Size-selection of cDNA libraries for the cloning of cDNAs after suppression subtractive hybridization
- Resource Type
- Authors
- Kalidou Ndiaye; Jacques G. Lussier; M. Nahé Diouf; Tania Fayad; Valérie Lévesque
- Source
- BioTechniques. 35(1)
- Subject
- Genetics
Cloning
endocrine system
Granulosa Cells
Library
cDNA library
Gene Expression Profiling
Nucleic Acid Hybridization
Biology
General Biochemistry, Genetics and Molecular Biology
Plasmid
Differentially expressed genes
Suppression, Genetic
Gene Expression Regulation
Suppression subtractive hybridization
Complementary DNA
Animals
Cattle
Female
Cloning, Molecular
Selection (genetic algorithm)
Cells, Cultured
Biotechnology
Gene Library
- Language
- ISSN
- 0736-6205
Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.