Patent ID: 7504238

Claim:
A process for directionally ligating a double-stranded nucleic acid to a first adaptor sequence, the process comprising: a. forming an amplification product from the double-stranded nucleic acid using a mixture comprising (i) a polymerase, (ii) a deoxynucleotidetriphosphate (dNTP) mixture, the dNTP mixture comprising non-modified dNTPs for at least one of the four nucleotide triphosphates comprising dATP, dGTP, dCTP, dTTP and analogs thereof which, when incorporated into a polynucleotide, do not impart resistance against enzymatic degradation by an exonuclease at the site of incorporation of the non-modified dNTPs, and modified dNTPs for at least one of the four nucleotide triphosphates comprising dATP, dGTP, dCTP, dTTP and analogs thereof which, when incorporated into a polynucleotide, impart resistance against enzymatic degradation by an exonuclease at the site of incorporation of the modified dNTPs, (iii) a first primer complementary to a first strand of the double-stranded nucleic acid, said first primer having a first terminus complementary to a first ligation site sequence of the first adaptor sequence, and (iv) a second primer complementary to a second strand of the double-stranded nucleic acid, said second primer having a second terminus complementary to a second ligation site sequence of a second adaptor sequence, wherein the first terminus of the first primer and the second terminus of the second primer are not identical; b. treating the amplification product with the exonuclease to form a digested amplicon having a first overhang sequence at a first termini and a second overhang sequence at a second termini, wherein the first and second termini of said digested amplicon terminate at the sites of incorporation of said modified dNTPs, and wherein the first overhang sequence at the first termini is complementary to the first ligation site sequence of the first adaptor sequence and the second overhang sequence at the second termini is complementary to the second ligation site sequence of the second adapter; and c. ligating the first overhang sequence of the digested amplicon to the first ligation site sequence of the first adaptor sequence.