B. Tews, J. Wilhelm, D. Summerer, M. Strerath, A. Marx, P. Friedhoff, A. Pingoud, M. Hahn
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 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.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 384, 11/2003
Pages: 1533 - 1541
Show full article (external site)
Show all available items of this journal
Search
