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

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.

• Authors: Mitsuhiro Sunohara, Masanori Kawakami, Hidenori Kage, Kousuke Watanabe, Noriko Emoto, Takahide Nagase, Nobuya Ohishi, Daiya Takai
• Journal: Biotechnology letters (Biotechnol Lett) Vol. 33 Issue 7 Pg. 1301-7 (Jul 2011) ISSN: 1573-6776 [Electronic] Netherlands
• PMID: 21384194 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA Primers
• DNA, Complementary
• DNA-Directed DNA Polymerase
• DNA Ligases

Topics

• DNA Ligases (metabolism)
• DNA Primers (genetics)
• DNA, Complementary (genetics, metabolism)
• DNA-Directed DNA Polymerase (metabolism)
• Molecular Biology (methods)
• Nucleic Acid Amplification Techniques (methods)