Enhanced evolutionary PCR using oligonucleotides with inosine at the 3�-terminus. Nucleic Acids Res

The polymerase chain reaction (PCR) is a powerful tool for the enzymatic amplification of individual DNA sequences from unique oligonucleotide primers and a small amount of target DNA (1). In order for a successful PCR to occur each of the oligonucleotide primers must hybridize to sequences flanking the gene of interest. Mismatched nucleotides at the 3'-ultimate and 3'-penultimate positions have previously been shown to be detrimental to the amplification process (2). This is primarily due to a need for a perfect 3 ' base-pair to allow enzymatic synthesis rather than any thermodynamic effect on duplex formation. Mismatch of the oligonucleotide primers is particularly important in evolutionary PCR since the oligonucleotide primer pair is usually based upon nucleotide sequence information from one organism which may have diverged from other species of interest many millions of years ago. Previously, deoxyinosine has bee