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Get the 5'-End

Rapid Amplification of cDNA ends (5'-RACE) is a polymerase chain reaction-based technique developed to facilitate the cloning of the 5'-ends of messages (see "Who Needs the 5'-End?"). Ambion's FirstChoice RLM-RACE Kit is a major improvement to the basic RACE protocol. The procedure specifically selects for capped transcripts and can be completed in a single day.


Shortcomings of cDNA Library Synthesis

Most cDNA synthesis reactions performed for cDNA library construction are initiated by oligo (dT) primed reverse transcription. This insures the inclusion of the 3'-end of most mRNA species in the library, and helps to orient the clones during subsequent analysis. However the majority of clones are truncated at their 5'-ends. This is due partly because it is difficult for reverse transcriptase to negotiate long RNA templates and stable secondary structure often encountered in RNA molecules. The predominant method for synthesizing double-stranded cDNA for cloning also contributes to under representation of the 5'-ends of mRNAs in cDNA libraries. Typically, RNase H is used to fragment the mRNA template and the fragments serve as primers for second strand synthesis. (1,2). Statistically, the further towards the 5'-end a sequence lies, the less likely a successful priming event will occur upstream from that end to make the region double-stranded and cloneable. In other words, you get only one chance to prime at the extreme 5'-end of the cDNA and make the 5'-end
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