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
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
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.