Fusion PCR via novel overlap sequences.
Overlap extension or fusion PCR is thought to be a simple and easy method to produce fusion DNA fragments without the need for restriction enzyme digestion and DNA ligation. However, this method has not been used frequently, probably as it is not always reliable. When natural sequences are used for overlap sequences, sometimes either no fusion DNA is produced or only faint DNA bands are detected owing to low annealing between the overlap sequences selected. Here, we introduce several artificial overlap sequences, most of which are GC-rich, that can be used for reliable fusion PCR. We describe how these overlap sequences can be used for fusion DNA construction, in-frame gene fusion, and cloning in yeast.
Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube, Japan.
Kamonchai
K
Cha-Aim
Hisashi
H
Hoshida
Tomoaki
T
Fukunaga
Rinji
R
Akada
D019677:Artificial Gene Fusion / Q000379:methods*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D020862:GC Rich Sequence; D005800:Genes, Fungal / Q000235:genetics; D005818:Genetic Engineering; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004271:DNA, Fungal