A simple method for generating full length cDNA from low abundance partial genomic clones

PCR amplification of target molecules involves sequence specific primers that flank the region to be amplified. While this technique is generally routine, its applicability may not be sufficient to generate a desired target molecule from two separate regions involving intron /exon boundaries. For th...

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Bibliographic Details
Published in:BMC molecular biology 2000-11, Vol.1 (1), p.2-2, Article 2
Main Authors: Wang, Y, Fugaro, J M, Siddiq, F, Goparaju, C M, Lonardo, F, Wali, A, Lechner, J F, Pass, H I
Format: Article
Language:English
Subjects:
Analysis
Cloning
DNA
DNA sequencing
Gene amplification
Genes
Genetic aspects
Genetic research
Health aspects
Methodology
Methods
Nucleotide sequencing
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Summary:PCR amplification of target molecules involves sequence specific primers that flank the region to be amplified. While this technique is generally routine, its applicability may not be sufficient to generate a desired target molecule from two separate regions involving intron /exon boundaries. For these situations, the generation of full-length complementary DNAs from two partial genomic clones becomes necessary for the family of low abundance genes. 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.
ISSN:1471-2199
1471-2199
DOI:10.1186/1471-2199-1-2