Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE)

Department of Respiratory Medicine, University of Tokyo Hospital, Tokyo, Japan.
Biotechnology Letters (Impact Factor: 1.59). 03/2011; 33(7):1301-7. DOI: 10.1007/s10529-011-0580-1
Source: PubMed


Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.

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    • "To construct the FER expression plasmid vector , full-length human FER was cloned using a previously reported method [29]. Briefly, the 5' and 3' sides of FER were first amplified from the 5' and 3' RACE products of FER, respectively, using the following primer pairs: 5'-AAA ATG GGG TTT GGG AGT GAC-3'/5'-CCA GAA GAT GAA TAC ACT CCA-3' for the 5' side of FER, and 5'-GAC AGC CTG TCT ACA TCA TTA TGG AAC- 3'/5'-CGC CAT CCT GTC ACT ATG TGA G-3' for the 3' side of FER. "
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