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.

Polymerases:

Topics:

Status:

new topics/pols set partial results complete validated

Results:

No results available for this paper.

Entry validated by:

Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.