Patent ID: 6420144
Filing Date: 2002-07-16
Classification: C12N,C12Q

Abstract:
A method for in vitro automated molecular cloning and amplification of a closed circular nucleic acid in a single reaction container, comprising:(a) mixing an effective amount of a donor nucleic acid comprising the donor sequence to be cloned, a recipient closed circular nucleic acid, a pair of 5â€²-phosphorylated cloning primers having the 3â€² and 5â€² portions complementary to different strands of the donor and recipient sequence, a thermostable DNA ligase, a thermostable DNA polymerase, all four deoxyribonucleoside triphosphates, and an appropriate buffer comprising all cofactors required for activity of both the ligase and polymerase in a single suitable reaction tube to form an automated in vitro cloning mixture which produces amplified closed circular recombinant clones; and (b) subjecting the cloning mixture tube to a single ligation during amplification (LDA) reaction process comprising a selected number of PCR cycles at a suitable denaturing temperature, then a suitable annealing temperature, and then a suitable extension temperature thereby forming a pair of insertion primers containing the donor nucleic acid to be inserted and causing said insertion primers to be extended by the polymerase, then ligated by the ligase for closing the extended insertion primers and then amplified exponentially to form closed circular nucleic acid recombinant clones containing the donor nucleic acid being inserted in the recipient closed circular nucleic acid in an inverted orientation.