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 method 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 nonspecific products that can hinder the cloning process. Here we report a new method that uses circularized first strand cDNA from mRNA and two gene specific 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. If 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 tested this new method on eight mRNAs that we have previously shown to respond 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 length sequence for the other two mRNAs. RNA folding program shows no iron responsive 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 enabled us to exclude the possibility that the expression of these iron responsive genes are regulated by IREs.
Ye
Z
Z
George M. Leader Family Laboratory for Alzheimer's Disease Research, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, 17033, USA.
Connor
J R
JR
D001254:Astrocytoma / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004270:DNA, Circular / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D016208:Databases, Factual; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007501:Iron / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004270:DNA, Circular; D018076:DNA, Complementary; D012333:RNA, Messenger; D007501:Iron