Patent ID: 6902914

Claim:
A kit for directionally ligating a double-stranded nucleic acid to a first adaptor sequence, the kit comprising: (A) a first unmodified deoxynucleotidetriphosphate and modified deoxynucleotidetriphosphate pair, unmodified dNTP 1 and modified dNTP 1 , respectively, selected from the group consisting of (1) an unmodified dATP and a modified dATP, (2) an unmodified dGTP and a modified dGTP, (3) an unmodified dCTP and a modified dCTP, (4) an unmodified dTTP and a modified dTTP, and (5) an unmodified dUTP and a modified UTP; (B) a second unmodified deoxynucleotidetriphosphate and modified deoxynucleotidetriphosphate pair, unmodified dNTP 2 and modified dNTP 2 , respectively, selected from the group consisting of (1) an unmodified dATP and the modified dATP, (2) an unmodified dGTP and a modified dGTP, (3) an unmodified dCTP and a modified dCTP, (4) an unmodified dTTP and a modified dTTP, and (5) an unmodified dUTP and a modified UTP, wherein said first and second pairs are different; and (C) instructions for using the deoxynucleotidetriphosphate mixture in a procedure for directionally ligating a nucleic acid into a first adaptor sequence, wherein the adaptor sequence is a duplex nucleotide sequence for cohesive ligation to an end of an exonuclease digested amplification product; wherein (1) said first and second pairs are different, (2) the ratio of the modified dNTP 1 to the modified dNTP 2 relative to the unmodified dNTP 1 to the unmodified dNTP 2 is less than 51; (3) the unmodified dATP is selected from the group consisting of dATP and analogs thereof, the unmodified dGTP is selected from the group consisting of dGTP and analogs thereof, the unmodified dCTP is selected from the group consisting of dCTP and analogs thereof, the unmodified dTTP is selected from the group consisting of dTTP and analogs thereof, and the unmodified dUTP is selected from the group consisting of dUTP and analogs thereof, (4) the analogs of dATP, dGTP, dCTP, dTTP, and dUTP do not impart resistance against enzymatic degradation by an exonuclease relative to dATP, dGTP, dCTP, dTTP, and dUTP, respectively, when incorporated into a polynucleotide, and (5) the modified dATP, modified dGTP, modified dCTP, modified dTTP, or modified dUTP when incorporated into a polynucleotide imparts resistance, relative to dATP, dGTP, dCTP, dTTP, and dUTP, respectively, against enzymatic degradation by an exonuclease at the site of incorporation.