cDNA Library Construction Using Streptavidin-Paramagnetic Beads and PCR
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cDNA clones of genes expressed in small amounts of material can be hard to obtain because the construction of conventional cDNA libraries requires microgram amounts of poly (A)+ RNA (1). The polymerase chain reaction (PCR), which is commonly used to amplify tiny amounts of DNA (2,3), has been adapted to facilitate the construction of cDNA libraries from small quantities of poly (A)+ RNA (4, 5, 6, 7). Most of these cDNA amplification methods require multiple purification or precipitation steps to remove primers and change buffers. These steps result in significant loss of material and compromise the quality of the final library. The method presented here eliminates precipitation and chromatography steps so that all cDNA synthesis and modification reactions are conducted in a single tube.
KeywordscDNA Library Total Nucleic Acid Paramagnetic Bead Oligonucleotide Adapter Polymerase Chain Reaction Reaction Buffer
- 11.Fernandez, J. M., Mc Atee, C., and Herrnstadt, C. (1990) Advances in cDNA technology. American Biotechnol. Lab. 8, 46–47.Google Scholar
- 12.Karrer, E. E., Lincoln, J. E., Hogenhout, S., Bennett, A. B., Bostock, R. M., Martineau, B., Lucas, W. J., Gilchrist, D. G., and Alexander, D. (1995) In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries. Proc. Natl. Acad. Sci. USA 92, 3814–3818.PubMedCrossRefGoogle Scholar