LONG AND ACCURATE PCR

总体目标是开发一个扩展我们最近的突破性发现 一种耐热的DNA聚合酶配方，可扩增至23kb 到目前为止。目前的模板是Lambda噬菌体DNA。伴随着 新实现的长长度聚合酶链式反应产物，保真度也同样高 或者比目前所描述的任何一种PCR扩增系统都要高。多个领域 分子生物学、基因分析和基因图谱的研究将受益于 这个系统，特别是如果它可以扩展到至少30-40 kb，并且如果 它可以进一步发展，使其适用于红豆杉的基因组DNA。 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 具有关键参数，同时放大当前附近的目标大小 可靠性边缘(现在为15-18 kb)。随着改进的进行，试验台 DNA的跨度将会更长。 将可实现的聚合酶链式反应产物的最大长度从 Lambda DNA模板包括：1)增加引物的长度以允许 最高温度和最大底漆选择性 聚合酶链式反应的退火/延伸步骤。2)提高材料的热稳定性 现在令人惊讶的是，该系统在94%的温度下仅被限制为20秒 每个聚合酶链式反应周期的变性阶段。3)扫描反应缓冲条件 和骑车协议。4)诱变黑曲霉的一种酶成分 混合，以测试它如何工作的理论，并可能改进它如何 行得通。 在用可靠的lambda DNA模拟基因组DNA扩增后 将模板与人DNA混合，将该系统应用于基因组学实验 DNA分析将利用5、10、15、20和25kb分离的引物 人类DNA的已知测序区域。一种待测试的长底漆 将是STS大分子，因为这个系统也被证明利用了 这种引物比其他已知的耐热DNA更有效 聚合酶。由STSS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.