Homologous-restraint polymerase chain reaction: an efficient and rapid protocol to clone multiple homologous genes.
In this article, we present a novel protocol, called homologous-restraint polymerase chain reaction (HRPCR), for cloning multiple homologous genes. One of the homologous genes was cloned by consensus-degenerate hybrid oligonucleotide (CODEHOP) polymerase chain reaction (PCR) and sequenced. Primers of HRPCR were designed with 20 to 30 nt inverted to the known gene before the 5' end of the CODEHOP primers. The amplification of the known gene was restricted owing to the loop of the PCR product or the incorrect binding of the primers and the template. As a result, only unknown genes could be cloned. This protocol proved to be simple, rapid, and efficient. We applied this protocol to clone the multiple homologous genes of beta-1,4-N,6-O-diacetylmuramidase from the genomic DNA of Streptomyces griseus.
Chen
Ming
M
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China.
Liu
Honglei
H
Bai
Yunfeng
Y
Zhang
Zhenghong
Z
Liu
Junjun
J
Zhang
Yuzhen
Y
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification
D001426:Bacterial Proteins; D017931:DNA Primers; C020026:N,O-diacetylmuramidase; D009113:Muramidase