title: 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.
abstract: This study describes a quantitative real-time PCR-based approach for discrimination of single nucleotide sequence variants, called CAPS (C4' alkylated primer system). To increase the discrimination potential of DNA polymerases against competing sequence variants of single nucleotides, 3'-terminally modified primers were designed carrying a methyl residue bound to the C4' of the thymidylate deoxyribose. In a model sequence system positional dependencies of modified thymidylate (at -1, -2, -3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modification at the ultimate 3'-position. In a comparison between Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrimination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a desired target among discriminated templates with a dynamic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence variants 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. Targeting of SNPs assures a universal exertion of this method, since these markers are gender-independent, highly abundant and ubiquitous.
authors.affiliation: Institut für Biochemie, FB 08, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Tews
authors.forename: Jochen
authors.initials: J
authors.lastname: Wilhelm
authors.forename: Daniel
authors.initials: D
authors.lastname: Summerer
authors.forename: Michael
authors.initials: M
authors.lastname: Strerath
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
authors.forename: Peter
authors.initials: P
authors.lastname: Friedhoff
authors.forename: Alfred
authors.initials: A
authors.lastname: Pingoud
authors.forename: Meinhard
authors.initials: M
authors.lastname: Hahn
mesh_terms: D000478:Alkylation; D000483:Alleles; D020029:Base Pairing; D001483:Base Sequence; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003855:Deoxyribose / Q000737:chemistry / Q000235:genetics*; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D019914:Taq Polymerase / Q000378:metabolism; D013696:Temperature; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000235:genetics
chemical_list: D017931:DNA Primers; D013942:Thymine Nucleotides; D003855:Deoxyribose; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase