Skip to main content
OSTI.GOVU.S. Department of Energy Office of Scientific and Technical Information
U.S. Department of Energy
Office of Scientific and Technical Information
 

Sequential cloning of chromosomes

Patent ·
OSTI ID:869986
 [1]
  1. Brookhaven, NY
+ Show Author Affiliations

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 to 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.

Research Organization:
ASSOC UNIVERSITIES INC
DOE Contract Number:
AC02-76CH00016
Assignee:
United States of America as represented by United States (Washington, DC)
Patent Number(s):
US 5434063
Application Number:
07/811,221
OSTI ID:
869986
Country of Publication:
United States
Language:
English

References (5)

Rapid cloning of specific DNA fragments of Streptococcus pneumoniae by vector integration into the chromosome followed by endonucleolytic excision journal November 1981
Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase journal January 1982
Sequential cloning by a vector walking along the chromosome journal July 1991
Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning journal October 1982
Isolation of a rat parvalbumin gene and full length cDNA. journal May 1986

Similar Records

Sequential cloning of chromosomes
Patent · Tue Jul 18 00:00:00 EDT 1995 · OSTI ID:87757
Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology
Journal Article · Sat May 01 00:00:00 EDT 1982 · J. Bacteriol.; (United States) · OSTI ID:6710460
Cloning of chromosomal genes in Streptococcus pneumoniae
Journal Article · Sat Oct 31 23:00:00 EST 1981 · Proc. Natl. Acad. Sci. U.S.A.; (United States) · OSTI ID:5729349

Related Subjects

/435/
alternately
carried
chromosomal
chromosomal dna
chromosomes
circular
circularly
cleaved
cleaved dna
cloned
cloning
containing
continues
cycles
designed
disclosed
distal
dna
dna segment
enzyme
enzymes
excision
form
formed
homologous
host
iis
integrated
integration
isolated
isolated dna
ligated
ligating
method
organism
permissive
plasmid
portion
previously
process
process continues
product
repeated
repeated cycles
replicated
replication
restriction
restriction enzyme
resulting
retrorestriction
segment
sequential
sequential cloning
sequentially
site
suitable
suitable host
suitably
target
third
transform
treated
vector
vector-containing