Patent ID: 7754451

Claim:
A method of appending a first oligonucleotide and a second oligonucleotide directly to a nucleic acid template, the method comprising: annealing the template, the first oligonucleotide and the second oligonucleotide to a probe, wherein said probe includes at least a first template complementarity region and at least a first oligo positioning region directly adjacent thereto, the nucleotide of the first template complementarity region and the nucleotide of the first oligo positioning region that are directly adjacent within said probe being first junctional nucleotides that define a first probe junction therebetween; wherein said probe further includes a second oligo positioning region directly adjacent to said first template complementarity region, the nucleotide of the first template complementarity region and the nucleotide of the second oligo positioning region that are directly adjacent within said probe being second junctional nucleotides that define a second probe junction therebetween; wherein said first oligonucleotide includes a terminal region that is complementary to the first oligo positioning region of said probe, the terminal nucleotide of said terminal oligonucleotide region being annealed to the junctional nucleotide of the probe's first oligo positioning region; and wherein said second oligonucleotide includes a terminal region that is complementary to the second oligo positioning region of said probe, the terminal nucleotide of said terminal oligonucleotide region being annealed to the junctional nucleotide of the second oligo positioning region; creating a first ligatable free end at the template nucleotide that is annealed to the junctional nucleotide of the probe's first template complementarity region; and then ligating said first oligonucleotide to said first free ligatable end to append said first oligonucleotide to the nucleic acid template; and creating a second ligatable free end at the template nucleotide that is annealed to the second junctional nucleotide of the first template complementarity region; and ligating said second oligonucleotide to said second ligatable free end; and then separating said template from said probe and said oligonucleotides.