Sticky PCR: A PCR-based protocol for targeted protein engineering.
This article describes a simple but powerful PCR-based protocol for the generation of cohesive ends on linear DNA fragments, permitting the precise engineering of DNA constructs for a variety of applications. These include the introduction of deletion mutations, domain swapping, creating hybrid DNA fusions, or targeted protein engineering. This novel method can also facilitate the cloning of large or complex DNA fragments into a relevant cloning vector independent of the use of internal restriction endonuclease sites. The protocol involves the amplification of the required fragments by polymerase chain reaction through the use of two sets of overlapping desalted oligonucleotide primers. The subsequent mixing, denaturation and re-annealing of these products present correct cohesive terminal ends for ligation. There is no requirement for special vectors, enzymes or bases, suggesting that this protocol provides a unique way of engineering constructs in a rapid and cost-effective way for specific applications, such as precise deletion or swapping of various domains of the epidermal growth factor receptor to determine their role in membrane localization.
Yamabhai
Montarop
M
Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand. montarop@sut.ac.th
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics
D017931:DNA Primers; D004274:DNA, Recombinant; D004247:DNA; D004262:DNA Restriction Enzymes