Reliable fusion PCR mediated by GC-rich overlap sequences.
Recombinant DNA technology largely depends upon Escherichia coli plasmid construction via restriction enzyme digestion and DNA ligation. Overlap extension PCR is another simple technique for constructing recombinant DNA but is not commonly used. This is likely due to the inefficiency of fusion after the annealing of overlaps that are generally designed from authentic sequences in the DNA fragments. In our current study, we describe the development of novel overlap sequences that can be used for the construction of fusion DNA fragments, including the one-step fusion of three fragments in a single PCR and also for in-frame fusions. Novel poly G or C stretches showed strong and also specific annealing to the complementary sequences in the fusion PCR. This DNA fusion method is thus both a simple and versatile recombinant DNA technique.
Cha-aim
Kamonchai
K
Department of Applied Chemistry, Graduate School of Science and Engineering, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan.
Fukunaga
Tomoaki
T
Hoshida
Hisashi
H
Akada
Rinji
R
D001483:Base Sequence; D020862:GC Rich Sequence / Q000235:genetics*; D018390:Gene Targeting; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics