Multiplex Single Nucleotide Polymorphism Genotyping Utilizing Ligase Detection Reaction Coupled Surface Enhanced Raman Spectroscopy Adam J. Lowe,* ,† Yun Suk Huh, ‡,| Aaron D. Strickland, § David Erickson, ‡ and Carl A. Batt § Graduate Field of Microbiology, Sibley School of Mechanical and Aerospace Engineering and Department of Food Science, Cornell University, Ithaca, New York 14853, and Division of Materials Science, Korea Basic Science Institute, Daejeon 305-333, Korea Single nucleotide polymorphisms (SNPs) are one of the key diagnostic markers for genetic disease, cancer pro- gression, and pharmcogenomics. The ligase detection reaction (LDR) is an excellent method to identify SNPs, combining low detection limits and high speciﬁcity. We present the ﬁrst multiplex LDR-surface enhanced Raman spectroscopy (SERS) SNP genotyping scheme. The plat- form has the advantage in that the diagnostic peaks of Raman are more distinct than ﬂuorescence, and in theory, a clinically signiﬁcant number of markers can be multi- plexed in a single sample using different SERS reporters. Here we report LDR-SERS multiplex SNP genotyping of K-Ras oncogene alleles at 10 pM detection levels, opti- mization of DNA labeling as well as Raman conditions, and the linear correlation of diagnostic peak intensity to SNP target concentration in heterozygous samples. Ge- nomic DNA from typed cells lines was obtained and scored for the K-Ras genotype. These advances are signiﬁcant as we have further developed our new SNP genotyping platform and have demonstrated the ability to correlate genotype ratios directly to diagnostic Raman peak signal intensity. SNPs are clinically useful for disease diagnosis and the selection of the appropriate therapies. 1-3 The ability to genotype multiple SNPs in limited clinical samples is important due to their potential for heterogeneous distribution. For example, oncogenic K-Ras alleles have been detected at G12V, G12A, G13D, and Q61R. 4 The K-Ras genotype from a patient’s tumor is highly informative, as tumors with different genotypes respond differently to treatment regiments. 5-7 Many methodologies have been previously developed for SNP genotyping. Strategies include primer elongation via PCR, enzymatic cleavage, hybridization, and LDR (oligonucelotide ligation), with most of them relying on a ﬂuorescent spectra or mass spectrometry for signal output. 8 Fluorescence is limited as a multiplex reporter due to spectral overlap. Mass spectrometry is able to deconvolute more complex mixtures since different mass tags can be used, 9,10 but the equipment is cumbersome and difﬁcult to integrate into a diagnostic device with a small footprint. We have recently developed a technology that utilizes SERS to circumvent the spectral overlap of ﬂuorescence spectroscopy while retaining sensitivity and accuracy of LDR for SNP detec- tion. 11 Detection schemes utilizing SERS are advantageous over ﬂuorescence as Raman peaks are approximately 1 nm 12 full width half-maximum (fwhm) while ﬂuorescent labels can be 100 times larger fwhm. 13 In addition to our LDR-SERS platform, Raman spectroscopy has been utilized for DNA identiﬁcation and SNP detection using hybridization platforms 14,15 as well as PCR based systems. 16 Importantly, multiplex identiﬁcation systems have been developed utilizing SERS technologies that require no additional data processing other than simple peak recognition. 16-18 * Corresponding author. E-mail: ajl248@cornell.edu. Fax: 607-255-8741. Phone: 607-255-7902. † Graduate Field of Microbiology, Cornell University. ‡ Sibley School of Mechanical and Aerospace Engineering, Cornell University. | Korea Basic Science Institute. § Department of Food Science, Cornell University. (1) Ching, A.; Caldwell, K. S.; Jung, M.; Dolan, M.; Smith, O. S.; Tingey, S.; Morgante, M.; Rafalski, A. J. BMC Genet. 2002, 3, 19. (2) Mehta, A. M.; Jordanova, E. S.; Corver, W. E.; van Wezel, T.; Uh, H. W.; Kenter, G. G.; Jan Fleuren, G. Genes Chromosomes Cancer 2009, 48, 410- 418. (3) Nam, R. K.; Zhang, W. W.; Trachtenberg, J.; Seth, A.; Klotz, L. H.; Stanimirovic, A.; Punnen, S.; Venkateswaran, V.; Toi, A.; Loblaw, D. A.; Sugar, L.; Siminovitch, K. A.; Narod, S. A. Clin. Cancer Res. 2009. (4) Khanna, M.; Park, P.; Zirvi, M.; Cao, W.; Picon, A.; Day, J.; Paty, P.; Barany, F. Oncogene 1999, 18, 27–38. (5) Colomer, R.; Monzo, M.; Tusquets, I.; Rifa, J.; Baena, J. 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