IN-VITRO HEADFUL PACKAGING SYSTEM FOR CLONING DNA FRAGMENTS AS LARGE AS 95 KB

摘要

This invention relates to an in vitro headful packaging process for cloning foreign DNA fragments as large as 95 kb comprising: modifying vector DNA by inserting a stuffer fragment into a blunt end producing site which is proximal to a pac site; digesting the product of step (a) to produce two vector arms each of which contains (i) a blunt end, (ii) another end which is compatible with the foreign DNA fragment which is to be cloned, and (iii) a loxP site; ligating the foreign DNA to the product of step (b) without generating concatemers; reacting the product of step (c) with pac cleavage proficient extract and head-tail proficient extract wherein the ratio of large heads to small heads in the head-tail extract is at least 5:1; infecting a Cre+ bacterial strain with the product of step (d); and recovering the cloned DNA.