Construction of linear functional expression elements with DNA and RNA hybrid primers: a flexible and fast method for proteomics.
Two methods of constructing linear functional expression elements (LFEE) using hybrid DNA and RNA primers in DNA amplification for rapid gene expression are described. In both methods, it is not necessary to have additional transformation or bacterial propagation. The promoter, open reading frame (ORF) and terminator are amplified using Pfu or Taq DNA polymerase. Three elements containing DNA or RNA overhang are covalently ligated by T4 DNA ligase. The recombinant molecule is amplified with element-specific primers. The LFEE can be generated by both methods in a few hours and can be expressed in mammalian cells.
Xin
Wen
W
Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, PR China.
Ma
Jing
J
Huang
Da-Wei
DW
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000096:biosynthesis / Q000138:chemical synthesis*; D015342:DNA Probes; D020869:Gene Expression Profiling / Q000379:methods*; D046911:Macromolecular Substances; D016366:Open Reading Frames / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D040901:Proteomics / Q000379:methods; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D015342:DNA Probes; D046911:Macromolecular Substances; D012313:RNA; D004247:DNA