HUMAN MUTATION 28(9), 922^927, 2007 METHODS Novel Plexor TM SNP Genotyping Technology: Comparisons With TaqMan s and Homogenous MassEXTEND TM MALDI-TOF Mass Spectrometry E.A. Tindall, 1,2 G. Speight, 1 D.C. Petersen, 1 E.J.D. Padilla, 1 and V.M. Hayes 1,2Ã 1 Cancer Research Program, Garvan Institute of Medical Research, St Vincent’s Hospital, Sydney, Australia; 2 Faculty of Medicine, University of New South Wales, Sydney, Australia Communicated by Ulf Landegren Analysis of SNPs for association, linkage, haplotype, and pharmacogenetic studies has led to a dramatic increase in the number and evolution of medium- to high-throughput genotyping technologies. This study introduces Plexor TM as a new method for medium-throughput (single SNP) genotyping. We compare this fluorescent-based chemistry for call rate, accuracy, affordability, throughput, and overall efficiency against two commonly used technologies. These include fluorescent-based TaqMan s allelic discrimination for single SNP analysis (medium-throughput) and the homogenous MassEXTEND TM (hME TM ) chemistry using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for multiple SNP analysis (high- throughput). Analysis of 11 SNPs, including all six possible nucleotide substitutions, showed Plexor TM to be highly comparable for both call rate (94.7%) and accuracy (99.2%) to the TaqMan s (94.6% and 99.8%, respectively) and hME TM (91.9% and 98.1%, respectively) chemistries. We demonstrate that this novel method is an efficient, cost-effective alternative to TaqMan s genotyping commonly used in diagnostic settings. Hum Mutat 28(9), 922–927, 2007. r r 2007 Wiley-Liss, Inc. KEY WORDS: SNP; genotyping; Plexor; TaqMan; MassEXTEND; hME INTRODUCTION The most common form of human genetic variation is the dimorphic single nucleotide polymorphism (SNP) [Cargill et al., 1999]. SNPs may contribute either directly or indirectly to an individual’s predisposition to complex disease, such as, cardiovas- cular disease, cancer, obesity, diabetes, psychiatric illness, and inflammatory disease, as well as individual differences in pharmacological responsiveness and disease progression [Evans and McLeod, 2003; Becker, 2004]. The completion of the Human Genome Project [Venter et al., 2001], followed by the introduction of the International HapMap Project in 2003 [International Hap Map Consortium, 2003], has resulted in an explosion in the identification and availability of human SNPs for genetic association, linkage, and haplotype studies. This has encouraged the development of medium- to high-throughput SNP genotyping technologies to enable rapid, reliable, accurate, and cost-effective genotyping. There are currently a large number of genotyping technologies available, which generally employ one of two mechanisms for allelic discrimination, namely allele-specific hybridization or primer extension. Amplifluor s (Chemicon International, Inc., Temecula, CA; www.chemicon.com) [Myakishev et al., 2001], GeneChip s (Affymetrix Inc., Santa Clara, CA; www.affymetrix.com), GoldenGate s (Illumina, Inc., San Diego, CA; www.illumina.com) [Engle et al., 2006], NanoChip s (Nanogen Corp., San Diego, CA; www.nanogen.com) [Sosnowski et al., 2002], SNPlex TM and TaqMan s (Applied Biosystems [ABI], Foster City, CA; www. appliedbiosystems.com) [De La Vega et al., 2005] use an allele- specific hybridization approach to genotyping. Alternatively, SNaPShot s (ABI) [Pati et al., 2004], Pyrosequencing TM (Biotage, Uppsala, Sweden; www.biotage.com) [Fakhrai-Rad et al., 2002], homogenous MassEXTEND TM (hME TM ; Sequenom, San Diego, CA; www.sequenom.com) [Gut, 2004], and iPLEX TM (Sequenom) [Engle et al., 2006], including the recently released iPLEX TM ! Gold (Sequenom), use primer extension. A keyword search for the term ‘‘SNP genotyping’’ using the PubMed database (www.ncbi.nlm. nih.gov) revealed that the most commonly used genotyping technologies for the period spanning June 2004 to December 2006 included ABI TaqMan s (83/434), Affymetrix GeneChip s (60/434), and Sequenom hME TM (50/434). Although RFLP analysis was the third most commonly used SNP genotyping technology (55/ 434), this gel-based method lacks automation and is therefore generally not classified as medium- or high-throughput [Gut, 2001]. Two of the most commonly used genotyping technologies, TaqMan s and hME TM , are frequently used in our laboratory. Published online 25 April 2007 inWiley InterScience (www.interscience. wiley.com). DOI 10.1002/humu.20533 Received 31 August 2006; accepted revised manuscript 5 March 2007. Grant sponsors: Cancer Institute of New South Wales, Australia; Australian Rotary Health Research Fund; Australian Cancer Research Foundation; Freedman Foundation, Australia; Armati Family Founda- tion, Australia; Medical Research Council of South Africa. Ã Correspondence to: Dr. Vanessa M. Hayes, Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia. E-mail: v.hayes@garvan.org.au r r 2007 WILEY-LISS, INC.