An Alternative Approach to Synthesize cDNA Bypassing Traditional Reverse Transcription

Authors: Li, Xiao-Xia¹; Zheng, Fang¹; Jiao, Yan-Li¹; Guo, Gang¹; Wang, Bao-Li²; Yao, Zhi³

Source: Molecular Biotechnology, Volume 39, Number 3, July 2008 , pp. 201-206(6)

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Abstract:

cDNAs of certain target genes are difficult to obtain by traditional reverse transcription. Herein we describe a novel method to synthesize cDNA based upon the use of the class IIS restriction enzymes. Briefly, the exons of a certain gene are separately PCR-amplified, each using the primers containing a recognition sequence of a certain class IIS restriction enzyme. All the fragments are restricted using the enzyme(s), resulting in the cohesive end of each exon that is complementary to the one in its adjacent exon. Then the fragments can be assembled together in their naturally occurring order. We successfully applied this method to acquire the coding sequence of Hoxa7 gene. This approach is simple, highly efficient, less error prone and cost-effective, and can also be used to fuse different PCR-fragments from distinct genes to create a chimeric gene or to perform site-directed mutagenesis.

Keywords: cDNA synthesis; DNA assembly; Class IIS restriction enzymes; Reverse transcription; Polymerase chain reaction

Document Type: Research article

DOI: http://dx.doi.org/10.1007/s12033-008-9035-x

Affiliations: 1: Key Research Lab of Hormone and Development Affiliated to the Ministry of Health, Tianjin Medical University, Tianjin, 300070, P.R. China, 2: Key Research Lab of Hormone and Development Affiliated to the Ministry of Health, Tianjin Medical University, Tianjin, 300070, P.R. China, Email: blwang7212@yahoo.com.cn 3: College of Basic Medical Science, Tianjin Medical University, Tianjin, 300070, P.R. China, Email: yaozhi23@hotmail.com

Publication date: 2008-07-01