Yang M, Sytkowski A J
Laboratory for Cell and Molecular Biology, Harvard Medical School, Boston, Massachusetts, 02215, USA.
Anal Biochem. 1996 May 15;237(1):109-14. doi: 10.1006/abio.1996.0207.
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.
我们开发了一种简单有效的方法，称为接头捕获消减（LCS），用于克隆两种细胞类型之间或两种不同处理方式的细胞之间差异表达的基因。在该方法的第一步中，将两个mRNA群体转化为双链cDNA，进行片段化，并连接接头用于PCR扩增。在第二步中，将来自一个mRNA群体的带接头DNA（测试者）与来自另一个mRNA群体的过量无接头DNA（驱动者）杂交，随后用绿豆核酸酶孵育，该酶特异性消化单链DNA。这使得在接下来的步骤中只有测试者-测试者同源杂交体能够通过PCR扩增，从而实现测试者特异性序列的富集。扩增的PCR产物随后用作下一轮消减的测试者。消减过程进行三次，最终的PCR产物插入载体进行克隆分析。我们已使用该策略开始克隆和鉴定在具有不同致瘤和转移潜能的人前列腺癌细胞系LNCaP和PC-3之间差异表达的基因。我们证明了靶序列的强烈富集。我们还报告了在这些细胞系中差异表达的两个基因的身份。一个是前列腺特异性抗原（PSA），已知在LNCaP中表达而在PC-3中不表达。另一个是波形蛋白，其差异表达此前在这些前列腺癌细胞中尚未见报道。
