野生类型阻止PCR结合直接测序作为低频体细胞突变的检测的高灵敏度的方法
Summary
Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.
Abstract
治疗后评估残留病时，筛选治疗期间出现的抗性突变精确检测和低频突变的鉴定可能是有问题时，或者当患者有几个循环肿瘤细胞。野生型阻断PCR，接着测序分析提供了高灵敏度，灵活性和简单性作为用于检测这些低频突变的方法。通过加入设计锁核酸寡核苷酸到一个新的或先前建立的常规的基于PCR的测序测定法自定义，在1,000 WT等位基因背景大约1突变等位基因的灵敏度，可以实现（1：1,000）。与脱氨事件相关联的伪影的测序在福尔马林固定的石蜡包埋的组织中发现可以部分地通过使用尿嘧啶DNA糖基化酶的过程中提取步骤补救。这里的优化的协议是特定用于检测MYD88突变，但可以作为一个模板来设计任何WTB-PCR测定。的WTB-PCR测定相对于其他常用的测定法，用于检测低频突变，包括等位基因特异性PCR和实时定量PCR包括假阳性的发生较少，更大的灵活性和易于实施的优点，并同时检测已知的能力和未知的突变。
Introduction
Sanger测序传统上一直是测试已知和未知的体细胞突变的金标准。一个Sanger测序的局限性是其检测极限（〜10 -在WT的背景20％突变等位基因）1。灵敏度的这个水平是不合适的检测低的水平的体细胞突变可存在于样品从癌前组织或患者几个循环肿瘤细胞，或当骨髓（BM）是片状。这也使得评估治疗后残留病或由单独2常规测序疗法难以期间检测新兴的抗性突变。通过与锁核酸（LNA）代替传统的PCR介导的野生型在Sanger测序阻断PCR（WTB-PCR），在WT的背景高达0.1％的突变体等位基因的灵敏度，可以实现2，3，4。在（ – 14 NT〜10）阻塞（LNA）的寡核苷酸优先结合WT DNA的DNA WT从而防止扩增WTB-PCR，富集突变体等位基因是通过添加短来实现的。然后将突变体富集WTB-PCR产物进行测序。通过阻断WT DNA，而不是选择用于特定突变WTB-PCR允许存在于分钟的细胞级分的已知和未知突变富集。
多种方法目前用于在小小区的级分检测突变。这包括等位基因特异性PCR，扩增的受阻突变系统（ARMS），变性高效液相色谱（DHPLC），小珠，乳液，放大，和磁性（整经），电场诱导释放和测量（EFIRM），高分辨率熔点等。然而，这些方法大多是由假阳性，并仅检测试验的目的是为4个突变的能力有限</SUP>。 WTB-PCR，但是，允许用户可视化测序痕迹使多个突变类型的检测，并且可以在排除由于伪像或脱氨基事件误报帮助。下一代测序（NGS）可以提供一个合适的替代常规测序，但是，显着更大的成本，复杂性和较长测定时间使其对于许多疾病类型的几个不同的分子标记物或用于监测患者对治疗的新兴的不必要的选项耐药性突变。具有小于5％的突变体等位基因频率。此外，当检测到高的假阳性率变体可造成问题对于基于扩增子-NGS 5,6。
在这里，我们证明在由WTB-PCR在通过Albitar 等人描述筛选在髓样分化因子88基因的突变来实现灵敏度的增加。 3 MYD88 mutatioNS是在华氏巨球蛋白血症（WM）未知的意义（IGM-MGUS）的，IgM单克隆丙种球蛋白病，脾边缘区淋巴瘤（SMZL）重要的诊断和预后因素，和弥漫性大B细胞淋巴瘤（DLBCL）。 MYD88突变在几乎所有情况下的WM和患者的免疫球蛋白M（IgM抗体）分泌型MGUS约50％的发现。与此相反，MYD88突变仅在0-6％的患者SMZL发现是多发性骨髓瘤7，8缺席。因为重叠形态学，免疫表型，细胞遗传学，和WM和SMZL或IgM多发性骨髓瘤通常可以鉴别诊断复杂之间临床特点，一个MYD88突变的存在可作为一种有用的鉴定因子9。 MYD88突变也与较大的疾病负担在DLBCL患者和总生存期差治疗后7相关， </SUP> 10。此外，因为MYD88突变在活化的B细胞样（ABC）DLBCL比更频繁地发现生发中心B细胞样（GCB）DLBCL或原发性纵隔B细胞淋巴瘤（PMBL），MYD88突变状态可以用作替代标记为ABC亚型11,12。
这里提供的详细协议用作从该新测定法可以开发或大多数现有的测序测定法可以容易地适合于准确地检测各种样品类型低频突变的模板。该方法也可用于监测和检测可能在肿瘤中或甚至同时患者正在与靶向治疗或抗生素治疗可能发展细菌发展抗性突变。此外，它解决和补救许多与突变富集特别是在福尔马林固定石蜡包埋（FFPE）组织相关的问题。
Protocol
Representative Results
Discussion
这里所描述的WTB-PCR测定法使用与设计的延伸部（ 图1）期间阻断WT DNA的扩增阻断寡核苷酸的通用引物组。然后将WTB-PCR产物测序突变分析。 WTB-PCR /桑格的效用在于它的简单性，高灵敏度，以及高通量。使用此处描述的准则，大多数现有的基于桑格测定可简单地通过加入阻断寡核苷酸，大大提高了灵敏度的修改。在这里介绍的例子测定中添加单一阻断寡核苷酸进行PCR的增加的检测的从约…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors have no acknowledgements.
