Application of the C4′-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants

This study describes a quantitative real-time PCR-based approach for discrimination of single nu-cleotide sequence variants, called CAPS ~4 ' i!lkylat-ed grimer ~ystem). To increase the discrimination potential of DNA polymerases against competing se-quence variants of single nucleotides, 3'-terminally modified primers were designed carrying a methyl residue bound to the C4 ' of the thymidylate deoxyri-bose. In a model sequence system positional depend-encies of modified thymidylate (at-1,- 2,-3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modifi-cation at the ultimate 3 '-position. In a comparison be-tween Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrim-ination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a de-sired target among discriminated templates with a dy-namic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence vari-ants only differing in a single base (e.g., SNP alleles or point mutations). The range of applications of this easy to perform, fast and reliable technique reaches from medical diagnostics, transplantation medicine, molecular and cell biology to human genetics. Target-Present addresses