Construction a vector system for trapping open-reading-frame DNA sequences

• Main Authors: Chou, Ai-Yu, 周愛玉
• Other Authors: Lang-Yang Ch'ange
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/75190038640805950099

碩士 === 國立臺灣大學 === 醫事技術學系 === 86 === We propose that nine different open-reading-frame combinations can be obtained when cleaving the 5'end and 3' end of an exprssed gene with the restriction endonuclease enzymes. In an effort to unambiguously explain this, we decided to use "the Code of Restriction Enyme Site (CRES) " which should help elucidate the relative combination of the constrction. The derivative of a CRES of the 5' end or 3'end is dependent upon the start codon (ATG) and the 5' end and 3' end cutting sites, repectively. Because of the codon usag being restrcted as three nucleotides for one amino acid translated the combination for trapping open reading frame cDNA fragment are calculated to be nine. In the work described in this thesis , ten ORF-trapping vectors, pCAY-0 and pCAY-1~pCAY-9,are constructed and characterized. We used pCAY-0 as the prototype vector and future developed nine different ORF trapping vectors(pCAY-1~pCAY-9). Bactreia contain pCAY-0 will grow on the x-Gal counting plate as blue colnies because of the frameshift design in the lacZ a-protein region. Upon receiving the correct ORF cDNA fragment ,pCAY-1~ pCAY-9 will produce in frame lacZ a-fusion protein with the inserted cDNA fragment and bacteria carrying these ORF fragment vectors will produce blue coloies again. We casll this as "phenotypic reversion". This will us easily screen for ORF fragment containing bacteria coloies. We further test plasmid pCAY-1 ~pCAY-9 for trapping known ORF fragments. When the individual combination of CRES DNA fragment its respective ORF-trapping vector is tested ,each of theORF-trapping vector showed 47% phenotypic reversion, because of the dual direction cloning site .In contrast , if the insert CRES DNA fragments are the mixture of nine combintions,each ofthe ORF-trapping vector showed 1/8 phenotypic reversion, which is close to the estimated 1/9 possibility. We further isolated and sequenced the cDNA inserts form a mouse amplicon library. Between different database ,me can easily identity the protein coding sequence from these cDNA inserts, Therfore ,we demonstate the effectiveness of this ORF-trapping vector system in identifying cDNA fragments within the ORF region.