| 摘要 |
Preparation of a linear, covalently closed double-stranded DNA molecule (I) comprises: (a) replicating DNA (Ia) that essentially comprises (I) as part of a construct that can be replicated in bacteria; (b) isolating the construct; (c) cutting out (Ia) with at least one restriction endonuclease (RE1); (d) ligating the product with a hairpin-forming, self-complementary deoxyoligonucleotide (II) to generate a single-strand, closed at both ends; (e) incubating the ligation mixture with a restriction endonuclease (RE2) and (vi) subsequently, or simultaneously with (v), incubating with an exonuclease (Exo) that is practically specific for free 3' and 5' ends. RE1 has recognition sequences on both sides of (Ia) and cuts to leave short overhanging ends of single-stranded DNA. (II) has short overhanging ends complementary to those produced by RE1. RE2 recognizes and cuts a sequence that is absent from (Ia) but is present, at least once, in the remainder of the replicable construct.
|
