Patent ID: 6770462
Filing Date: 2004-08-03
Classification: C07K,C12N

Abstract:
A method for inserting a cassette into a DNA molecule to produce a DNA sequence fusion cassette without requiring ligation, said method comprising the steps of:(a) providing a selected DNA molecule comprising a first region of DNA sequences upstream of a site targeted for disruption and a second region of DNA sequences downstream of the site targeted for disruption, wherein said first region of DNA sequences upstream of a site targeted for disruption further comprises a first strand having a first and a second end, and said second region of DNA sequences downstream of the site targeted for disruption further comprises a first strand having a first and a second end; (b) providing a cassette comprising a first strand of DNA, wherein the first strand of said cassette comprises at its 5â€² end DNA sequences which overlap with sequences at the second end of the first region of DNA sequences upstream of a site targeted for disruption, and at its 3â€² end DNA sequences which overlap with sequences of the first end of the second region of DNA sequences downstream of the site targeted for disruption; (c) amplifying the first region of DNA sequences upstream of a site targeted for disruption using primers for said first region and amplifying the second region of DNA sequences downstream of the site targeted for disruption using primers for said second region thereby producing amplified first and second regions; (d) mixing the cassette with the amplified first and second regions thereby producing a mixture; (e) amplifying the mixture of (d) using polymerase chain reaction, thereby producing without ligation a DNA sequence fusion cassette comprising the first region of DNA sequences and second region of DNA sequences flanking the cassette, wherein said amplifying further comprises the steps of: (i) heating the mixture of (d) for about 5 minutes in the absence of polymerase or primers at about 94Â° C.; (ii) cooling the mixture of (i) to 50Â° C. over about 30 minutes; (iii) maintaining the mixture of (ii) at about 50Â° C. for about 5 minutes; (iv) adding a thermostable polymerase to the mixture of (iii); (v) adding a proof-reading polymerase with 3â€² exonuclease activity to the mixture of (iv); (vi) heating the mixture of (v) to about 72Â° C. for about 5 minutes; and (vii) adding to the mixture of (vi) primers comprising a 5â€² forward primer P1 complementary to said first region and a 3â€² reverse primer P4 complementary to said second region.