Highly multiplexed and mutation-sensitive quantitative PCR for cancer diagnostics

？ 描述：检测和定量携带罕见癌症驱动基因突变的核酸序列目前被用于指导临床治疗决策，如使用或停用或治疗。DNA诊断也可以用于基于无细胞核酸的非侵入性早期癌症筛查，但需要开发一种廉价、多重和突变敏感的平台。目前检测罕见突变的方法要么突变敏感度低(NGS)，要么多重(数字PCR)差，而且往往昂贵和/或劳动密集型(NGS和数字PCR)。PI建议开发一个聚合酶链式反应平台，能够以0.1%的单碱基突变敏感度从单个10 ng DNA样本中同时分析1000个点突变。聚合酶链式反应(PCR)仍然是DNA诊断中最可靠、最简单、最敏感和最受欢迎的方法。等位基因特异的聚合酶链式反应(PCR)引物可以区分大约1/100的单核苷酸变异(SNV)，但很难设计出能够在高度(&gt；10)多重环境中发挥作用同时保持高SNV选择性的引物。NGS已被研究人员和临床医生用于癌症组织样本的高度多重基因分析，但要获得足够的读取深度以允许&lt；0.1%的突变敏感性是不现实的。针对聚合酶链式反应的多重局限性的不完善的解决方案包括(1)样品分离，这需要相当大的样品输入量以保持临床灵敏度，以及(2)套式聚合酶链式反应/预扩增，这需要复杂的设备或有污染风险的开管步骤。高度多重等位基因特异性聚合酶链式反应的两个主要挑战是(1)温度稳定性和(2)引物-二聚体抑制。等位基因特异的聚合酶链式反应只能在约1C的狭窄窗口内检测/扩增罕见的等位基因，其精确值不能从序列中准确预测。PI正在开发对8摄氏度窗口内0.1%等位基因频率的单碱基突变敏感的新型引物；这种广泛的温度稳定性克服了通过经验优化许多引物以具有相同熔化温度的困难。一般情况下，聚合酶链式反应的引物可能会虚假地相互杂交，形成引物-二聚体，从而导致无意义的扩增产物；随着同时在溶液中的引物数量的增加，引物设计的难度急剧增加。PI正在开发大多数是双链的新型引物，因此大大减少了导致引物-二聚体相互作用的可能性；非特异性引物杂交的抑制使PCR具有显著更高的复制率。最后，研究小组将开发一种新型的片上对流聚合酶链式反应仪器，该仪器具有集成的多路复用阵列读数。重要的是，不需要对PCR扩增进行开管转移，从而防止了导致假阳性的污染。拟议研究项目的所有三个方面都已获得重要的初步数据，研究团队计划将开发的技术初步应用于非小细胞肺癌(NSCLC)患者血液样本的回顾分析。

? DESCRIPTION: Detection and quantitation of nucleic acid sequences bearing rare cancer driver mutations is currently used to inform clinical treatment decisions such as use or discontinuation or therapeutics. DNA diagnostics can also be used to perform noninvasive early-stage cancer screening based on cell-free nucleic acids, but requires the development of an inexpensive, multiplex, and mutation-sensitive platform. Current methods to detect rare mutations exhibit either low mutation sensitivity (NGS) or poor multiplexing (digital PCR), and are frequently expensive and/or labor-intensive (both NGS and digital PCR). The PI proposes to develop a PCR platform capable of simultaneous analysis of 1000 point mutations at 0.1% single-base mutation sensitivity from a single 10 ng DNA sample. The polymerase chain reaction (PCR) remains most robust, simple, sensitive, and popular method for DNA diagnostics. Allele-specific PCR primers can discriminate single nucleotide variants (SNVs) at roughly 1 part in 100, but it is difficult to design primers that can function in a highly (>10) multiplexed setting while preserving high SNV selectivity. NGS has been adopted by researchers and clinicians for highly multiplexed genetic analysis of cancer tissue samples, but it is not practical to obtain sufficient read depth to allow <0.1% mutation sensitivity. Imperfect solutions to the multiplexing limitation of PCR include (1) sample splitting, which requires significantly larger sample input quantities to preserve clinical sensitivity, and (2) nested PCR/pre-amplification which necessitates complex equipment or an open-tube step that risks contamination. The two primary challenges for highly multiplexed allele-specific PCR are (1) temperature robustness and (2) primer-dimer suppression. Allele-specific PCR primers are capable of rare allele detection/amplification only within a narrow window of about 1 C, the exact value of which cannot be accurately predicted from sequence. The PI is developing novel primers that are sensitive to single-base mutations at 0.1% allele frequency across a window of 8 C; this broad temperature robustness overcomes the difficulty of empirically optimizing many primers to have the same melting temperature. PCR primers in general can spuriously hybridize to each other and form primer-dimers that result in nonsense amplification products; primer design grows sharply more difficult as the number of primers simultaneously in solution increases. The PI is developing novel primers that are mostly double-stranded and thus significantly less likely to result in primer-dimer interactions; the suppression of nonspecific primer hybridization enables PCR with significantly higher multiplexing. Finally, the research team will develop a novel on-chip convection PCR instrument with an integrated multiplexed array-based readout. Importantly, no open-tube transfer of PCR amplicons is necessary, preventing contamination that leads to false positive. Significant preliminary data has been obtained for all three aspects of the proposed research project, and the research team plans to apply the developed technology initially to retrospective analysis of non-small cell lung cancer (NSCLC) patient blood samples.