Microscale cDNA subtraction method for detection of regulators for maxillo-facial development.

消减杂交方法被用来检测在特定组织或细胞中表达但在其他组织或细胞中不表达的基因。通常，包含差异表达基因的被称为“测试者”的cdna与不包含此类基因的被称为“驱动程序”的过量RNA杂交。结果表明，差异表达的基因片段中富含未杂交的基因片段。传统的消减杂交方法虽然在某些情况下得到了成功的应用，但它们需要大量的检测和驱动程序，因此当只有少量的组织(如小鼠牙芽等)时，很难识别差异表达的基因。在本研究中，我们用两种不同的方法构建了软骨细胞特异性消减cDNA文库：[1]常规单链消减(tester cdna-DRIVER m RNA)和[2]双链cDNAd消减(tester dscDNA-DscDNA)。在这两种方法中，我们使用了聚合酶链式反应来扩增测试器cDNA，我们发现这些文库的质量是相似的，但方法[2]中构建文库所需的测试器mRNA量比文献[1]中的要少得多。在这两个文库中，大多数克隆编码3‘非翻译序列和部分序列，插入的cDNA平均大小约为550bp。我们推测这是由于聚合酶链式反应扩增的限制。虽然部分基因可以作为探针来研究基因在组织中的表达模式，但需要筛选未消减的文库来获得全长的基因。

Subtractive hybridization methods are used to detect genes that are expressed in a specific tissue or cell but not in the other. Usually cDNA called "tester" that contains differentially expressed genes is hybridized with an excess amount of RNA called "driver" that does not contain such genes. As a result, differentially expressed cDNA in enriched in unhybridized cDNA fraction. Although traditional subtractive hybridization methods have been successfully used in some cases, they require a lot of tester and driver mRNA.Therefore, it has been difficult to identify differentially expressed genes when only small amount of tissue (e.g.mouse tooth bud etc.) is available as a starting material.In the present study, we constructed chondrocytes specific subtraction cDNA library by two different methods : [1] conventional single strand subtraction (tester cDNA-driver mRNA) and [2] double strand cDNA subtraction (tester dscDNA-driver dscDNA). In both methods, we used PCR for amplifying tester cDNA.We found the quality of those libraries was similar but the amount of tester mRNA that was required for library construction was much smaller in the method [2] than that of [1]. In both libraries, most of cDNA clones encodes 3' untranslated sequence plus partial sequence and the average size of inserted cDNAs was about 550bp. We speculated this was due to the limitation of PCR amplification. Although partial cDNA may be useful as a probe to examine expression pattern of the gene in the tissue, It is necessary screen the unsubtracted library to obtain full length cDNA.