A method for functional mapping of protein-protein binding domain by preferential amplification of the shortest amplicon using PCR.
We have developed a novel method for rapid and empirical mapping of the protein interaction domain using a unique and atypical PCR-based amplification and a conventional yeast two-hybrid system. The modified PCR, designated as PASA-PCR, enables preferential amplification of the shortest amplicon from a complex expression library. PASA-PCR consists of reiterative cycles of denaturation of template DNAs and extremely abbreviated annealing/extension of primers to prevent their complete extension in a single cycle, followed by conventional amplification cycles. In PASA-PCR, the shortest (ranging from 400 to 1000 bp) amplicon is amplified almost exclusively from templates of various amplicon sizes. In addition, the frequency of in vitro recombination can be increased using low cooling rates (<0.5 degrees C/s) between the denaturation and annealing/extension steps, which was helpful in generating precisely trimmed protein-coding regions. Identification of Spc19-binding region of Spc34, which is a component of yeast's spindle pole body, was achieved by a combination of the yeast two-hybrid system and PASA-PCR.
Kawarasaki
Yasuaki
Y
Division of Molecular Cell Mechanisms, Department of Biological Mechanisms and Functions, Graduate School of Biological and Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. kawaras@agr.nagoya-u.ac.jp
Sasaki
Yoko
Y
Ikeuchi
Akinori
A
Yamamoto
Shoji
S
Yamane
Tsuneo
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism*; D018385:Centrosome / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D025941:Protein Interaction Mapping / Q000379:methods*; D011995:Recombination, Genetic; D008941:Spindle Apparatus / Q000737:chemistry*; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D018797:Cell Cycle Proteins; D005656:Fungal Proteins