Highly multiplexed and mutation-sensitive quantitative PCR for cancer diagnostics

用于癌症诊断的高度多重且突变敏感的定量 PCR

• 批准号: 9896788
• 负责人: David Yu Zhang
• 金额: $ 61.24万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896788
• 关键词: Adopted; Alleles; Base Sequence; Biological Assay; Blood specimen; Cancer Diagnostics; Cancer Patient; Cells; Clinical Sensitivity; Clinical Treatment; Complex; Convection; DNA; DNA Sequence; Data; Detection; Development; Diagnostic; Dimerization; Effectiveness; Equipment; Evolution; Exhibits; Fluorescence; Frequencies; Gene Frequency; Genomic DNA; Human; Hybrids; In Situ; Malignant Neoplasms; Methods; Microarray Analysis; Molecular; Monitoring for Recurrence; Mutation; Nested PCR; Non-Small-Cell Lung Carcinoma; Nucleic Acids; Point Mutation; Polymerase Chain Reaction; RNA Sequences; Reaction; Recurrence; Research; Research Personnel; Research Project Grants; Resistance; Risk; Sampling; Screening for cancer; Single Nucleotide Polymorphism; Surface; System; Tail; Technology; Temperature; Therapeutic; Tissue Sample; Tube; Validation; amplification detection; base; cancer biomarkers; cancer recurrence; cost; design; digital; dimer; driver mutation; experimental study; fluorophore; genetic analysis; genetic variant; graph theory; improved; instrument; instrumentation; lung cancer screening; melting; next generation sequencing; novel; preservation; prevent; public health relevance; rare cancer; rare variant; representation theory; tumor progression

? 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.

? 产品说明： 携带罕见癌症驱动突变的核酸序列的检测和定量目前用于告知临床治疗决策，例如使用或中止或治疗。DNA诊断也可用于基于无细胞核酸进行非侵入性早期癌症筛查，但需要开发廉价、多重和突变敏感的平台。目前检测罕见突变的方法表现出低突变灵敏度（NGS）或差的多重（数字PCR），并且通常是昂贵的和/或劳动密集型的（NGS和数字PCR两者）。PI建议开发一种PCR平台，能够以0.1%的单碱基突变灵敏度从单个10 ng DNA样本中同时分析1000个点突变。 聚合酶链反应（PCR）仍然是最强大的，简单的，敏感的，和流行的DNA诊断方法。等位基因特异性PCR引物可以以大约1/100的比例区分单核苷酸变体（SNV），但是难以设计可以在高度（>10）多重设置中起作用同时保持高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遗传分析，但获得足够的读取深度以允许<0.1%的突变灵敏度是不切实际的。PCR多重限制的不完美解决方案包括（1）样品分离，其需要显著更大的样品输入量以保持临床灵敏度，和（2）巢式PCR/预扩增，其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PCR的两个主要挑战是（1）温度稳健性和（2）引物二聚体抑制。等位基因特异性PCR引物仅能够在约1C的窄窗口内进行稀有等位基因检测/扩增，其精确值不能从序列准确预测。PI正在开发新的引物，这些引物在8 ℃的窗口内对0.1%等位基因频率的单碱基突变敏感;这种广泛的温度稳定性克服了经验优化许多引物以具有相同解链温度的困难。PCR引物通常可以虚假地彼此杂交并形成引物二聚体，其导致无义扩增产物;随着溶液中同时存在的引物数量增加，引物设计急剧地变得更加困难。PI正在开发新型引物，这些引物大多是双链的，因此不太可能导致引物-二聚体相互作用;抑制非特异性引物杂交使PCR具有显著更高的多重性。 最后，研究小组将开发一种新型的芯片上对流PCR仪器，该仪器具有集成的基于多路复用阵列的读出。重要的是，不需要PCR扩增子的开管转移，从而防止导致假阳性的污染。该研究项目的所有三个方面都获得了重要的初步数据，研究团队计划将开发的技术最初应用于非小细胞肺癌（NSCLC）患者血液样本的回顾性分析。

项目成果

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.

• DOI: 10.1038/s41551-017-0126-5
• 发表时间: 2017
• 期刊: Nature biomedical engineering
• 影响因子: 28.1
• 作者: Wu LR;Chen SX;Wu Y;Patel AA;Zhang DY
• 通讯作者: Zhang DY

Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing.

• DOI: 10.1038/s41551-021-00713-0
• 发表时间: 2021-07
• 期刊: Nature biomedical engineering
• 影响因子: 28.1

Oncogene Concatenated Enriched Amplicon Nanopore Sequencing for rapid, accurate, and affordable somatic mutation detection.

• DOI: 10.1186/s13059-021-02449-1
• 发表时间: 2021-09-06
• 期刊: Genome biology
• 影响因子: 12.3
• 作者: Thirunavukarasu D;Cheng LY;Song P;Chen SX;Borad MJ;Kwong L;James P;Turner DJ;Zhang DY
• 通讯作者: Zhang DY

A recurrent 8 bp frameshifting indel in FOXF1 defines a novel mutation hotspot associated with alveolar capillary dysplasia with misalignment of pulmonary veins.

FOXF1 中反复出现的 8 bp 移码插入缺失定义了与肺泡毛细血管发育不良伴肺静脉错位相关的新突变热点。

• DOI: 10.1002/ajmg.a.61338
• 发表时间: 2019
• 期刊: American journal of medical genetics. Part A
• 作者: Karolak,JustynaA;Bacolla,Albino;Liu,Qian;Lantz,PatrickE;Petty,John;Trapane,Pamela;Panzer,Karin;Totapally,BalagangadharR;Niu,Zhiyv;Xiao,Rui;Xie,NinaG;Wu,LuciaR;Szafranski,Przemyslaw;Zhang,DavidY;Stankiewicz,Paweł
• 通讯作者: Stankiewicz,Paweł

Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE).

• DOI: 10.1038/s41467-022-29500-4
• 发表时间: 2022-04-11
• 期刊: Nature communications
• 影响因子: 16.6