3' iCLIP Primer

Description

PO DNA, 5' (rApp) AGA TCG GAA GAG CGG TTC AG (ddC) 3'UV crosslinking and immunoprecipitation (CLIP) was developed to identify RNA-protein interactions (Ule J et. al. 2003). The method involves irreversible crosslinking of the RNA to protein, immunoprecipitation, ligation of 5' and 3' adapters, reverse transcription and finally PCR amplification. Unfortunately, CLIP is limited because reverse-transcriptase cannot read through crosslinked areas. This creates truncated cDNA without the 3' and 5' adapters needed to initiate PCR amplification.While several versions of the CLIP method now exist, individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) has a number of advantages. iCLIP circumvents the problem of poor amplification of the truncated cDNA. This is accomplished through the use of a preadenylated primer (iCLIP primer) and circularization of the reverse transcription product. This method also resolves the binding site to individual nucleotides and increases accuracy for binding sites located within repetitive motifs (Konig J et al. 2010., Konig J et, al. 2011).

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