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专利名称：Method for purification of covalently closed
DNA-molecules
发明人：WITTIG, BURGHARDT,JUNGHANS,
CLAAS,SCHROFF, MATTHIAS
申请号：EP99250187.4申请日：19990614公开号：EP0967274A3公开日：20000412
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 constructthat can be replicated in bacteria; (b) isolating the construct; (c) cutting out (Ia) with atleast one restriction endonuclease (RE1); (d) ligating the product with a hairpin-forming,self-complementary deoxyoligonucleotide (II) to generate a single-strand, closed at bothends; (e) incubating the ligation mixture with a restriction endonuclease (RE2) and (vi)subsequently, or simultaneously with (v), incubating with an exonuclease (Exo) that ispractically 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 shortoverhanging ends complementary to those produced by RE1. RE2 recognizes and cuts asequence that is absent from (Ia) but is present, at least once, in the remainder of thereplicable construct.
申请人：MOLOGEN GMBH
地址：Fabeckstrasse 30 14195 Berlin DE
国籍：DE
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