Direct cloning of cell differential expression genes with full-length by a new strategy based on the multiple rounds of 'long distance' polymerase chain reaction and magnetic beads mediated subtraction.
The paper described a new cDNA subtractive cloning strategy. This strategy was based on the 'cap-finder' method, 'long distance' polymerase chain reaction (PCR), streptavidin magnetic beads mediated subtraction, and spin column chromatography. When PCR products were resolved on agarose gel after three rounds of subtraction, the 'single gene difference' group displayed a predominantly enriched band, but the 'multiple gene difference' group did not display any apparent difference. Of 200 clones inserted with 0.7-2 kb fragments from the 'multiple gene difference' group, 50% were identified to be new sequences and 35% were known. Of 100 new sequences, 35% contained coding regions and 75% were confirmed by dot-blotting to be differentially expressed 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.
Zhao
Z
Z
Department of Immunology, Second Military Medical University, Shanghai, People's Republic of China.
Huang
X
X
Li
N
N
Zhu
X
X
Chen
S
S
Cao
X
X
D003001:Cloning, Molecular / Q000379:methods*; D003713:Dendritic Cells / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction