總之,自Gall 和Pardue 1969年建立雜交免疫細胞化學以來的20余年中,已有不少新的改良的方法出現(xiàn),但基本原則仍為Gall和Pardue所建立的相仿。作者個人體會是任何方法需在本人的實驗實踐中加以體驗和改良。比如目前對乙;饔,即浸入醋酸酐和三乙醇胺是否能減低背景染色的問題有作者對此提出異議。在科技工作者認為在原位雜交實驗的所有應用溶液中加入0.1%~0.2%的二乙基焦碳酸乙酯(diethyl pyrocarbonate, DEPC)能有效的減低背景染色,但有些作者對此持懷疑態(tài)度。蛋白酶K的消化作用曾被認為是增加細胞或組織滲透性的關鍵步驟,但有作用在自身的實驗室實踐中體會除結(jié)構較致密的組織如腦、脊髓等外,對培養(yǎng)或涂片的單層細胞、蛋白酶K的消化及預雜交均是可以省略的。本文列舉各作者的操作步驟略有不同,可能是基于這個原因。
目前,原位雜交技術主要應用于基因組圖(Gene mapping),基因表達定位(localization of gene expression),核DNA和RNA的排列,mRNA的排列和運輸(arrangement and transport of mRNA),復制(replication)和細胞的分類(sorting of cells)。臨床研究應用在細胞遺傳學(Cytogenetics),生前診斷(Prenatal diagnosis),腫瘤和傳染性疾病的診斷,生物學劑量測定(biological dosimetry)和病毒學的病原學診斷等。隨著核酸探針的制備,標記方法和基本操作方法的不斷改進,新的技術不斷涌現(xiàn),相信在不久的將來,原位雜交化學技術將會更廣泛的被應用于各個學科,并不斷為生命科學提供新的資料,開拓新的領域。
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