Robust and reliable amplification of long unknown sequence by RACE. Obtaining a full-length cDNA is of critical importance for structural and expression studies. The amplification of DNA sequences from a messenger RNA template between a defined internal site and unknown sequences of either the 3' or the 5' end of the mRNA are often referred to as RACE (rapid amplification of cDNA ends), “anchored” PCR, or “one-sided” PCR. Transcriptor Reverse Transcriptase is supplied with the kit to transcribe full-length cDNA up to 14 kb in length and, due to the thermostability of Transcriptor Reverse Transcriptase (up to 65°C), to work with GC-rich templates with high secondary structure. In addition, with Transcriptor Reverse Transcriptase, high sensitivity can be achieved. This results in highly efficient cDNA synthesis and the generation of long RACE products. Purification of the first-strand cDNA is performed with the High Pure PCR Product Purification Kit. Recombinant Terminal Transferase is used to add a homopolymeric A-tail to the 3' end of the cDNA. The use of poly(A) tail decreases the likelihood of inappropriate trunction by the oligo dT-anchor primer, and due to the weaker A/T binding compared to the G/C binding, longer stretches of A residues are required before the oligo dT-anchor primer will bind to an internal site and truncate the amplification product. Tailed cDNA is amplified by PCR using a gene-specific primer and the oligo dT-anchor primer. The obtained cDNA is further amplified by a second PCR using a nested specific primer and the PCR anchor primer. As a result, the RACE products can be cloned into an appropriate vector for subsequent characterization studies.
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