Oligonucleotide Primers and Probes: Use of Chemical Modifications to Increase or Decrease the Specificity of qPCR

Abstract

Although the vast majority of primers and probes employed in qPCR applications today are synthesized using unmodi!ed DNA bases, selective use of chemically modi!ed bases and non-base modifying groups can prevent primer dimer artefacts, improve speci!city, and allow for selective ampli!cation of sequences that di"er by as li#le as a single base. A wide variety of chemical modi!cations have been characterized for use in qPCR. As a general class, the modi!cations that are in greatest use today increase the binding a$nity of the oligonucleotides (i.e. increase the melting temperature, Tm). Tm-enhancing modi!cations allows both primers and probes to be shorter, improving the di"erential Tm (ΔTm = Tm match – Tm mismatch) between perfect match and mismatch hybridization. &ese modi!cations have widespread application in allele-speci!c PCR and in the detection of single nucleotide polymorphisms (SNPs). Conversely, a second class of base modi!cations are in common use that decrease speci!city and improve duplex formation in the presence of base mismatches. Although these modi!cations lower Tm, they have less of an impact on primer stability than do actual mismatched bases. Universal bases permit use of primers and probes in polymorphic loci when it is desirable to detect all sequence variants and minimize mismatch discrimination. Introduction &e primers and probes used in real-time quantitative PCR (qPCR) are synthetic oligonucleotides and can be manufactured using natural DNA bases or can comprise RNA bases, arti!cially modi!ed bases, or a variety of pendant chemical groups not found in nature. While most needs are well met using unmodi!ed oligodeoxynucleotides, the use of chemical modi!cations can improve qPCR performance in a number of areas. &is chapter will cover three speci!c areas of interest, with a primary focus on increasing speci!city and allelic discrimination methods: 1 increasing speci!city, with a focus on SNP discrimination; 2 preventing primer-dimer or other PCR artefacts; 3 decreasing speci!city using universal bases. DOI: https://doi.org/10.21775/9781912530243.09