Nucleotide Incorporation Opposite Degenerate Bases by Taq DNA Polymerase

Oligodeoxyribonucleotides containing degenerate bases, 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P), 2-amino-6-methoxyaminopurine (K), and N6-methoxyadenine (Z) were made on a DNA synthesizer. The oligonucleotides were used for the preparation of DNA templates by enzymatic ligation of DNA fragments. The templates were annealed with a primer and extended with Taq DNA polymerase and dNTPs to reveal the nucleotide(s) incorporated opposite the modified bases. Analysis of the products showed that A was inserted opposite the pyrimidine P and T opposite the purines, K and Z. Incorporation of a small amount of C opposite K was also observed.