Dr. Peter M. Vallone
Identity Project Team
DNA Measurements Group
Biochemical Science Division
Chemical Science & Technology Laboratory
National Institute of Standards and Technology
100 Bureau Drive, Mail Stop 8311
Gaithersburg, MD 20899-8311, USA
B. S. Chemistry, University of Illinois at Chicago, 1992
Ph. D. Chemistry, University of Illinois at Chicago, 1999
Postdoctoral Fellow, Biotechnology Division, National Institute of Standards and Technology, 1999-2001
Multiplex Assay Development. The ability to detect multiple genetic markers in a single tube or reaction is of great importance. It is much easier to run one reaction that detects 20 markers rather than 20 separate reactions. The advantages of multiplexing an assay are: a reduction in the amount of sample consumed (important for forensic applications), a reduction in the amount of enzyme/reagents consumed (cost reduction), the collection of more information per unit time and a simplification of data analysis. There are however, challenges in designing a multiplexed assay. The greater number of components required in a multiplexed assay can result in an increased likelihood of side reactions or failure in detecting certain markers. We are primarily concerned with multiplexing the polymerase chain reaction (PCR). In the PCR specific regions of DNA located in a genome are amplified. The amplification process allows for detection by current instrumental techniques such as capillary electrophoresis, mass spectrometry, and HPLC. We are developing tools such as primer screening software and multiplex assay condition guidelines in order to design assays with a greater success rate and in a shorter period of time.
Quantitative Real Time PCR (qPCR). In order obtain optimal fluorescent signal on a 310/3100/3130 capillary electrophoresis instrument the starting amount of DNA input into a multiplex PCR must be known. Commercial STR typing kits (PowerPlex 16, Identifiler, Yfiler, PowerPlex Y etc) typically work well with approximately 1 ng of genomic template DNA. In the forensic community several in house and commercially available qPCR assays have been described. We are running these assays on the AB 7500 Real Time PCR platform. We are evaluating the assays for potential bias (method and calibrant-bsaed) and as part of a study on candidates for the new Human DNA Quantitation Standard 2372. In July of 2006 I co-taught a qPCR workshop as part of the President’s DNA Initiative, NIJ, and NFSTC. The workshop presentation slides can be found here: [Introduction] [Quantitation Using PCR] [Instrumentation] [Signals and Probes] Selected Forensic qPCR Assays: [VT Alu SYBR Green and Quantifiler] [CA DOJ nuclear/mtDNA duplex and CA DOJ degradation triplex] [Maintenance, Validation, etc.] [Data Analysis and Troubleshooting] [qPCR Analysis Software for ABI 7000 & 7500]
AutoDimer Software. A The capability of copying multiple regions of DNA simultaneously, a process referred to as multiplex PCR, has enabled simultaneous amplification and detection technologies that permit more rapid data gathering in an analogous fashion to multitasking now routinely performed by computers. Multiplex PCR requires addition of at least two primers for each region of DNA being amplified. For every n regions amplified, there are (2n2 + n) possible primer-primer comparisons that need to be made. Prior to the development of AutoDimer, there were no efficient ways to compare large numbers of PCR primers to one another through any public or commercial software. Although AutoDimer was originally created to assist in development of multiplex PCR assays for probing short tandem repeat (STR) and single nucleotide polymorphism (SNP) markers used in human identity applications, the program enables development of any multiplex PCR assay. Other applications include medical diagnostics, pathogen detection, and disease association studies. The AutoDimer program performs primer-primer intercomparisons while evaluating interactions according to traditional Watson-Crick base pair rules. The results can be visually inspected or saved to a text file. The information output consists of a visual component along with a score that represents the degree of interaction. Predicted values of the transition melting temperature (Tm) and the standard molar Gibbs free energy of melting (ΔG) are also obtained for each of the potential primer-primer interactions.
http://www.cstl.nist.gov/biotech/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm hosts the AutoDimer program. This multiplex PCR assay design and development tool is freely available to the DNA diagnostic community. A web-based interface is now available at http://yellow.nist.gov:8444/dnaAnalysis/. The web-based version of AutoDimer provides an additional functionality for calculating DNA related chemical properties.
