Creating seamless junctions independent of restriction sites in PCR cloning.

摘要: A method is described for the efficient cloning of any given DNA sequence into desired location without limitation naturally occurring restriction sites. The technique employs polymerase chain reaction (PCR) combined with capacity type-IIS endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain site (5'-CTCTTC) are used amplify fragments being manipulated. Because ENase inhibited by site-specific methylation in sequence, all internal sites present can be protected performing PCR amplification presence 5-methyldeoxycytosine (m5dCTP). primer-encoded not affected modified nucleotides (nt) since newly synthesized strand does cytosine residues In addition, ENase's ability cleave several bases downstream from allows removal superfluous, terminal sequences amplified fragments, resulting 5' overhangs defined nt within cleavage site. Thus, elimination extraneous and generation unique, non-palindromic sticky ends permits formation seamless junctions a directional fashion during subsequent ligation event.