Open AccessSubtractive Hybridization Strategy Using Paramagnetic Oligo(dT) Beads and PCR
Author(s) -
Melinda Mészáros,
David B. Morton
Publication year - 1996
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/19962003413
Subject(s) - suppression subtractive hybridization , microbiology and biotechnology , polymerase chain reaction , biology , chemistry , computational biology , genetics , complementary dna , gene , cdna library
Subtractive hybridization has been widely used for the identification of differentially expressed genes. Here we describe a simple, sensitive strategy of subtractive hybridization that involves binding the driver poly(A) + RNA pool to paramagnetic Dynabeads ® Oligo (dT)25. After hybridization with target cDNA, the molecules common to both pools are removed. The subtracted cDNA is then amplified with PCR and used for library screening. Using this method, we have identified four cDNA clones that represent developmentally regulated transcripts in the central nervous system of the tobacco hornworm Manduca sexta. All four transcripts are of low abundance, comprising only 0.001%–0.5% of the poly(A) + RNA pool.