Typing SNP markers. The most common form of genetic variation in the human genome is the single nucleotide polymorphism or SNP (snip). A SNP can be defined as insertion, deletion, or sequence variation of a single base. A SNP is present in approximately every 1000 bases of the human genome. The typing of SNPs throughout the genome can facilitate genetic mapping, disease association studies, and evolutionary studies. Recent analysis of SNPs markers located on the non-combining region of the Y chromosome provides information on tracing human migration patterns and evolution. Primer extension assays to type SNPs located on the Y chromosome as well as in the mitochondrial genome are being designed in order to evaluate their usefulness in forensic applications. The primary methods of SNP typing in our laboratory is Allele Specific Primer Extension (ASPE). ASPE relyied on multiplex PCR amplification of target region of DNA containing the SNP of interest. The SNP is then probed using a minisequencing assay in conjunction woth the SNaPshot reagent. The extended DNA fragments are then separated and detected on a capillary electrophoresis platform (AB 310/3100/3130). We also have some experience using the Luminex bead-based SNP typing technologies (see Marligen Y-SNP assays kits)
Affymetrix Microarrays. The GeneChip Mitochondrial Resequencing Array is a means to perform full genome sequencing on an array-based platform. The amount of DNA needed for the resequencing array is much greater than that required for autosomal DNA typing (1 ng versus 10-30 ng). Because of this relatively high sample requirement the array may have limitations for running a limited quantity of casework sample. However the platform should have utility in running family reference samples for the elucidation of SNPs that will help resolve individuals. These array-determined polymorphisms found in reference sample can then be probed in the limited casework sample. We are running arrays in tandem with fluorescent sequencing experiments to evaluate the performance of the array platform.
Representative Publications (see NIST human identity team publications)
Butler, J.M., Coble, M.D., Vallone, P.M. (2007) STRs vs SNPs: thoughts on the future of forensic DNA testing. Forensic Science, Medicine and Pathology, in press.
Vallone, P.M., Jakupciak, J.P., Coble, M.D. (2007) Forensic application of the Affymetrix human mitochondrial resequencing array. FSI Genetics 1:196-198.
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., 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. and Butler, J.M. (2004) AutoDimer: a screening tool for primer-dimer and hairpin structures. Biotechniques, 37(2): 226-231.
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) 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.
Butler, J.M., Schoske, R., Vallone, P.M., Kline, M.C., Redd, A.J., and Hammer, M.F. (2002) A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers. Forensic Sci Int. 129: 10-24.
Vallone, P.M., Devaney, J.M., Marino, M.A., Butler, J.M. (2002) A strategy for examining complex mixtures of deoxyoligonucleotides using IP-RP HPLC, MALDI-TOF MS, and informatics. Anal. Biochem. 304: 257-265.
Benight, A.S., Pancoska P., Owczarzy, R., Vallone, P.M., Nesetril, J., and Riccelli, P.V. (2001) Calculating sequence-dependent melting stability of duplex DNA oligomers and multiplex sequence analysis by graphs. Methods Enzymol 340:165-92.
Butler, J.M., Devaney, J.M., Marino, M.A., and Vallone, P.M. (2001) Quality control of PCR primers used in multiplex STR amplification reactions. Forensic Sci Int 119: 87-96.
Devaney, J.M., Pettit, E.L., Kaler, S.G., Vallone, P.M., Butler, J.M., and 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.
Butler, J.M., Ruitberg, C.M., and Vallone, P.M. (2001) Capillary electrophoresis as a tool for optimization of multiplex PCR reactions. Fresenius J Anal Chem. 369: 200-205.
For list of recent presentations (numerous invited talks and training workshops given since 2000), see http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm and http://www.cstl.nist.gov/biotech/strbase/training.htm.