DNA-DNA subtractive cDNA cloning using oligo(dT)30-Latex and PCR: identification of cellular genes which are overexpressed in senescent human diploid fibroblasts.
We previously reported an efficient method for subtractive cDNA cloning using oligo(dT)30-Latex and polymerase chain reaction (PCR) (E. Hara et al., Nucleic 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 cDNA-oligo(dT)30-Latex by asymmetric PCR. Using the improved method, a subtractive cDNA library with longer cDNA inserts was successfully constructed with higher probability than the original method.
Hara
E
E
Department of Applied Biological Science, Science University of Tokyo, Noda, Japan.
Yamaguchi
T
T
Tahara
H
H
Tsuyama
N
N
Tsurui
H
H
Ide
T
T
Oda
K
K
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D016922:Cellular Senescence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000378:metabolism*; D004171:Diploidy; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005796:Genes; D006801:Humans; D007840:Latex; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary; D007840:Latex; D009838:Oligodeoxyribonucleotides; C084566:oligo(dT)-latex