Constructing recombinant DNA molecules by PCR.
This unit describes the use of PCR to construct hybrid DNA molecules. The unit provides an overview of how PCR can be exploited to accomplish numerous cloning strategies. The Basic Protocol outlines the PCR amplification and cloning strategies. The Commentary includes a troubleshooting guide for problems most frequently encountered in PCR cloning, plus four specific examples of the application of this technique to create in-frame fusion proteins, to create recombinant DNA products, to generate deletions and inversions by inverse PCR, and to introduce mutagenized PCR products or specific mutations or fusions by gap repair in yeast.
Elion
Elaine A
EA
Harvard Medical School, Boston, Massachusetts, USA.
Marina
Pablo
P
Yu
Lu
L
D004262:DNA Restriction Enzymes / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism*; D005822:Genetic Vectors; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D004262:DNA Restriction Enzymes