LONG AND ACCURATE PCR

总的目标是发展一个扩展我们最近的突破性发现 一种热稳定的DNA聚合酶制剂， 迄今 目前的模板是λ噬菌体DNA。 伴随着 新实现的长长度的PCR产物，保真度也同样高 或高于迄今为止描述的任何PCR扩增系统。 许多领域 分子生物学、基因分析和基因定位将受益于 这个系统，特别是如果它可以扩展到至少30-40 kb， 它可以进一步发展，使其适用于基因组DNA的 细菌或人类的复杂性。 将通过实验来寻求对放大系统的改进 同时放大电流附近的目标尺寸 Edge of reliability（现在为15-18 kb）。 随着改进的进行，试验台 DNA的长度会更长。 实验以延长可实现的PCR产物的最大长度， λ DNA模板包括：1）增加引物的长度以允许 最高温度和最大引物选择性 PCR的退火/延伸步骤。 2)提高热稳定性 该系统，现在令人惊讶地限制在94%C下仅20秒， 每个PCR循环的变性阶段。 3)扫描反应缓冲液条件 和循环方案。 4)突变的酶组成部分之一， 混合物，以测试它如何工作的理论，并可能改善它如何 工程. 在用可靠的λ DNA进行模拟基因组DNA扩增后， 模板与人类DNA混合，将该系统应用于基因组的实验 DNA分析将使用5、10、15、20和25 kb间隔的引物， 已知的人类DNA序列。 一种待测长引物 将是STS大引物，因为该系统也已被证明利用 这种引物比其它已知的热稳定DNA更有效 聚合酶 由STS定义的基因组图谱将很容易受益于 这一进步。

The overall aim is to develop an extend our recent breakthrough discovery of a thermostable DNA polymerase formulation that can amplify up to 23 kb so far. The current template is lambda phage DNA. Concomitant with the newly achievable long lengths of PCR product, the fidelity is also as high or higher than any PCR amplification system so far described. Many areas of molecular biology, gene analysis, and gene mapping will benefit from this system, especially if it can be extended to at least 30-40 kb and if it can be further developed to make it applicable to genomic DNA of bacterial or human complexity. Improvements to the amplification system will be sought by experimenting with critical parameters while amplifying target sizes near the current edge of reliability (now 15-18 kb). As improvements are made, the test bed DNA spans will be longer. Experiments to extend the maximum length of achievable PCR product from lambda DNA template include; 1) Increase the length of primers to allow maximum temperature and maximum primer selectivity during the annealling/extension step of the PCR. 2) Increase the thermostability of the system, now surprisingly limited to only 20 seconds at 94% C during the denaturation phase of each PCR cycle. 3) Scan reaction buffer condiitons and cycling protocols. 4) Mutagenize one of the enzyme components of the mixture to test the theory of how it works, and to possible improve how it works. After simulated genomic DNA amplifications with reliable lambda DNA template mixed with human DNA, experiments to apply the system to genomic DNA analysis will utilize primers of 5, 10, 15, 20, and 25 kb separation on known sequenced regions of human DNA. A form of long primer to be tested will be STS megaprimers, since this syst em has also been shown to utilize such primers more effectively than other known thermostable DNA polymerases. Genomic maps defined by STSs will then benefit easily from this advance.