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Bifunctional thermostable DNA polymerases with dual DNA polymerase and reverse transcriptase activities for use in qRT-PCR

具有双 DNA 聚合酶和逆转录酶活性的双功能热稳定 DNA 聚合酶，用于 qRT-PCR

基本信息

批准号：
BB/F00687X/1
负责人：
Bernard Connolly
金额：
$ 56.27万
依托单位：
Newcastle University
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2008
资助国家：
英国
起止时间：
2008 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FF00687X%2F1
关键词：
Bifunctional thermostable DNA polymerases dual

项目摘要

Reverse transcription PCR (RT-PCR) is a protocol that involves copying RNA, most commonly messenger RNA (mRNA), to DNA and then amplifying the DNA produced. In this way a tiny amount of RNA can be converted to large quantities of DNA, with retention of the sequence information encoded in the RNA. In the past RT-PCR has mainly been used to prepare cDNA libraries from eukaryotes such as humans and yeast. A cDNA library is a snapshot of every mRNA present in a cell or tissue at a particular instance and can give valuable insights about the status and functioning of a cell. With eukaryotes it is also necessary to produce cDNA in order to use recombinant DNA techniques to obtain large quantities of proteins, for example, human insulin, growth hormones and blood clotting factors, used for disease treatment. More recently RT-PCR has undergone enormous expansion with the development of quantitative or real time PCR (denoted here, qRT-PCR), a technology that allows the accurate measurement of the amount of any mRNA present in a cell/tissue. qRT-PCR is amenable to a high degree of automation and can be used to simultaneously quantify a large number of mRNA levels. The technique is of immense application in fundamental research for example to determine at what levels mRNAs are expressed in particular cells and organs, how and why distinct cell types differ in their mRNA levels and how these levels respond to differing conditions such as age, nutritional status and stress levels. qRT-PCR is of high medicinal significance and can determine mRNA levels in cancerous tumours or tissues subject to autoimmune disease. The method can assess how cells respond to bacterial and viral infections or how drugs influence the expression of particular mRNAs. Thus qRT-PCR is used both to monitor disease progression and to suggest therapeutic intervention. Normally RT-PCR requires two enzymes, reverse-transcriptase to copy the RNA to DNA and DNA polymerase to amplify the DNA produced. However, the use of two enzymes often gives rise to problems, especially in automated high throughput qRT-PCR. It is frequently difficult to optimise conditions to simultaneously meet the requirements of both enzymes, and inconvenient two step protocols are often required. Furthermore the reverse transcriptase often gives low yields of product and is intrinsically error prone, leading to loss of genetic information. There are some single enzyme systems for RT-PCR, typically using a DNA polymerase under conditions were it is able to copy RNA to DNA. Such conditions are invariably sub-optimal both in regards to final product yield and in the fidelity with which genetic information is transmitted from the starting RNA to the amplified DNA product. The aim of this grant is to produce a single bifunctional enzyme with both reverse transcriptase and DNA polymerase activities. The starting enzyme will be a thermostable DNA polymerase from an archaeon (Pyrococcus furiosus), capable of rapid and accurate amplification of DNA. A directed evolution method will be used to endow these enzymes with an additional reverse transcriptase activity. The evolution strategy randomises amino acids in the starting enzymes and then uses a protocol that specifically selects for novel variants with both DNA polymerase and reverse transcriptase activities. It is anticipated that the bifunctional polymerase produced will possess high thermostability, rapid amplification rates and high fidelity and, it is hoped, should make RT-PCR, an especially qRT-PCR, simple, robust and straightforward. Such an enzyme will be applicable to in the many, and expanding, real-time PCR applications described above. It should be noted that the real time PCR market is currently worth ~ $1000 million, and growing rapidly.

逆转录PCR（RT-PCR）是一种涉及将RNA（最常见的是信使RNA（mRNA））复制到DNA，然后扩增产生的DNA的方案。通过这种方式，少量的RNA可以转化为大量的DNA，并保留RNA中编码的序列信息。在过去，RT-PCR主要用于从真核生物如人和酵母制备cDNA文库。cDNA文库是在特定情况下存在于细胞或组织中的每个mRNA的快照，可以提供有关细胞状态和功能的有价值的见解。对于真核生物，为了使用重组DNA技术获得大量蛋白质，例如用于疾病治疗的人胰岛素、生长激素和凝血因子，还需要产生cDNA。最近，随着定量或真实的时间PCR（在此表示为qRT-PCR）的发展，RT-PCR经历了巨大的扩展，该技术允许精确测量存在于细胞/组织中的任何mRNA的量。qRT-PCR适合高度自动化，可用于同时定量大量mRNA水平。该技术在基础研究中具有巨大的应用，例如确定特定细胞和器官中mRNA的表达水平，不同细胞类型的mRNA水平如何以及为什么不同，以及这些水平如何响应不同的条件，如年龄，营养状况和压力水平。qRT-PCR具有很高的医学意义，并且可以确定癌性肿瘤或患有自身免疫性疾病的组织中的mRNA水平。该方法可以评估细胞如何对细菌和病毒感染做出反应，或者药物如何影响特定mRNA的表达。因此，qRT-PCR用于监测疾病进展和建议治疗干预。通常RT-PCR需要两种酶，逆转录酶将RNA复制为DNA，DNA聚合酶扩增产生的DNA。然而，使用两种酶通常会产生问题，特别是在自动化高通量qRT-PCR中。通常难以优化条件以同时满足两种酶的要求，并且通常需要不方便的两步方案。此外，逆转录酶通常产生低产量的产物，并且本质上容易出错，导致遗传信息的丢失。有一些用于RT-PCR的单酶系统，通常在能够将RNA复制到DNA的条件下使用DNA聚合酶。这样的条件在最终产物产率和遗传信息从起始RNA传递到扩增的DNA产物的保真度方面总是次优的。这项资助的目的是生产一种具有逆转录酶和DNA聚合酶活性的单一双功能酶。起始酶将是来自古细菌（激烈火球菌）的热稳定DNA聚合酶，能够快速准确地扩增DNA。定向进化方法将用于赋予这些酶额外的逆转录酶活性。进化策略使起始酶中的氨基酸随机化，然后使用一种方案，该方案专门选择具有DNA聚合酶和逆转录酶活性的新变体。预期所产生的双功能聚合酶将具有高热稳定性、快速扩增速率和高保真度，并且希望使RT-PCR（尤其是qRT-PCR）简单、稳健和直接。这种酶将适用于上述许多且不断扩大的实时PCR应用。应该注意的是，真实的时间PCR市场目前价值约10亿美元，并且增长迅速。