%0 Journal Article
%T A simple method for generating full length cDNA from low abundance partial genomic clones
%A Yongxin Wang
%A Joseph M Fugaro
%A Fauzia Siddiq
%A Chandra Mouli V Goparaju
%A Fulvio Lonardo
%A Anil Wali
%A John F Lechner
%A Harvey I Pass
%J BMC Molecular Biology
%D 2000
%I BioMed Central
%R 10.1186/1471-2199-1-2
%X The first approach we used for the isolation of full-length cDNA from two known genomic clones of Hox genes was based on fusion PCR. Here we describe a simple and efficient method of amplification for homeobox D13 (HOXD13) full length cDNA from two partial genomic clones. Specific 5' and 3' untranslated region (UTR) primer pairs and website program (primer3_www.cgv0.2) were key steps involved in this process.We have devised a simple, rapid and easy method for generating cDNA clone from genomic sequences. The full length HOXD13 clone (1.1 kb) generated with this technique was confirmed by sequence analysis. This simple approach can be utilized to generate full-length cDNA clones from available partial genomic sequences.The first approach we used for the isolation of full length cDNA from two known genomic clones of Hox genes was based on fusion PCR (1). It involved selection of primers at the 5' and 3' ends of the two exons with a complimentary set of primers in the middle of the clone. This approach was time consuming and labor intensive, especially with the number of different combinations of primer pair sets and series of PCR amplifications. We were successful in generating the 3' half of the clone with this procedure, but the high GC content in the 5' end of the clone proved to be a challenge with this strategy. To circumvent this problem, we used 5'-RACE (Rapid amplification of cDNA ends) procedure to generate 5'-end of the clone (3). This method involved use of many more specific primers, nested gene-specific primers, followed by PCR amplifications of homopolymer-containing anchor/adaptor priming steps. While considerably more efficient than fusion PCR, this technique was still unable to generate the specific high GC rich 5' end of our gene of interest i.e., the homeobox D13 cDNA (4). Here we describe a novel and efficient PCR-based method for the generation of full length cDNA of low abundance transcripts from two partial genomic clones.Our approach took advan
%U http://www.biomedcentral.com/1471-2199/1/2