title: [A reverse transcriptase-polymerase chain reaction method improving the specific amplification of caveolin-1 gene sequences].
abstract: OBJECTIVE
  To construct a reverse transcriptase-polymerase chain reaction (RT-PCR)approach that can improve the specificity of primers while dropping down the nonspecific amplification.
  
  
  METHODS
  In the recent study we reported a new RT-PCR assay which improved markedly the specificity. However its efficiency of regressing nonspecific amplification remains to be accurately checked and further documented. In primer design, we looked over again some sequences that showed differences at 5' or 3' ends between human CAV1 and mouse Cav1 genes. cDNAs and the diluted plasmids which harbored the sequence of human CAV1 or mouse Cav1 gene were chosen as the templates. The ordinary PCR compared with one, of which primers modified by phosphorothioate and combined with proofreading polymerase, for their efficiencies of nonspecific amplification inhibited.
  
  
  RESULTS
  Taq DNA polymerase without proofreading activity could efficiently catalyze the extension of primers with a single or multiple mismatched base pairs at the 3' terminus, but the kind of primer extension can be effectively blocked by phosphorothioate modified primers combined with proofreading polymerase. Compared with ordinary PCR reaction, this new PCR method can effectively regress the primer mismatched amplification of 50 ng DNA almost equaling to 2 x 10(4) unmatched template copies in a final volume of 50 microL.
  
  
  CONCLUSIONS
  Compared with the first generation of polymerases with or without proofreading activities mediating RT-PCR reaction, the introduction of nuclease-resistant 3' modified primers (3' phosphorothioate primer extension) can offer more simplicity, accuracy, and also decrease cost.
authors.affiliation: Institute of Pharmacology and Pharmacy, Nanhua University, Hengyang, Hunan 421001, P.R. China.
authors.forename: Yang-yan
authors.initials: YY
authors.lastname: Xu
authors.forename: Hui-ling
authors.initials: HL
authors.lastname: Yang
authors.forename: Yong
authors.initials: Y
authors.lastname: You
authors.forename: Li
authors.initials: L
authors.lastname: Qin
authors.forename: Jian
authors.initials: J
authors.lastname: Tu
mesh_terms: D000818:Animals; D051242:Caveolin 1 / Q000235:genetics*; D003851:Deoxyribonucleases / Q000378:metabolism; D005784:Gene Amplification; D006801:Humans; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013873:Thionucleotides / Q000378:metabolism
chemical_list: D051242:Caveolin 1; D013873:Thionucleotides; D003851:Deoxyribonucleases