A universal method for the direct cloning of PCR amplified nucleic acid.
We have devised a simple, universal cloning strategy that permits the direct ligation of PCR amplified nucleic acid to a compatible vector preparation. The method does not require that special restriction sites or additional sequences be appended onto the amplification primers, nor the use of restriction endonucleases, modifying enzymes, or any purification procedures. This approach takes advantage of the single 3' deoxyadenylate extension that Thermus aquaticus, Thermus flavus, and Thermococcus litoralis DNA polymerases add to the termini of amplified nucleic acid. A new type of plasmid was constructed that allows the preparation of ends containing a single complementary 3' deoxythymidylate extension. Cloning PCR products by this method is approximately 50 times more efficient than blunt-ended ligation reactions. This direct PCR ligation technique has been engineered in-phase with a truncated lacZ gene to facilitate the discrimination of recombinants from non-recombinants. We have applied this method to directly clone amplified RNA and DNA from as little as 1 microliter of a PCR reaction, without prior modification or purification steps.
Mead
D A
DA
Department of Chemistry, University of Wisconsin, Madison 53706.
Pey
N K
NK
Herrnstadt
C
C
Marcil
R A
RA
Smith
L M
LM
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant / Q000032:analysis; D004277:DNA, Single-Stranded / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic / Q000235:genetics
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D009696:Nucleic Acids