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

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

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

Steps Involved Collection Specimen Storage Extraction Quantitation Multiplex PCR STR Typing Interpretation of Results Usage: Human ID Paternity Test Missing Person Mass Disaster

Steps in DNA Analysis

Usually 1-2 day process (a minimum of ~5 hours)

qPCR Blood Stain Buccal swab Sample Collection & Storage DNA Extraction DNA Quantitation

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 Multiplex PCR Amplification DNA separation and sizing DNA Database Search STR Typing Interpretation of Results

What Type of Genetic Variation?

•Length Variation

short tandem repeats

( STRs ) CTAGTCGT

(GATA)(GATA)(GATA)

GCGATCGT •Sequence Variation

single nucleotide polymorphisms

( SNPs ) insertions/deletions GCTAGTCGATGCTC

(G/A)

GCGTATGCTGTAGC

Example Profile of a Short Tandem Repeat Assay Information is tied together with multiplex PCR and data analysis

D21S11 D8S1179 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 VWA TPOX D18S51 AMEL D5S818 FGA

16 STR loci amplified in a single reaction 0.5 ng of human genomic DNA

Identifiler STR Kit from Applied Biosystems

SNP Genotyping Allele-Specific Primer Extension

SNP Primer is extended by one base unit

“tail” used to vary electrophoretic mobility Oligonucleotide primer 18-28 bases

5’ 3’ G C T A

PCR Amplified DNA Template

ABI PRISM ® SNaPshot™ Multiplex System G Fluorescently labeled ddNTPs + polymerase 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

Autosomal 12-plex SNP Assay

125 pg 63 pg 31 pg

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

In House Assay Design

Selection of Loci

• From – Literature – Collaborators – Sequencing • Characterize sequence – Map in software (Lasergene) – NCBI accession number – ~1000 bp/locus

Selection of Loci

High quality sequence information Standardize nomenclature Strand orientation Keep track of primer design

Primer Selection

• Primer3 ( http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

) – Stand alone version on Mac OSX • Visual OMP ( www.dnasoftware.com

) • Standard design parameters (T m – Amplicon length (minimize) ~60 o C) – Restrict primers to flank target region • Take advantage of mis-priming libraries to screen primers

Further Primer Screening

• AutoDimer software – Web based version http://yellow.nist.gov:8444/dnaAnalysis/ – Stand alone version http://www.cstl.nist.gov/div831/strbase//AutoDimerHomepage/AutoDimer ProgramHomepage.htm

• BLAST – http://www.ncbi.nlm.nih.gov/BLAST/ – Avoid significant homology with other chromosomal locations and/or organisms – Save search results for further review – Confirm primer binding sites

Tools for Primer Selection

Screens oligos for primer-dimers interactions Provides T m , D G, etc (oligo calculator) Design primers for ASPE SNP assays Freely available http://yellow.nist.gov:8444/dnaAnalysis/primerToolsPage.do

http://yellow.nist.gov:8444/dnaAnalysis/

Simple Oligo Calculator

Primer-Dimer Screening Open to new ideas and additional functionalities

http://yellow.nist.gov:8444/dnaAnalysis

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

General PCR Conditions

• Attempt to keep conditions a constant – 1 x PCR buffer – 1 Unit of polymerase (TaqGold) – 2 mM Mg ++ – 250 m M dNTPs – 0.16 mg/mL BSA – 0.2 m M of each PCR primer – 0.5 – 1 ng of template DNA (80-200 copies of target)

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

• • • • • • • • • • • • • • • • • • • •

Literature from our Mplex Work

Butler, J.M. (2005) Constructing STR multiplex assays.

Methods in Molecular Biology: Forensic DNA Typing Protocols

Press: Totowa, New Jersey, 297: 53-66. [preprint] (Carracedo, A., ed.), Humana 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. http://www.promega.com/profiles/502/ProfilesInDNA_502_07.pdf

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. 301-305 .

Butler, J.M., Shen, Y., McCord, B.R. (2003) The development of reduced size STR amplicons as tools for analysis of degraded DNA.

48(5) 1054-1064

.

J. Forensic Sci

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: 10-24 .

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.

Multiplex Assays Developed at NIST Coble, M.D. and Butler, J.M. (2005) Characterization of new miniSTR loci to aid analysis of degraded DNA.

J. Forensic Sci.

50: 43-53.

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 Various Y-SNPs (6-plexes) 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 domestic cat (

Felis catus

Y-STR 10-plex & 20-plex 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. Mitochondrial SNP 11-plex 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: 333-343 .

270 pp.

Autosomal SNP 12-plexes , American University, 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: 107-121 .

Coming soon: SNPs distributed throughout the mitochondrial genome.

Int. J. Legal Med.,

118: 147-157

.

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

, extension.

J. Forensic Sci.

Autosomal STR 26-plex 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: 279-286 .

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, 61-63 .

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: 169-178.

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

6FAM

Autosomal 26-plex

Under Development (

19plex so far

)

2 1 3 4 5 6 VIC 7 8 9 10 11 12 13 14 NED 15 16 18 PET 17 19 1 ng DNA; 30 cycles

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

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

Mitochondrial SNPs

Closely spaced polymorphisms Haplotypes will vary from person to person

Summary

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

Acknowledgments

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 Jan Redman Amy Decker Becky Hill Dave Duewer

[email protected]