Materials
| 1.5 or 2 ml Safe-Lock microcentrifuge tubes | Eppendorf | 05-402-25 | |
| 100% alcohol | VWR | 89370-084 | Histology grade; 91.5% Ethanol, 5% Isopropyl alcohol, 4.5% Methyl alcohol |
| 3730XL sequencer | ABI | or equivalent | |
| Agencourt AMPure XP | Beckman Coulter | A63881 | For magnetic bead PCR purification |
| Aluminum sealing foils | GeneMate | T-2451-1 | For PCR and cold storage |
| BigDye Terminator v3.1 Cycle sequencing kit | Life Technologies | 4337455 | For bi-directional sequencing. With 5X Sequencing Buffer |
| Centrifuge 5804 Series | Eppendorf | A-2-DWP rotor (for PCR plate) | |
| Cold plate for 96 well plates | Eppendorf | Z606634 | |
| DNAse, RNAse-free, ultra-pure water | |||
| dNTPs (100mM) | Invitrogen | 10297-117 | |
| DynaMag-96 Side-Skirted Magnet | Thermo Fisher Scientific | 12027 | For use in PCR Purification. |
| Ethanol Absolute | Sigma | E7023 | 200 proof, for molecular biology |
| Exiqon website Oligo Tools | www.exiqon.com/oligo-tools | ||
| FastStart Taq DNA polymerase (5 U/ul) | Roche | 12032937001 | With10X concentrated PCR reaction buffer, with 20 mM MgCl2 |
| Gel electrophoresis apparatus | 2% agarose gel | ||
| GeneRead DNA FFPE extraction Kit | Qiagen | 180134 | Contains uracil DNA glycosylase necessary for reducing sequencing artifacts |
| Hi-Di Formamide | ABI | 4311320 | For sequencing. |
| LNA oligonucleotide | Exiqon | 500100 | 5'-TCAGA+AG+C+G+A+C+T+G+A+T+CC/invdT/ (+N = LNA bases) |
| M13-F Sequencing Primer | ABI | 5'-tgt aaa acg acg gcc agt | |
| M13-R Sequencing Primer | ABI | 5'-cag gaa aca gct atg acc | |
| Mastercycler Pro S Thermocycler | Eppendorf | E950030020 | |
| Microcentrifuge Model 5430 | Eppendorf | FA-45-30-11 rotor (for 1.5/2 ml microcentrifuge tubes) | |
| NanoDrop 2000 Spectrophotometer | Thermo Fisher Scientific | ||
| PCR forward primer | IDT | 5'-tgt aaa acg acg gcc agt TGC CAG GGG TAC TTA GAT GG | |
| PCR reverse primer | IDT | 5'-cag gaa aca gct atg acc GGT TGG TGT AGT CGC AGA CA | |
| PCR plates | GeneMate | T-3107-1 | |
| Pipettors | 20, 200, 1000 µl | ||
| Plate septa, 96 well | ABI | 4315933 | |
| QIAamp DNA Mini Kit | Qiagen | 51304 | For BM aspirate and peripheral blood |
| SeqScape Sortware v3.0 | ABI | 4474978 | For sequencing analysis |
| Slide basket | |||
| Sodium Acetate (3M, pH 5.2) | Sigma | S7899 | |
| Sterile filtered pipette tips | 20, 200, 1000 µl | ||
| Thermomixer C | Eppendorf | 5382000023 | |
| Vortex genie | Scientific Industries | SI-0236 | |
| Wash reservoir | ~1000 ml | ||
| Xylene | VWR | 89370-088 | Histology grade |
Referências
- Vogelstein, B., Kinzler, K. W. Digital PCR. Proc Natl Acad of Sci U S A. 96 (16), 9236-9241 (1999).
- Milbury, C. A., Li, J., Makrigiorgos, G. M. PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem. 55 (4), 632-640 (2009).
