Patent ID: 7414118

Claim:
A purified single-stranded polynucleotide primer comprising 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, at least one said modified pyrimidine nucleobase is no more than 4 nucleotides from the 3′ terminus of the primer, and said purified single-stranded polynucleotide primer comprising said modified pyrimidine nucleobase now complementary to a target sequence.