Cloning differentially expressed genes by linker capture subtraction.
We have developed a simple and effective method, designated linker capture subtraction (LCS), for cloning differentially expressed genes between two cell types or between cells treated in two different ways. In the first step of the method, two mRNA populations are converted to double-stranded cDNAs, fragmented, and ligated to linkers for PCR amplification. In the second step, the linkered DNA (tester) from one mRNA population is hybridized to an excess of the unlinkered DNA (driver) from the other mRNA population, followed by incubation with mung bean nuclease which digests single-stranded DNA specifically. This leaves only tester-tester homohybrids to be amplified by PCR in the following step, so as to achieve an enrichment of tester-specific sequences. The amplified PCR products are then used as tester for another round of subtraction. The process of subtraction is carried out three times, and the final PCR products are inserted into a vector for clonal analysis. We have used the strategy to begin to clone and identify the genes expressed differentially between the human prostate cancer cell lines LNCaP and PC-3, which have different tumorigenic and metastatic potentials. We demonstrated strong enrichment of target sequences. We also report the identities of two of the genes expressed differentially in these cell lines. One is prostate-specific antigen (PSA) which is known to be expressed in LNCaP but not in PC-3. The other is vimentin, the differential expression of which has not been reported previously in these prostate cancer cells.
Yang
M
M
Laboratory for Cell and Molecular Biology, Harvard Medical School, Boston, Massachusetts, 02215, USA.
Sytkowski
A J
AJ
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017430:Prostate-Specific Antigen / Q000235:genetics; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000235:genetics
D014746:Vimentin; D017430:Prostate-Specific Antigen