Patent ID: 7393635

Claim:
A method of cloning in vitro a double-stranded target DNA into a double stranded vector DNA, said method comprising: a) generating a double-stranded target nucleic acid molecule by extension of at least two target-specifying primers that each contain at least one ribonucleotide, at one or more positions within at most 5 nucleotides from a 3′-end, wherein the first target-specifying primer is complementary to a first vector-specifying primer that would direct amplification of vector; the second target primer is complementary to a second vector-specifying primer; and the extension of primers is performed using a polymerase mixture comprising (i) an Archael polymerase and (ii) a Taq polymerase, a Klentaq polymerase, or Taq polymerase and a Klentaq polymerase; and b) contacting the target DNA and an RNAse so as to cleave a phosphodiester bond between a ribonucleotide and a deoxyribonucleotide in a double stranded nucleic acid and to generate a cleaved target DNA with 3′overhangs; c) providing a double-stranded vector DNA with 3′-overhangs that are complementary to the 3′-overhangs of the cleaved target DNA; and d) incubating said cleaved target DNA and said vector DNA together under conditions that result in annealing of the complementary ends thereof to form a vector comprising the double-stranded target DNA; wherein, said cloning method does not require the use of a restriction enzyme, a restriction site sequence, a joining enzyme, or a topoisomerase to form a stable vector comprising the double-stranded target DNA that is capable of transformation into a host cell without further enzymatic processing in vitro; and the stable vector comprising the double-stranded target DNA formed in (d) is not contacted with a joining enzyme in vitro prior to use for transformation of a host cell.