Patent ID: 6168918
Filing Date: 2001-01-02
Classification: C12N,C12Q,Y10S

Abstract:
A method of detecting the presence of a foreign DNA sequence integrated in a chromosomal DNA molecule of a eukaryotic cell in a sample that contains chromosomal DNA molecules of eukaryotic cells and non-integrated foreign DNA, said method comprising the steps of: a) digesting the DNA molecules of said sample with one or more restriction enzymes that cleave chromosomal DNA, foreign DNA sequences integrated in said chromosomal DNA molecules and non-integrated foreign DNA to produce DNA digestion segments, wherein said eukaryotic cells are free of deoxyadenosine methyltransferase, said foreign DNA sequence contains at least one DpnI site, and said non-integrated foreign DNA molecules are produced in cells that contain deoxyadenosine methyltransferase; b) fractionating said DNA digestion segments of a) to produce a plurality of fractions of DNA digestion segments containing chromosomal DNA and non-integrated foreign DNA; c) forming a reaction mixture comprising (i) a fraction of (b) containing DNA digestion segments and (ii) restriction enzyme DpnI in PCR buffer containing DTT; d) heating the reaction mixture to remove functional nuclease and protease contamination without inactivating DpnI; e) digesting said DNA digestion segments with restriction enzyme DpnI in the reaction mixture, whereby non-integrated DNA is digested and integrated DNA remains undigested; f) amplifying fragments of said integrated foreign DNA molecules using said DNA digestion segments of e) and sets of primers that flank said DpnI site in said foreign DNA molecules in conditions free of functional nuclease and protease activity; and, g) detecting the presence of fragments of said foreign DNA molecules amplified in step f), wherein the presence of said amplified fragments indicates the presence of a foreign DNA sequence integrated into eukaryotic chromosomal DNA of eukaryotic cells.