Patent ID: 7807376

Claim:
A method of oligonucleotide ligation comprising: i) forming a complex comprising a first and a second polynucleotide strand annealed to a DNA template such that, the first polynucleotide strand anneals to a first complementary polynucleotide sequence on the strand of the denatured DNA template and the second polynucleotide strand anneals to a second complementary polynucleotide sequence on the strand of the denatured DNA template, wherein the second complementary polynucleotide sequence on the strand of the denatured DNA template is located 5′ to the first complementary polynucleotide sequence on the strand of the denatured DNA template, and ii) forming a stable covalent bond between the first and second polynucleotide strands, wherein at least one of the first polynucleotide strand or the second polynucleotide strand comprises at least one modified pyrimidine nucleobase of the structure wherein X is N or C, R 1 is selected from —H, —F, —Cl, —Br, C 1 -C 6 alkyl, C 1 -C 6 substituted alkyl, C 3 -C 10 aryl, C 3 -C 10 substituted aryl, —CF 3 , —CF 2 H, —CF 2 CH 3 , —CF 2 CF 3 , —CCl 3 , —CN, —CHO, —CO 2 R, —SO 3 R, —PO 3 RR, —C(O)NRR, azido, and —NO 2 where each R is independently —H, C 1 -C 6 alkyl or C 3 -C 10 aryl or alkylaryl, and R 2 is selected from —H, —F, —Cl, —Br, C 1 -C 6 alkyl, C 1 -C 6 substituted alkyl, C 3 -C 10 aryl, C 3 -C 10 substituted aryl, —CF 3 , —CF 2 H, —CF 2 CH 3 , —CF 2 CF 3 , —CCl 3 , —CN, —CHO, —CO 2 R, —SO 3 R, —PO 3 RR, —C(O)NRR, azido, and —NO 2 where each R is independently —H, C 1 -C 6 alkyl or C 3 -C 10 aryl or alkylaryl, wherein at least one of R 1 or R 2 is an electron withdrawing substituent or X is N, such that when X is N, R 2 is absent, and at least one said modified pyrimidine nucleobase is no more than 4 nucleotides from the 3′ terminus of the first or second polynucleotide strand.