基于人工模拟核酸的新型低丰度基因突变富集方法的研究

The detection of low-abundance DNA variants or mutations is important in many regards, but especially for early cancer detection, determining personalized treatment and therapy, monitoring of therapy outcome and cancer remission/relapse, and noninvasive prenatal diagnosis. Because the sensitivity of downstream analysis techniques (such as Sanger sequencing, pyrosequencing, and HRM analysis) is too low to meet kinds of requirements form basic and clinical studies, many enrichment methods for known or unknown mutations have been developed over the past two decades. Among these methods, a novel method called ICE COLD-PCR developed by Makrigiorgos GM and colleagues is considered as a paragon of low-abundance unknown mutation enrichment methods, because it has high selectivity, is simple in performance, and more importantly it can be used combined with almost all existing downstream analysis methods with no additional cost, time, and labor. However, there still exists some shortcomings: firstly, it is hard to achieve real exponential amplification of variants; secondly, the selectivity is intimately associated with the type of mutation, the position of the mutation located, and the length of the test DNA segment; finally, according to the basic design idea, even under ideal conditions, it is impossible to block the amplification of wild-type completely. To overcome these shortcomings, in this work, the artificial nucleic acid analogues technology, especially 'self-avoiding molecular-recognition system' and 'locked nucleic acid', will be introduced, and incorporated with the existing enrichment method.The execution of this project will develop 1-2 novel low-abundance unknown mutation enrichment methods with higher selectivity and intellectual property rights.

检测样本中的低丰度突变基因在癌症早期诊断等诸多领域均有着非常诱人的前景。由于测序等下游分析技术本身灵敏度不能够满足各种科研及临床需求，人们发展了一系列可以对样本中的突变基因进行富集的方法。这其中，ICE COLD-PCR技术由于可以对样本中的各种未知突变进行富集，并且选择效率高，操作简单，还能和各种下游检测技术之间实现无缝衔接，被认为是现有未知突变基因富集技术之典范。然而，该技术自身也仍然存在着诸如无法真正实现对突变基因的指数扩增，检测效率与突变的类型、所处的位置和序列长度有关，以及即使在理想状态下，也无法完全抑制野生型基因的扩增等诸多不足，仍然有一些可以改进和提高的余地。针对上述不足，本研究拟将本实验室在模拟核酸领域的成果，尤其是自回避分子识别系统和锁核酸技术，与现有技术相整合，从而发展出具有自主知识产权的，同样使用方便，操作简单，但却更为高效的1-2种新型低丰度未知突变基因富集技术。

检测样本中的低丰度突变基因在癌症早期诊断等诸多领域均有着非常诱人的前景。由于测序等下游分析技术本身灵敏度不能够满足各种科研及临床需求，人们发展了一系列可以对样本中的突变基因进行富集的方法。这其中，ICE COLD PCR 技术由于可以对样本中的各种未知突变进行富集，并且选择效率较高，操作简单，还能和各种下游检测技术之间实现无缝衔接，被认为是现有未知突变基因富集技术之典范。然而，该技术自身也仍然存在着诸如针对突变基因和野生型基因的扩增差异程度有限，对PCR仪温控要求极高等诸多不足，仍然有一些可以改进和提高的余地。针对上述不足，本研究将锁核酸技术与现有技术相整合，初步建立了同样使用方便，操作简单，但却更为高效的新型低丰度未知突变基因富集技术LNA COLD PCR。通过引入锁核酸替代原来的天然核酸作为封闭序列，LNA COLD PCR较现有技术实现了更大幅度的差异扩增，对PCR仪温控精度的要求也不再苛刻。即使普通PCR仪上运行，结合常规Sanger测序，我们的LNA COLD PCR也可以清晰检出样本中低至1%的未知突变基因。