INTRODUCTIONThe objective of a polymerase chain reaction (PCR) is to amplify a specific DNA segment without any nonspecific by-products. In principle, each physical and chemical component of PCR can be modified to produce a potential increase in yield, specificity, or sensitivity. Yet the most critical parameter for successful PCR is optimal primer design. A poorly designed primer can result in little or no product, due to nonspecific amplification and/or primer-dimer formation leading to reaction failure, even when all the other parameters are properly optimized. This article provides general guidelines for PCR primer design, tips for development of primer pairs for more complex applications, and advice on the development of probes for real-time PCR.
RNA interference (RNAi) is silencing of gene expression by double-stranded RNA (dsRNA) having complementary sequence to the target gene to be silenced. This phenomenon has transformed into a complete technology for functional genomic studies. Small interfering RNAs (siRNAs) are 21- to 23-nucleotide dsRNAs, in which the sense strand is the same as the target mRNA and the antisense strand is the complement of the target mRNA sequence. These are the effector molecules for inducing RNAi, leading to posttranscriptional gene silencing with RNA-induced silencing complex. Besides siRNA, which can be chemically synthesized, various other systems in the form of potential effector molecules for posttranscriptional gene silencing are available, such as short hairpin RNAs (shRNAs), long dsRNAs, short temporal RNAs, and micro RNAs (miRNAs). These effector molecules either are processed into siRNA such as in the case of shRNA or directly aid gene silencing as in the case of miRNA. RNAi for various unknown genes may facilitate to elucidate inherited genetic diseases and provide drug candidates for viral and oncogenic diseases. This can be achieved by targeting mRNA from oncogenic genes or mRNA for viral cellular receptor and viral structural proteins for RNAi. In this article, we evaluate various aspects and applications of RNAi technology and provide comprehensive information for the system currently available for inducing RNAi.
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