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[PMC..free..article]..[PubMed]...SLIM..hybridization..The..conditions..for..SLIM..heteroduplex..formation..were..adapted..from..Tillett..and..Neilan..(17)...Nucleic..Acids..Res...When..using..a..plasmid..larger..than..12..kb,..amplification..succeeded..when..using..a..partially..complementary..primer..pair..(Figure..2,..lane..14),..but..not..with..a..completely..overlapping..primers...Wallace..RB,..Johnson..PF,..Tanaka..S,..Schld..M,..Itakura..K,..Abelson..J...1978...Sep...25;253(18):65516560....The..exponential..amplification..in..PCR..produces..a..fragment..containing..the..desired..mutation..in..sufficient..quantity..to..be..separated..from..the..original,..unmutated..plasmid..by..gel..electrophoresis,..which..may..then..be..inserted..in..the..original..context..using..standard..recombinant..molecular..biology..techniques...The.region.to.be.deleted.is.denoted.by.a.gray.rectangle..To..demonstrate..the..validity..of..the..suggested..method,..we..designed..a..point..mutation..in..plasmid..pcDNA3.1-pIgR,..which..is..approximately..8..kb..in..size..and..expresses..human..polymeric..immunoglobulin..receptor..(pIgR)..by..the..cytomegalovirus..(CMV)..early..promoter..and..enhancer...For...plasmid...manipulations,...other...site-directed...mutagenesis...techniques...have...been...supplanted...largely...by...techniques...that...are...highly...efficient...but...relatively...simple,...easy...to...use,...and...commercially...available...as...a...kit....Hong..GF..."Local...mutagenesis:...a...method...for...generating...viral...mutants...with...base...substitutions...in...preselected...regions...of...the...viral...genome."....Science..doi:10.1073/pnas.75.5.2170...Oligonucleotide-directed...mutagenesis...as...a...general...and...powerful...method...for...studies...of...protein...function....

 

Methods.Enzymol.,.293,.5371..Back-to-back..primer..design..methods..not..only..have..the..advantage..of..transforming..non-nicked..plasmids,..but..also..allow..exponential..amplification..to..generate..significantly..more..of..the..desired..product..(Figure..2)...(1995)..[PubMed]...(1989)..Molecular..Cloning:..A..Laboratory..Manual,..2nd..edn... d680c458d3

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