Restriction site-dependent PCR: an efficient technique for fast cloning of new genes of microorganisms.
New bioactive proteins need to be screened from various microorganisms for the increasing need for industrial and pharmaceutical peptide, proteins, or enzymes. A novel polymerase chain reaction (PCR) method, restriction site-dependent PCR (RSD-PCR), was designed for rapid new genes cloning from genomic DNA. RSD-PCR strategy is based on these principles: (i) restriction sites disperse throughout genomes are candidacy for universal pairing; (ii) a universal primer is a combination of a 3'-end of selected restriction sites, and a 5'-end of degenerated sequence. A two-round PCR protocol was designed and optimized for the RSD-PCR: amplify the single strand target template from genomic DNA by a specific primer and amplify the target gene by using the specific primer and one of the universal RSD-primers. The optimized RSD-PCR was successfully applied in chromosome walking using specific internal primers, and cloning of new genes using degenerated primers derived from NH2-terminal amino acid sequence of protein.
Jiang
Yu
Y
Jiangsu Key Laboratory for Biodiversity and Bio-Resources, Nanjing Normal University, Nanjing, Jiangsu 210046, People's Republic of China.
Pei
Jianjun
J
Song
Xin
X
Shao
Weilan
W
D000595:Amino Acid Sequence; D001419:Bacteria; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D040461:Gene Components; D005798:Genes, Bacterial / Q000235:genetics*; D005800:Genes, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D015003:Yeasts
D017931:DNA Primers