Quantification of homologous alleles and methylation analysis by single-nucleotide primer extension and fluorescence-based DHPLC. H. Yoshihashi¹, R. Kosaki^1,2, T. Suzuki¹, Y. Ohashi³, N. Matsuo¹, K. Kosaki¹. 1) Department of Pediatrics, Keio University School of Medicine,Tokyo, Japan; 2) Health Center, Keio University, Tokyo, Japan; 3) Transgenomic Japan, Inc, Tokyo, Japan.
   The single-nucleotide primer extension [SNuPE] is widely used for the analysis of the differential expression of homologous alleles involved in genomic imprinting, X-chromosome inactivation, and allelic imbalances associated with cancer. In the SNuPE reaction, a primer that binds adjacent to the nucleotide polymorphism is extended by a single base in the presence of a dNTP and/or ddNTP specific for one of the variant alleles. The purpose of the present study was to evaluate whether quantitative SNuPE assay could be accomplished by fluorescence-based DHPLC. ROX-labeled dideoxy CTP (ROX-ddCTP) was incorporated at the 3' end of the primer annealed to the template in the SNuPE reaction. Then the product was loaded onto a DNASep column with a linear gradient of acetonitrile in triethylammonium acetate. Two separate peaks, one from the primer extension product, and the other from the unincorporated dye terminator, were detected. When mixed template with variable amounts of the T and C alleles was analyzed, the signal intensity from incorporated ROX-ddCTP correlated with the relative molar proportion of the C- allele in the genomic DNA template. Furthermore, fluorescent SNuPE reaction has been successfully utilized to assess degree of cytosine methylation. Genomic DNA was subjected to bisulfite treatment, by which unmethylated cytosines are converted to uracils whereas methylated cytosines are not. Using the principle outlined above, the relative molar ratio of the methylated alleles versus the unmethylated allele was accurately determined. Unlike the original SNuPE assay based on radio-labeling, chromatographic separation can be monitored on-line, and completed in less than ten minutes after automated loading of the samples. The development of high-throughput technology for quantitative analysis of allele-specific expression and methylation should significantly expand our ability to derive molecular information from clinical specimens.