Selective protection of restriction endonuclease sites.
A difficulty that is encountered when attempting to insert a PCR-amplified product or DNA fragment of interest into a particular vector is the presence within the insert of one or more internal restriction endonuclease (RE) sites identical to those selected for the flanks of the insert. Our method circumvents this problem by partially protecting internal RE sites while flanking sites for the same RE are cleaved. The amplified product is first heat denatured in the presence of excess amounts of perfectly complementary oligonucleotides that can anneal to the flanks of the insert. The mixture is allowed to anneal and is subsequently digested with the appropriate endonucleases. This results in the cleavage of the flanking RE sites while digestion at the internal RE site is not efficient. The mixture is subsequently heat denatured and column purified to remove the oligonucleotides. The product is then allowed to anneal and can be used directly in a ligation reaction with the plasmid vector. This method facilitates the construction of recombinant molecules by creating desired flanks while preserving internal RE sites.
Gebreyesus
Kagnow H
KH
National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Department of Health and Human Services, Bethesda, MD, USA.
Owens
Roland A
RA
D003001:Cloning, Molecular / Q000379:methods; D053938:DNA Fragmentation; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D017384:Sequence Deletion / Q000235:genetics
D017931:DNA Primers; D004262:DNA Restriction Enzymes