y-STRs
SRM 2372 – DNA quantitation
Helps meet DAB Std. 9.5 and ISO 17025
Update materials for new loci
Lab 1
Lab 2
Calibration with SRMs enables confidence in comparisons of results between laboratories
Standards Reference Material

30. Summary Evolving strategy for developing multiplex PCR assays
Robust assay development assists in interlaboratory testing
What aspects can be applied to pathogen screening?

31. Acknowledgments petev@nist.gov
Funding from interagency agreement 2003-IJ-R-029 between the National Institute of Justice and the NIST Office of Law Enforcement Standards
NIST Human Identity Project Team – Leading the Way in Forensic DNA…
John Butler
Margaret Kline
Amy Decker
Becky Hill
Dave Duewer
Jan Redman

32. SRM 2372 Human DNA Quant 3 genomic DNA samples highly purified
Certified for UV absorbance
assume 1 OD = 50 ng/uL of genomic DNA
Determine performance (is there bias)
Commercial qPCR
in house qPCR assays
NIST SRM 2372 Human DNA Quantitation Standard
Assists with the calibration of commercial and in house standards
Monitor lot to lot variation of qPCR standards
Candidate 2372 materials were sent out for interlaboratory testing of various qPCR methods in the community before release

33. Human Identity Testing
NIST SRM 2391b PCR-Based DNA Standard
Cell Lines and Genomic DNAs
Ensures accurate and reproducible testing throughout the human ID community
Samples extracted (methods vary)
need ~1ng of genomic DNA
inhibitors, degradation
Multiplex PCR (commercial kits) no in house assays
microsatellites
16 loci in a single reaction
high degree of reproducibility
Protocols for PCR the same across the community
Products separated on gel or CE instrumentation
Protocols similar across the community
Data review
Using the same software package
Interpretation guidelines

34. Two Adjacent Mitochondrial SNPs
16223 (C/T) and (A/G)
Relative Intensity
m/z
16223
16224
Initial control region 10plex data
16223t
primer
extension product
16224b
primer
extension product
297 Da
ddA
288 Da
ddT

35. Single PCR product containing 5 polymorphisms
Multiple PCR products containing 5 polymorphisms