Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information

Abstract

The performance of oligonucleotide primers containing deoxyinosine (dI) at all ambiguous positions for polymerase chain reaction, based on ambiguous sequence information derived either from compilations of consensus nucleotide sequences or from amino acid sequences, has been evaluated in two model systems represented respectively by amplification of conserved genomic regions from different types of human papillomavirus and by amplification of a region of the human lysozyme cDNA on the basis of the protein amino acid sequence. In both instances the dI-containing primers obtained the expected amplification products. When using short primers or primers with very high dI contents, however, peculiar reaction conditions had to be adopted to obtain successful amplification and, in the latter case, performance remained suboptimal. Comparison of results with those obtained using corresponding degenerate primers showed that the use of dI-containing primers can be advantageous in terms of both specificity and yield of the amplification product. Sequence analysis of amplification products showed that dG residues are always found at positions corresponding to the dI residues of the primers.