cDNA cloning by amplification of circularized first strand cDNAs reveals non-IRE-regulated iron-responsive mRNAs

Currently, the rapid amplification of cDNA ends (RACE) is the most common m ethod for PCR cloning of cDNA Because RACE uses a gene specific primer and one adaptor primer that is shared by all cDNAs may result in numerous nonsp ecific products that can hinder the cloning process. Here we report a new m ethod that uses circularized first strand cDNA from mRNA and two gene speci fic primers to amplify both the 5' and 3' cDNA ends in one reaction. A cDNA band of correct size can be obtained on the first pass in this approach. I f the correct size is not obtained on the first pass, amplification of cDNA ends can be repeated until the correct size of the cDNA is obtained. We te sted this new method on eight mRNAs that we have previously shown to respon d to cellular iron levels. We obtained sequences for six mRNAs that were 43 bp to 1324 bp longer than that reported in GenBank and obtained the same l ength sequence for the other two mRNAs. RNA folding program shows no iron r esponsive elements (IRE) on these mRNA, In conclusion, our cloning approach offers a more efficient method for cloning full-length cDNA and it may be used to replace the existing method of 5' end cDNA extension. The data enab led us to exclude the possibility that the expression of these iron respons ive genes are regulated by IREs. (C) 2000 Academic Press.