Oligonucleotide Primers and Probes: Use of Chemical Modifications to Increase or Decrease the Specificity of qPCR
Scott D. Rose, Richard Owczarzy, Joseph R. Dobosy and Mark A. Behlke
from: Real-Time PCR: Advanced Technologies and Applications (Edited by: Nick A. Saunders and Martin A. Lee). Caister Academic Press, U.K. (2013)
Although the vast majority of primers and probes employed in qPCR applications today are synthesized using unmodified DNA bases, selective use of chemically-modified bases and non-base modifying groups can prevent primer-dimer artifacts, improve specificity, and allow for selective amplification of sequences that differ by as little as a single base. A wide variety of chemical modifications have been characterized for use in qPCR. As a general class, the modifications that are in greatest use today increase the binding affinity of the oligonucleotides (i.e., increase the melting temperature, Tm). Tm-enhancing modifications allows both primers and probes to be shorter, improving the differential Tm (DTm=Tm match-Tm mismatch) between perfect match and mismatch hybridization. These modifications have widespread application in allele-specific PCR and in the detection of single nucleotide polymorphisms (SNPs). Conversely, a second class of base modifications are in common use that decrease specificity and improve duplex formation in the presence of base mismatches. Although these modifications 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 read more ...