title: Restriction site-dependent PCR: an efficient technique for fast cloning of new genes of microorganisms.
abstract: 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.
authors: Jiangsu Key Laboratory for Biodiversity and Bio-Resources, Nanjing Normal University, Nanjing, Jiangsu 210046, People's Republic of China.
authors: Yu
authors: Y
authors: Jiang
authors: Jianjun
authors: J
authors: Pei
authors: Xin
authors: X
authors: Song
authors: Weilan
authors: W
authors: Shao
mesh_terms: 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
chemical_list: D017931:DNA Primers