Article

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

ABSTRACT

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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    Preview · Article · Apr 2013 · International journal of clinical and experimental pathology
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    ABSTRACT: Previously, we reported that the overexpression of fer tyrosine kinase (FER), a non-receptor tyrosine kinase, is correlated with poor postoperative prognosis and cancer-cell survival in non-small cell lung cancer (NSCLC). In the present study, we further analyzed FER-overexpressed NSCLC cases and identified various patterns of chimeric mRNAs, composed of paraja ring finger 2 (PJA2) and FER. We detected no genomic rearrangements between PJA2 and FER and attributed these chimeric mRNAs to alterations at the transcriptome level: i.e., trans-splicing. Several chimeric patterns were detected concurrently in each patient, and the pattern sets varied among patients, although the pattern in which PJA2 exon 1 was fused to FER exon 3 (designated as Pe1-Fe3 mRNA) was detected constantly. Therefore, in a wide screening for PJA2-FER mRNAs in NSCLC, we focused on this chimeric pattern as a representative chimera. In analyses of 167 NSCLC samples, Pe1-Fe3 mRNA was identified in about 10% of the patients, and the presence of chimeric mRNA was significantly correlated with a high expression level of parental FER mRNA. Furthermore, we found that the detection of Pe1-Fe3 mRNA was correlated with poor postoperative survival periods in NSCLC, consistent with a previous finding in which FER overexpression was correlated with poor postoperative prognosis in NSCLC. This report is the first to suggest a correlation between chimeric mRNA and the expression level of parental mRNA. Furthermore, our findings may be clinically beneficial, suggesting that PJA2-FER mRNAs might serve as a novel prognostic biomarker in NSCLC.
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