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A PCR BASED HIGH THROUGHPUT METHOD FOR CONSTRUCTION OF FULL SITES SMALL INTERFERING RNA (SIRNA) POLYNUCLEOTIDES AND RELATED COMPOSITIONS
A PCR BASED HIGH THROUGHPUT METHOD FOR CONSTRUCTION OF FULL SITES SMALL INTERFERING RNA (SIRNA) POLYNUCLEOTIDES AND RELATED COMPOSITIONS
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机译:基于PCR的高通量方法构建全位小分子干扰RNA(SIRNA)核苷酸及相关成分
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摘要
The present invention provides a PCR based high-throughput method for preparing full-sites siRNA polynucleotide pool, comprising: DNase I random digestion; Loop-1 phosphate linker ligation; single PCR amplification; a type III restriction/modification enzyme digestion; blunt ending; Loop-2 phosphate linker ligation; double primer PCR; Fokl digestion and cloning into an siRNA expression vector. The present invention enables the use of a type III restriction/modification enzyme linkers mediated PCR method for high-throughput preparing an siRNA polynucleotide pool, in which the functional length of siRNAs can be controllably distributed from 19-23bp, thus completely mimic the natural siRNA length diversity, specially suitable for RNAi therapeutic targets screening. The present invention overcomes the bottlenecks and drawbacks of conventional siRNA polynucleotide pool construction technologies.
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