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AIM:To construct a differentially-expressed gene subtractedcDNA library from two colorectal carcinoma (CRC) cell lineswith different metastatic phenotypes by suppressionsubtractive hybridization.METHODS:Two cell lines of human CRC from the samepatient were used.SW620 cell line showing highlymetastatic potential was regarded as tester in the forwardsubtractive hybridization,while SW480 cell line with lowlymetastatic potential was treated as tester in the reversehybridization.Suppression subtractive hybridization (SSH)was employed to obtain cDNA fragments of differentiallyexpressed genes for the metastasis of CRC.These fragmentswere ligated with T vectors,screened through the blue-white screening system to establish cDNA library.RESULTS:After the blue-white screening,235 white cloneswere picked out from the positive-going hybridization and232 from the reverse.PCR results showed that 200-700 bpinserts were seen in 98% and 91% clones from the forwardand reverse hybridizations,respectively.CONCLUSIONS:A subtractive cDNA library of differentiallyexpressed genes specific for metastasis of CRC can beconstructed with SSH and T/A cloning techniques.
AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppressionsubtractive hybridization. METHODS: Two cell lines of human CRC from the samepatient were used. SW620 cell line showed highly quantified was found as tester in the forwardsubtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Shuppression subtractive hybridization (SSH) was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened Through the blue-white screening system to establish cDNA library .RESULTS: After the blue-white screening, 235 white cloneswere picked out from the positive-going hybridization and232 from the reverse. PCR results showed that 200-700 bpinserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively. CONCLUSIONS: A subtractive cDNA library of differentiallyexpressed genes specific for metastasis of CRC can be constructed with SSH and T / A cloning techniques.