Laboratory of Molecular and Cellular Biology. Department of Materials and Biosystem Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan

Correspondence: Masaharu Isobe, Laboratory of Molecular and Cellular Biology. Department of Materials and Biosystem Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan. Fax: +81-76-4456874. E-mail: isobe@eng.toyama-u.ac.jp

Received 29 September 2003; Accepted 31 October 2003; Published online 22 January 2004.

Abstract

The 3' rapid amplification of cDNA ends (3' RACE) is widely used to isolate the cDNA of unknown 3' flanking sequences. However, the conventional 3' RACE often fails to amplify cDNA from a large transcript if there is a long distance between the 5' gene-specific primer and poly(A) stretch, since the conventional 3' RACE utilizes 3' oligo-dT-containing primer complementary to the poly(A) tail of mRNA at the first strand cDNA synthesis. To overcome this problem, we have developed an improved 3' RACE method suitable for the isolation of cDNA derived from very large transcripts. By using the oligonucleotide-containing random 9mer together with the GC-rich sequence for the suppression PCR technology at the first strand of cDNA synthesis, we have been able to amplify the cDNA from a very large transcript, such as the microtubule-actin crosslinking factor 1 (MACF1) gene, which codes a transcript of 20 kb in size. When there is no splicing variant, our highly specific amplification allows us to perform the direct sequencing of 3' RACE products without requiring cloning in bacterial hosts. Thus, this stepwise 3' RACE walking will help rapid characterization of the 3' structure of a gene, even when it encodes a very large transcript.