Has the polymerase chain reaction come of age for ophthalmology?
Has the polymerase chain reaction come of age for ophthalmology?
复制标题
聚合酶链式反应在眼科领域已经成熟了吗？
DOI：
10.1016/j.ajo.2008.09.023
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发表时间：
2009
影响因子：
4.2
通讯作者：
VanGelder,RussellN
中科院分区：
文献类型：
--
作者：
VanGelder,RussellN
记住描述现代聚合酶链式反应(PCR)的文章发表于1988年的那一周。我当时正在写博士论文，这个技巧简直是天赐之物。第一次，我们有了一种生物化学方法，可以从无限小的起始量中产生分析数量的脱氧核糖核酸(DNA)，这种方法不需要生长大量的细菌或酵母。在最初的日子里，聚合酶链式反应是一项繁琐的工作--坐在长凳上三个小时，在保持不同温度的三个加热块之间传递管子，每轮都添加酶。不到一年，自动热循环仪的出现和耐热DNA聚合酶的商业化应用使这一技术成为分子生物学中最容易的技术之一。进行聚合酶链式反应变得如此简单，以至于成为新加入我们团队的本科生的第一个任务。要进行这项技术，需要含有要扩增的DNA的材料(即玻璃体、房水或组织活检)、要扩增的序列的一些知识(作为用于启动复制的寡核苷酸引物捕获)、耐热的DNA聚合酶、DNA构建块(脱氧核苷酸三磷酸盐)、含有适当盐的缓冲液、热循环仪和几个小时。DNA与其互补序列的结合具有非凡的特异性，这使得引物能够在模板中快速找到它们的同源序列，并启动DNA复制。在合成子DNA链后，用热来分离父链和子链，每条链都成为下一轮扩增的模板。因此，聚合酶链式反应是一个指数过程。经过30个循环的聚合酶链式反应，一个模板分子可以被扩增超过十亿倍。这种高灵敏度使聚合酶链式反应成为检测复杂混合物中属于潜在病原体的DNA的理想选择。在其描述之后不久，聚合酶链式反应就被用于这一目的。Dlugoch和他的同事在1991年使用这项技术检测疱疹病毒DNA；在接下来的十年里，对影响眼睛的各种病毒、寄生虫、细菌和真菌进行了两次聚合酶链式反应检测。3.聚合酶链式反应的研究用途包括发现几种葡萄膜炎综合征的基础生物，包括热带葡萄膜炎
IVIVIDLY REMEMBER THE WEEK THE ARTICLE DESCRIBING the modern polymerase chain reaction (PCR) was published in 1988. 1 I was working on my PhD thesis, and this technique was a godsend. For the first time, we had a biochemical means for generating analytic amounts of deoxyribonucleic acid (DNA) from infinitesimal starting quantities, one which did not involve growing prodigious quantities of bacteria or yeast. In those early days, PCR was tedious work–three hours sitting at the bench, transferring tubes between three heat blocks kept at different temperatures, adding enzymes each round. Within a year, the advent of automated thermal cyclers and commercial availability of thermostable DNA polymerase turned this into one of the easiest techniques in molecular biology. Performing PCR became so easy it was the first task given to new undergraduates joining our group. To perform the technique, one needs material containing the DNA to be amplified (ie, a vitreous, aqueous, or tissue biopsy), some knowledge of the sequence to be amplified (captured as the oligonucleotide primers used to initiate replication), a thermostable DNA polymerase, DNA building blocks (deoxynucleotide triphosphates), a buffer with appropriate salts, a thermal cycler, and a few hours. The phenomenal specificity of DNA binding to its complementary sequence allows the primers to rapidly find their cognate sequences in the template and initiate DNA replication. After synthesis of the daughter DNA strand, heat is used to separate parent and daughter strands, and each becomes the template for the next round of amplification. PCR is thus an exponential process. After 30 cycles of PCR, one template molecule may be amplified over a billion times.This high sensitivity makes PCR ideal for the detection of DNA belonging to a potential pathogen in a complex mixture. Soon after its description, PCR was being used for this purpose. Dlugosch and associates used the technique to detect herpes virus DNA in 1991; 2 PCR assays for a variety of viruses, parasites, bacteria, and fungi affecting the eye have followed over the next decade. 3 Research uses of PCR have included discovery of the organisms underlying several uveitic syndromes, including Tropheryma