Developing multiplex PCR assays for human identity testing - is there overlap with pathogen screening? Dr. Peter M Vallone Human Identification Project.

Developing multiplex PCR assays for human identity testing - is there overlap with

pathogen screening?

Dr. Peter M ValloneHuman Identification Project

U.S. National Institute of Standards and Technology

SoGAT XXWarsaw, 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 in DNA Analysis

DNA Extraction

Multiplex PCR Amplification

Interpretation of Results

Sample Collection & Storage

Buccal swabBlood Stain

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

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

STR Typing

DNA separation and sizing

Tech

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DNA Database Search

Collection

Extraction

Quantitation

STR Typing

Interpretation of Results

Usage:Human ID

Paternity TestMissing PersonMass Disaster

Specimen Storage

Multiplex PCR

Steps Involved

DNA Quantitation

qPCR

What Type of Genetic Variation?

CTAGTCGT(GATA)(GATA)(GATA)GCGATCGT

GCTAGTCGATGCTC(G/A)GCGTATGCTGTAGC

•Length Variationshort tandem repeats (STRs)

•Sequence Variation single nucleotide polymorphisms (SNPs)

insertions/deletions

Example Profile of a Short Tandem Repeat Assay

D8S1179D21S11

D7S820 CSF1PO

D13S317D16S539 D2S1338

D18S51TPOXVWA

FGAD5S818AMEL

D19S433

TH01D3S1358

Information is tied together with multiplex PCR and data analysis

16 STR loci amplified in a single reaction0.5 ng of human genomic DNA

Identifiler STR Kit from Applied Biosystems

PCR Amplified DNA Template

G

SNP GenotypingAllele-Specific Primer Extension

SNP Primer is extended by one base unit

Oligonucleotide primer 18-28 bases

5’ 3’

“tail” used to vary electrophoretic mobility G

CA

T

Fluorescently labeled ddNTPs + polymerase

ABI PRISM® SNaPshot™ Multiplex System

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

250 pg

125 pg

63 pg

31 pg

Autosomal 12-plex SNP Assay

1 2 3 4 5 6 7 8 9 10 11 12

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 informationStandardize nomenclatureStrand orientationKeep 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 (Tm ~60oC)

– Amplicon length (minimize)– 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/AutoDimerProgramHomepage.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

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

Screens oligos for primer-dimers interactions

Provides Tm, G, etc (oligo calculator)

Design primers for ASPE SNP assays

Freely available

Simple Oligo Calculator

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

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 dNTPs– 0.16 mg/mL BSA– 0.2 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 (Carracedo, A., ed.), Humana Press:

Totowa, New Jersey, 297: 53-66. [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. 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. J. Forensic Sci

48(5) 1054-1064.• 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.

369: 200-205.• 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 extension

and high-performance liquid chromatography. Anal. Chem. 73: 620-624.• 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): 1061-1070. • 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. http://www.promega.com/geneticidproc/ussymp12proc/contents/butler.PDF• 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.• 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: 107-121.• 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: 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, Elsevier

Science: Amsterdam, The Netherlands, International Congress Series 1261, 85-87.• 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: 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.

Multiplex Assays Developed at NISTVarious Y-SNPs (6-plexes)Y-STR 10-plex & 20-plex

Mitochondrial SNP 11-plex Autosomal SNP 12-plexes

Coming soon:Autosomal STR 26-plex

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

Autosomal 26-plex Under Development (19plex so far)

1

910 11 12

13 1415 16

17 18

5432

7

6

8

19

6FAM

VIC

NED

PET 1 ng DNA; 30 cycles

Rapid PCR with Short Amplicon STRs

Initial results amplifying a STR 3-plex in 36 minutes

Could show promise for rapid screening

1 ng of genomic DNA

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 polymorphismsHaplotypes 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

[email protected]

John Butler

Margaret Kline

Amy Decker

Becky Hill

Dave Duewer

Jan Redman

NIST Human Identity Project Team – Leading the Way in Forensic DNA…