- Albitar, A., Ma, W., DeDios, I., Estella, J., Agersborg, S., Albitar, M. Positive selection and high sensitivity test for MYD88 mutations using locked nucleic acid. Int J Lab Hematol. 38 (2), 133-140 (2016).
- Dominguez, P. L., Kolodney, M. S. Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens. Oncogene. 24 (45), 6830-6834 (2005).
- Gray, P. N., Dunlop, C. L. M., Elliott, A. M. Not All Next Generation Sequencing Diagnostics are Created Equal: Understanding the Nuances of Solid Tumor Assay Design for Somatic Mutation Detection. Cancers. 7 (3), 1313-1332 (2015).
- Smith, E. N., et al. Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments. Genome Biol. 15 (8), 420 (2014).
- Varettoni, M., et al. Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenström’s macroglobulinemia and related lymphoid neoplasms. Blood. 121 (13), 2522-2528 (2013).
- Xu, L., et al. MYD88 L265P in Waldenström macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction. Blood. 121 (11), 2051-2058 (2013).
- Gertz, M. A. Waldenström macroglobulinemia: 2012 update on diagnosis, risk stratification, and management. Amer J Hematol. 87 (5), 503-510 (2012).
- Salar, A., et al. MYD88 (L265P) Mutation Confers Very Poor Response and Outcome after Second-Line Therapy in Patients with Diffuse Large B-Cell Lymphoma (DLBCL). Blood. 124 (21), 1690-1690 (2014).
- Ngo, V. N., et al. Oncogenically active MYD88 mutations in human lymphoma. Nature. 470 (7332), 115-119 (2011).
- Pasqualucci, L., et al. Analysis of the coding genome of diffuse large B-cell lymphoma. Nat Genet. 43 (9), 830-837 (2011).
- Dieffenbach, C., Lowe, T., Dveksler, G. General concepts for PCR primer design. PCR Methods Appl. 3 (3), S30-S37 (1993).
- Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. Agarose Gel Electrophoresis for the Separation of DNA Fragments. J Vis Exp. (62), e3923 (2012).
- Applied Biosystems. . User Guide for Applied Biosystems 3730/3730xl DNA Analyzer. , (2014).
- Applied Biosystems. . User Guide for SeqScape Software 3. , (2012).
- Mouritzen, P., Nielsen, A. T., Pfundheller, H. M., Choleva, Y., Kongsbak, L., Møller, S. Single nucleotide polymorphism genotyping using locked nucleic acid (LNA). Expert Rev Mol Diagn. 3 (1), 27-38 (2003).
- Quach, N., Goodman, M. F., Shibata, D. In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BMC Clin Pathol. 4 (1), (2004).
- Gallegos Ruiz, M. I., Floor, K., Rijmen, F., Grünberg, K., Rodriguez, J. A., Giaccone, G. EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen specimens. Anal Cell Pathol. 29 (3), 257-264 (2007).
- Solassol, J., et al. KRAS mutation detection in paired frozen and formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Int J Mol Sci. 12 (5), 3191-3204 (2011).
- Yost, S. E., et al. Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens. Nucleic Acids Res. 40 (14), e107-e107 (2012).
- Do, H., Wong, S. Q., Li, J., Dobrovic, A. Reducing sequence artifacts in amplicon-based massively parallel sequencing of formalin-fixed paraffin-embedded DNA by enzymatic depletion of uracil-containing templates. Clin Chem. 59 (9), 1376-1383 (2013).
- Oldenburg, R. P., Liu, M. S., Kolodney, M. S. Selective amplification of rare mutations using locked nucleic acid oligonucleotides that competitively inhibit primer binding to wild-type DNA. J Invest Dermatol. 128 (2), 398-402 (2008).
- Mancini, M., et al. Two novel methods for rapid detection and quantification of DNMT3A R882 mutations in acute myeloid leukemia. J Mol Diagn. 17 (2), 179-184 (2015).
- Parsons, B. L., Heflich, R. H. Genotypic selection methods for the direct analysis of point mutations. Mutat Res Rev Muta Res. 387 (2), 97-121 (1997).
- Liu, Q., Swiderski, P., Sommer, S. S. Truncated amplification: a method for high-fidelity template-driven nucleic acid amplification. BioTechniques. 33 (1), 129-139 (2002).
- Kaur, M., Zhang, Y., Liu, W. H., Tetradis, S., Price, B. D., Makrigiorgos, G. M. Ligation of a primer at a mutation: a method to detect low level mutations in DNA. Mutagenesis. 17 (5), 365-374 (2002).
- Albitar, A., et al. High Sensitivity Testing Shows Multiclonal Mutations in Patients with CLL Treated with BTK Inhibitor and Lack of Mutations in Ibrutinib-Naive Patients. Blood. 126 (23), 716 (2015).
