Multiplex single nucleotide polymorphism genotyping utilizing ligase detection reaction coupled surface enhanced Raman spectroscopy.
Single nucleotide polymorphisms (SNPs) are one of the key diagnostic markers for genetic disease, cancer progression, 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 platform 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 multiplexed 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, optimization of DNA labeling as well as Raman conditions, and the linear correlation of diagnostic peak intensity to SNP target concentration in heterozygous samples. Genomic 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.
Graduate Field of Microbiology, Cornell University, Ithaca, New York 14853, USA. ajl248@cornell.edu
Adam J
AJ
Lowe
Yun Suk
YS
Huh
Aaron D
AD
Strickland
David
D
Erickson
Carl A
CA
Batt
D005838:Genotype; D022521:Ligase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D013059:Spectrum Analysis, Raman / Q000379:methods*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D018631:ras Proteins