总之,自Gall 和Pardue 1969年建立杂交免疫细胞化学以来的20余年中,已有不少新的改良的方法出现,但基本原则仍为Gall和Pardue所建立的相仿。作者个人体会是任何方法需在本人的实验实践中加以体验和改良。比如目前对乙酰基化作用,即浸入醋酸酐和三乙醇胺是否能减低背景染色的问题有作者对此提出异议。在科技工作者认为在原位杂交实验的所有应用溶液中加入0.1%~0.2%的二乙基焦碳酸乙酯(diethyl pyrocarbonate, DEPC)能有效的减低背景染色,但有些作者对此持怀疑态度。蛋白酶K的消化作用曾被认为是增加细胞或组织渗透性的关键步骤,但有作用在自身的实验室实践中体会除结构较致密的组织如脑、脊髓等外,对培养或涂片的单层细胞、蛋白酶K的消化及预杂交均是可以省略的。本文列举各作者的操作步骤略有不同,可能是基于这个原因。
目前,原位杂交技术主要应用于基因组图(Gene mapping),基因表达定位(localization of gene expression),核DNA和RNA的排列,mRNA的排列和运输(arrangement and transport of mRNA),复制(replication)和细胞的分类(sorting of cells)。临床研究应用在细胞遗传学(Cytogenetics),生前诊断(Prenatal diagnosis),肿瘤和传染性疾病的诊断,生物学剂量测定(biological dosimetry)和病毒学的病原学诊断等。随着核酸探针的制备,标记方法和基本操作方法的不断改进,新的技术不断涌现,相信在不久的将来,原位杂交化学技术将会更广泛的被应用于各个学科,并不断为生命科学提供新的资料,开拓新的领域。
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