Amplification And Isolation Of cDna Ends By 5 RACE

5'-RACE stands for rapid amplification of 5'-cDNA ends and is widely utilized to amplify or isolate partial-length cDNA transcribed from some rare mRNAs. As shown in Figure 2.4, the procedures and principles are quite similar to those described previously except for the addition of oligo(dC) and the use of oligo(dG) as an anchor primer or forward primer. A reverse primer can be designed according to the known sequence in the 5' portion of the cDNA to be amplified or the N-terminal amino acid of the protein of interest. Addition of oligo(dC) to the 3' end of the first strand cDNA can be facilitated using terminal deoxynucleotidyl transferase. The drawback of this approach is the potential nonspecific priming if the target cDNA sequence is GC rich. Nonetheless, this strategy provides a powerful tool for analysis of the expression of rare messages. If the 5'-UTR sequence is available or the N-terminal amino acid sequence is known, the addition of oligo(dC) and synthesis of (dG) anchor primer is not necessary. A forward primer can be designed in the 5'-UTR sequence or N-terminal amino acid sequence.

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