CLONING DIFFERENTIALLY EXPRESSED GENES BY LINKER CAPTURE SUBTRACTION

We have developed a simple and effective method, designated linker cap ture subtraction (LCS), for cloning differentially expressed genes bet ween two cell types or between cells treated in two different ways. In the first step of the method, two mRNA populations are converted to d ouble-stranded cDNAs, fragmented, and ligated to linkers for PCR ampli fication. In the second step, the linkered DNA (tester) from one mRNA population is hybridized to an excess of the unlinkered DNA (driver) f rom the other mRNA population, followed by incubation with mung bean n uclease which digests single-stranded DNA specifically. This leaves on ly tester-tester homohybrids to be amplified by PCR in the following s tep, so as to achieve an enrichment of tester-specific sequences. The amplified PCR products are then used as tester for another round of su btraction. 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 ex pressed differentially between the human prostate cancer cell lines LN CaP and PC-3, which have different tumorigenic and metastatic potentia ls. We demonstrated strong enrichment of target sequences. We also rep ort the identities of two of the genes expressed differentially in the se cell lines. One is prostate-specific antigen (PSA) which is known t o be expressed in LNCaP but not in PC-3. The other is vimentin, the di fferential expression of which has not been reported previously in the se prostate cancer cells. (C) 1996 Academic Press, Inc.