Enhanced allele-specific PCR discrimination in SNP genotyping using 3? locked nucleic acid (LNA) primers

Abstract

The specificity and reliability of locked nucleic acid (LNA) substitution at the 3' position of allele-specific PCR (AS-PCR) primers for SNP detection was investigated in direct comparison to DNA primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3' LNA mismatch combinations were tested in triplicate with the plasmid target. LNA primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3' LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3' LNA primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3' LNA primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications.

Prognostic Relevance of Tumor Purity and Interaction with MGMT Methylation in Glioblastoma

“…Notably, we also evaluated primers with a locked nucleic acid modified 3 0 base to improve discrimination (27,28). Although this greatly improved specificity for qualitative applications (i.e., genotyping), it reduced the dynamic performance of the assay with poor linearity (data not shown).…”

Genotyping of the PXR A11156C polymorphism with locked nucleic acid containing fluorogenic probes

“…Based on NMR studies of LNA/DNA duplexes, it has been concluded that the main factor contributing to the extraordinary high stability of LNA-containing duplexes is a local change of the phosphate backbone geometry that favours a much higher degree of base stacking. [6][7][8][9][10][11][12][13] Figure 1c shows these melting curves. The heterozygotes now yielded two separated peaks with a DTm of 7.51C (Table 1).…”

“…[6][7][8][9][10][11][12][13] This last mentioned possibility is based on some recently published reports, which documented the high discriminative power of fluorogenic probes containing LNA to differentiate between matched and mismatched duplexes in several techniques, but not yet applied in FRET assays. [14][15][16] However, FRET assays have become a major technique in the field of polymorphism analysis.…”

Genotyping—From Genomic DNA to Genotype in a Single Tube

“…[42] So called multiplex reactions in which two allele variants are differentiated in a single reaction tube are realized by adding a specific 5' tail sequences to each allele specific primer. The signal generation is thereby directly linked to the allele specific primer in the PCR amplification.…”