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Title: Sequential cloning of chromosomes

A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism's chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated tomore » give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.« less
  1. (Brookhaven, NY)
Issue Date:
OSTI Identifier:
United States of America as represented by United States (Washington, DC) BNL
Patent Number(s):
US 5434063
Application Number:
Contract Number:
Research Org:
Country of Publication:
United States
sequential; cloning; chromosomes; method; chromosomal; dna; target; organism; disclosed; segment; homologous; sequentially; cloned; isolated; restriction; enzyme; site; vector; product; formed; ligating; suitably; designed; circularly; integrated; resulting; cleaved; ligated; form; vector-containing; plasmid; replicated; host; containing; distal; portion; previously; suitable; treated; third; retrorestriction; iis; transform; permissive; replication; process; continues; repeated; cycles; circular; integration; excision; carried; alternately; enzymes; repeated cycles; cleaved dna; suitable host; isolated dna; chromosomal dna; dna segment; restriction enzyme; sequential cloning; process continues; /435/