An ultra-sensitive method to detect mutations in human RAS templates.
An ultra-sensitive method to detect mutations in human RAS templates.
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DOI：
10.1080/21541248.2022.2083895
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发表时间：
2022-01
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RAS家族的小GTP酶在大约五分之一的人类癌症中发生突变。密码子G12、G13和Q61处的热点点突变占所有这些突变的95%，这些突变被充分确立为使编码的蛋白质致癌。在人类中，这个家族包括三个基因：HRAS，NRAS和KRAS。越来越多的证据表明，致癌RAS点突变可能正在启动，因为它们在人类肿瘤中通常是截断的，并且能够诱导小鼠的肿瘤发生。因此，人们对检测RAS基因中的致癌突变以了解癌症的起源以及早期检测目的非常感兴趣。为此，我们之前采用了微生物超灵敏最大深度测序（MDS）检测小鼠Kras基因，该检测能够在致癌物暴露后几天检测小鼠组织中的致癌突变，基本上捕获了肿瘤起始的第一步。鉴于此，我们在此报告了该测定的适应性和细节，以检测人KRAS序列中的突变，分析灵敏度为一百万个独立条形码化模板中的一个突变。因此，这种MDS的人源化版本可以用于以令人难以置信的灵敏度检测KRAS中的致癌突变，并针对其他RAS基因进行相同目的的修饰。
The RAS family of small GTPases is mutated in roughly a fifth of human cancers. Hotspot point mutations at codons G12, G13, and Q61 account for 95% of all these mutations, which are well established to render the encoded proteins oncogenic. In humans, this family comprises three genes: HRAS, NRAS, and KRAS. Accumulating evidence argues that oncogenic RAS point mutations may be initiating, as they are often truncal in human tumours and capable of inducing tumorigenesis in mice. As such, there is great interest in detecting oncogenic mutation in the RAS genes to understand the origins of cancer, as well as for early detection purposes. To this end, we previously adapted the microbial ultra-sensitive Maximum Depth Sequencing (MDS) assay for the murine Kras gene, which was capable of detecting oncogenic mutations in the tissues of mice days after carcinogen exposure, essentially capturing the very first step in tumour initiation. Given this, we report here the adaption and details of this assay to detect mutations in a human KRAS sequence at an analytic sensitivity of one mutation in a million independently barcoded templates. This humanized version of MDS can thus be exploited to detect oncogenic mutations in KRAS at an incredible sensitivity and modified for the same purpose for the other RAS genes.