Direct cloning of cell differential expression genes with full-length by anew strategy based on the multiple rounds of 'long distance' polymerase chain reaction and magnetic beads mediated subtraction

Citation
Zl. Zhao et al., Direct cloning of cell differential expression genes with full-length by anew strategy based on the multiple rounds of 'long distance' polymerase chain reaction and magnetic beads mediated subtraction, J BIOTECH, 73(1), 1999, pp. 35-41
Citations number
9
Categorie Soggetti
Biotecnology & Applied Microbiology",Microbiology
Journal title
JOURNAL OF BIOTECHNOLOGY
ISSN journal
01681656 → ACNP
Volume
73
Issue
1
Year of publication
1999
Pages
35 - 41
Database
ISI
SICI code
0168-1656(19990730)73:1<35:DCOCDE>2.0.ZU;2-T
Abstract
The paper described a new cDNA subtractive cloning strategy. This strategy was based on the 'cap-finder' method, 'long distance' polymerase chain reac tion (PCR), streptavidin magnetic beads mediated subtraction, and spin colu mn chromatography. When PCR products were resolved on agarose gel after thr ee rounds of subtraction, the 'single gene difference' group displayed a pr edominantly enriched band, but the 'multiple gene difference' group did not display any apparent difference. Of 200 clones inserted with 0.7-2 kb frag ments from the 'multiple gene difference' group, 50% were identified to be new sequences and 35% were known. Of 100 new sequences, 35% contained codin g regions and 75% were confirmed by dot-blotting to be differentially expre ssed by genes of the target cell. The results suggested that this strategy might be very efficient for full-length cloning of differentially expressed genes of the cell. (C) 1999 Published by Elsevier Science B.V. All rights reserved.