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

Authors

Yongxin Wang, Barbara Ann Karmanos Cancer Institute, Wayne State University School of MedicineFollow
Joseph M. Fugaro, Barbara Ann Karmanos Cancer Institute, Wayne State University School of MedicineFollow
Fauzia Siddiq, Barbara Ann Karmanos Cancer Institute, Wayne State University School of MedicineFollow
Chandra Mouli V. Goparaju, Barbara Ann Karmanos Cancer Institute, Wayne State University School of MedicineFollow
Fulvio Lonardo, Barbara Ann Karmanos Cancer Institute, Wayne State University School of MedicineFollow
Anil Wali, Barbara Ann Karmanos Cancer Institute, Wayne State University School of MedicineFollow
John F. Lechner, Barbara Ann Karmanos Cancer Institute, Wayne State University School of MedicineFollow
Harvey I. Pass, Barbara Ann Karmanos Cancer Institute, Wayne State University School of MedicineFollow

Document Type

Article

Abstract

Abstract

Background

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.

Results

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.

Conclusions

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.

Disciplines

Genetics and Genomics | Investigative Techniques | Medical Genetics

Recommended Citation

Wang et al. BMC Molecular Biology 2000, 1:2
doi:10.1186/1471-2199-1-2