Universal restriction site-free cloning method using chimeric primers.
A universal restriction site-free cloning method has been developed to precisely insert a DNA fragment into a vector at any desired location without altering any nucleotide(s) in either the DNA fragment or the vector. The technique employs two pairs of chimeric primers, each containing a ribonucleotide. One pair of primers is used to amplify a target DNA fragment and another is used to prepare a linear vector. The ribonucleotide is used as a specific site for cleavage promoted by rare-earth metal ions such as La3+ or Lu3+. Therefore, blunt-ended PCR products can be converted into a dsDNA with single-stranded 3'overhangs for efficient ligation. The primers are designed so that both the target DNA fragment and vector PCR products create defined 3' overhangs to permit the formation of a seamless plasmid during the subsequent ligation. This method has been used successfully to clone the E. coli gene coding for peptidyl-tRNA hydrolase.
Chen
Guo Jun
GJ
F. Hoffmann-La Roche Ltd, Basel, Switzerland. guo_jun.chen@roche.com
Qiu
Nahong
N
Page
Malcolm P G
MP
D020413:3' Untranslated Regions / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics; D002678:Chimera; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics; D007811:Lanthanum; D008187:Lutetium; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics
D020413:3' Untranslated Regions; D017931:DNA Primers; D008187:Lutetium; D007811:Lanthanum; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase; D004262:DNA Restriction Enzymes