Patent ID: 7629120
Filing Date: 2009-12-08
Classification: C12Q

Abstract:
1. A method of assembling PCR fragments, comprising a) making a first PCR fragment with first and second primers, wherein the second primer comprises a modified nucleotide that can be removed by a DNA repair enzyme, resulting in a 3′ overhang, and wherein the first PCR fragment comprises a first site specific recombinase site; b) treating the first PCR fragment with a DNA repair enzyme to generate a 3′ overhang and immobilizing the first PCR fragment on a solid support or vice versa; c) making a second PCR fragment with third and fourth primers, wherein the third and fourth primers each comprises a modified nucleotide that can be removed by a DNA repair enzyme resulting in a 3′ overhang; d) treating the second PCR fragment with a DNA repair enzyme to generate a 3′ overhang; e) annealing and ligating the first and second PCR fragments; f) optionally repeating steps c, d and e until a last PCR fragment is added to the growing chain to produce an assembled fragment, wherein the last PCR fragment comprises a second site specific recombinase site; and g) simultaneously removing and circularizing the assembled fragment from the solid support with a site specific recombinase in a single step.