Primed Synthesis and Direct Sequencing in the Isolation of cDNA Clones Using Short Oligonucleotides
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With the development of improved techniques for the construction of cDNA libraries representing rare messenger species has come increased demand for screening techniques to isolate specific cDNAs. A variety of techniques has been developed:
| 1. | The use of exact and degenerate oligonucleotides complementary to RNAs coding for established protein sequence. |
| 2. | Bacterial expression of cDNA and immunological detection of the protein products. |
| 3. | Selection by hybrid-arrest or hybrid-select translation. |
| 4. | Differential hybridization using cDNAs obtained under different physiological conditions. |
| 5. | Bacterial expression of cDNA and selection by biological assay. |
| 6. | Transfer of cDNA libraries in eukaryotic expression vectors into mammalian cell lines and subsequent biological selection. |
| 7. | Expression of cloned cDNAs and mRNA in SP6 vectors with subsequent microinjection of RNA and biological selection of decreasing pools. |
