DNA-DNA Subtractive cDNA Cloning Using oligo(dT)30-Latex and PCR: Identification of Cellular Genes Which Are Overexpressed in Senescent Human Diploid Fibroblasts
✍ Scribed by E. Hara; T. Yamaguchi; H. Tahara; N. Tsuyama; H. Tsurui; T. Ide; K. Oda
- Publisher
- Elsevier Science
- Year
- 1993
- Tongue
- English
- Weight
- 552 KB
- Volume
- 214
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
We previously reported an efficient method for subtractive cDNA cloning using oligo(dT) (){30})-Latex and polymerase chain reaction (PCR) (E. Hara et al., (\mathrm{Nu}) cleic Acids Res. 19, 7097-7104, 1991). The subtraction was performed by hybridization between mRNA of cell type (B) and the cDNA made from mRNA of cell type A using an oligo(dT) (){30}) primer covalently linked to Latex particles in an Eppendorf tube. The mRNA common to both types of cells could be removed by a brief centrifugation. In the present paper, the method was improved by using the sense strand DNA instead of mRNA for hybridization to cDNA covalently linked to the particles to minimize mRNA degradation and by optimizing the hybridization condition. The sense strand DNA was made from (c) DNA-oligo (dT) ()_{30})-Latex by asymmetric PCR. Using the improved method, a subtractive cDNA library with longer eDNA inserts was successfully constructed with higher probability than the original method. c 1993 Academic Press, Inc.