Patent ID: 8728820
Filing Date: 2014-05-20
Classification: C12N

Abstract:
1. A method for inserting a single stranded replacement nucleic acid intermediate produced from a double stranded nucleic acid substrate into a target nucleic acid, said method comprising the steps of: a) generating a single stranded replacement nucleic acid intermediate from a double stranded nucleic acid substrate, wherein the double stranded nucleic acid substrate is adapted at one or both of its 5′ ends such that preferential degradation of one strand and/or strand separation generates the single stranded replacement nucleic acid intermediate, wherein the single stranded replacement nucleic acid intermediate comprises a 5′ region that is identical to sequence on the target nucleic acid, a 3′ region that is identical to sequence on the target nucleic acid and optionally a replacement region between the 5′ and 3′ regions that is not identical to sequence on the target nucleic acid, b) exposing the target nucleic acid to the single stranded replacement nucleic acid intermediate under conditions suitable for recombination to occur between the single stranded replacement nucleic acid intermediate and the target nucleic acid, and c) selecting a target nucleic acid whose sequence has been altered by inclusion of said single stranded replacement nucleic acid intermediate; wherein steps (a) and (b) take place within the same recombination reaction; wherein steps (a) and (b) are carried out in the presence of Red beta; wherein the Red beta comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 2 and is capable of mediating recombination between the single stranded replacement nucleic acid intermediate and the target nucleic acid; and wherein the double stranded nucleic acid substrate is adapted so that it is asymmetric at its 5′ ends, wherein the asymmetry causes one strand to be preferentially degraded.