Increased single-nucleotide discrimination in allele-specific polymerase chain reactions through primer probes bearing nucleobase and 2'-deoxyribose modifications.

The diagnosis of genetic dissimilarities between individuals is becoming increasingly important due to the discovery that these variations are related to complex phenotypes like the predisposition to certain diseases or compatibility with drugs. The most common among these sequence variations are single-nucleotide polymorphisms (SNPs). The availability of reliable and efficient methods for the interrogation of the respective genotypes is the basis for any progress along these lines. Many methods for the detection of nucleotide variations in genes exist, in which amplification of the target gene is required before analysis can take place. The allele-specific polymerase chain reaction (asPCR) combines target amplification and analysis in a single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes. The most important parameter in asPCR is the discrimination of these matched or mismatched pairs. In recent publications we have shown that the reliability of SNP detection through asPCR is increased by employing chemically modified primer probes. In particular, primer probes that bear a polar 4'-C-methoxymethylene residue at the 3' end have superior properties in discriminating single-nucleotide variations by PCR. Here we describe the synthesis of several primer probes that bear nucleobase modifications in addition to the 4'-C-methoxymethylenated 2'-deoxyriboses. We studied the effects of these alterations on single-nucleotide discrimination in allele-specific PCR promoted by a DNA polymerase and completed these results with single-nucleotide-incorporation kinetic studies. Moreover, we investigated thermal denaturing of the primer-probe-template complexes and recorded circular dichroism (CD) spectra for inspecting the thermodynamic and photophysical duplex behaviour of the introduced modifications. In short, we found that primer probes bearing a 4'-C-methoxymethylene residue at the 2'-deoxyribose moiety in combination with a thiolated thymidine moiety have synergistic effects and display significantly increased discrimination properties in asPCR.