Temperature-Tolerant COLD-PCR enables mutation-enriched targeted re-sequencing

耐温 COLD-PCR 可实现突变富集的靶向重测序

• 批准号: 8591934
• 负责人: G. Mike Makrigiorgos
• 金额: $ 20万
• 依托单位: TRANSGENOMIC, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8591934
• 关键词: 2,6-Diaminopurine; Alleles; Biological Markers; Blood; Cancer Detection; Cell Line; Cells; Clinical; Clinical Oncology; Collaborations; Colonic Neoplasms; DNA; DNA Damage; DNA Fingerprinting; DNA analysis; Detection; Development; Diagnosis; Dideoxy Chain Termination DNA Sequencing; Disease; Drug resistance; Early Diagnosis; Epidermal Growth Factor Receptor; Exons; Gene Mutation; Gene Targeting; Genes; Genomics; Heterogeneity; Inosine; Ions; KRAS2 gene; Lead; Licensing; Life; Liquid substance; Lung Neoplasms; Malignant Neoplasms; Marketing; Medicine; Methods; Modification; Mutation; Neoplasm Metastasis; Noise; Nucleotides; Oncogenes; Patients; Phase; Positioning Attribute; Preparation; Process; Radio; Reaction; Reading; Resistance development; Sampling; Screening for cancer; Small Business Technology Transfer Research; Somatic Mutation; Specimen; Steam; TP53 gene; Tail; Techniques; Technology; Temperature; Time; Tube; Validation; base; cancer cell; cancer diagnosis; cancer therapy; cell transformation; clinical practice; clinically relevant; follow-up; genetic profiling; improved; meetings; mutant; next generation sequencing; novel strategies; outcome forecast; public health relevance; research study; therapy resistant; tumor

DESCRIPTION (provided by applicant): Cancers develop from the life-long accumulation of critical somatic mutations due to DNA-damaging agents that lead to cells transforming into tumor-forming cells. These low-level tumor-associated somatic DNA mutations can have profound implications for development of metastasis, prognosis, choice of treatment, follow-up or early cancer detection. Unless they are effectively detected, these low-level mutations can misinform patient management decisions or become missed opportunities for personalized medicine. Widely-used technologies such as sequencing are not sensitive enough to detect these mutations when they are at very low percentages compared to normal DNA. Likewise the next generation sequencing technologies (NGS) are promising technology advances that can effectively detect prevalent somatic mutations in targeted gene panels; however due to the limited quantity of DNA in most patient samples and the abundance of normal DNA when analyzing blood, NGS 'loses steam' and its integration with clinical practice is problematic. For mutations at an abundance of ~2-5% or below, NGS generates false positives ('noise') independent of sequencing depth; yet these are often the clinically relevant mutations causing resistance to drug treatments. Commercial sample preparation kits for targeted re-sequencing of cancer gene panels have emerged1-3, however they are uniformly unable to detect mutations below a 2% abundance level. Thus, while targeted re-sequencing provides an opportunity for integration of NGS with clinical oncology, the technology is ineffective in detecting DNA mutations in heterogeneous cancers or in circulating DNA. We intend to use COLD-PCR, a new method that enriches unknown mutation-containing sequences over wild-type, normal alleles during PCR amplification. We have been able to show sequencing of mutations down to 0.02% abundance. However in its current form this method only can be used with single amplicon per reaction, limiting its efficient combination with NGS. In this project we propose a simple and powerful modification that enables COLD-PCR to be applied on hundreds or thousands of DNA targets in a single reaction, thus enabling mutation enrichment in cancer- specific gene panels prior to NGS. This would convert the rare mutations to high abundance mutations, overcoming the 'noise' and avoid the costly need for repeated sequence reads during NGS. This method, known as temperature-tolerant-COLD-PCR (TT-COLD-PCR), will be developed into kits for cancer-specific gene panels, to magnify rare mutations in multiple DNA targets thus enabling expanded application of targeted re-sequencing for heterogeneous cancers or circulating DNA. This project meets one of the stated aims of the NCI to support the development of new methods of diagnosis for the detection, discovery and validation of biomarkers for cancer detection, diagnosis and prognosis.

描述（由申请人提供）：癌症是由于导致细胞转化为形成肿瘤细胞的细胞而导致的临界体细胞突变终身积累而发展的。这些低水平肿瘤相关的体细胞DNA突变可能对转移，预后，治疗选择，随访或早期癌症检测具有深远的影响。除非有效检测到它们，否则这些低级突变可能会误导患者管理决策或成为个性化医学的错失机会。 与正常DNA相比，当这些突变的百分比非常低时，诸如测序等广泛使用的技术不足以检测到这些突变。同样，下一代测序技术（NGS）也是有希望的技术进步，可以有效地检测靶向基因面板中普遍的体细胞突变。但是，由于大多数患者样品中DNA的数量有限，并且在分析血液时的正常DNA丰度，因此NGS“失去蒸汽”及其与临床实践的整合是有问题的。 对于大约2-5％或以下的突变，NGS会产生误报（“噪声”），而与测序深度无关。然而，这些通常是临床上相关的突变，导致对药物治疗的抗性。用于靶向重新测量癌症基因面板的商业样品制备试剂盒已经出现了1-3，但是它们统一地无法检测到低于2％丰度水平的突变。因此，尽管有针对性的重新测序为将NG与临床肿瘤学的整合提供了机会，但该技术在检测异质性癌症或循环DNA中的DNA突变方面无效。 我们打算使用Cold-PCR，这是一种新方法，可在PCR扩增过程中富含野生型，正常等位基因的未知突变序列。我们已经能够显示出降低到0.02％丰度的突变测序。但是，在当前形式中，该方法只能与每个反应的单个扩增子一起使用，从而限制了其与NGS的有效组合。 在这个项目中，我们提出了一种简单而强大的修改，使Cold-PCR能够在单个反应中应用于数百或数千个DNA靶标上，从​​而在NGS之前可以在癌症特异性基因面板中富集突变。这将使罕见的突变转换为高丰度突变，克服“噪声”，并避免在NGS期间重复序列读取的昂贵需求。该方法称为温度耐温-Cold-PCR（TT-Cold-PCR），将开发到用于癌症特异性基因面板的试剂盒中，以放大多个DNA靶标的罕见突变，从而实现靶向重新序列的异质性癌症或循环DNA的靶向重新应用。 该项目符合NCI的一个既定目标，以支持开发新的诊断方法，以检测，发现和验证生物标志物用于癌症检测，诊断和预后。