Name: wild-type blocking pcr combined with direct sequencing as a highly sensitive method for detection of low-frequency somatic mutations
Uploaded: 2017-03-29T15:00:00
Description: Watch this Scientific Journal Video about Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations at JoVE.com

The authors have no acknowledgements.

伦理声明：获得机构审查委员会（IRB）的批准后进行人体样品所有测试。 1. DNA的提取FFPE组织，外周血和骨髓吸<ol><li>对于DNA FFPE提取试剂盒骨髓FFPE组织<ol><li>与来自未染色的载玻片FFPE组织开始（5 - 5 10部分 - 10微米厚）。 注意：如果开始与组织屑，使用3 - 6个切片在5 - 10微米厚，并跳到步骤1.1.6。 </li><li>放置在滑动的滑动篮和制备四种洗涤储层（两个用于二甲苯和...

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenically+active+MYD88+mutations+in+human+lymphoma.">Oncogenically active MYD88 mutations in human lymphoma.</a> Nature. 470 (7332), 115-119 (2011).</li><li>Pasqualucci, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+coding+genome+of+diffuse+large+B-cell+lymphoma.">Analysis of the coding genome of diffuse large B-cell lymphoma.</a> Nat Genet. 43 (9), 830-837 (2011).</li><li>Dieffenbach, C., Lowe, T., Dveksler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+concepts+for+PCR+primer+design.">General concepts for PCR primer design.</a> PCR Methods Appl. 3 (3), S30-S37 (1993).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Applied Biosystems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> User Guide for Applied Biosystems 3730/3730xl DNA Analyzer. , (2014).</li><li>Applied Biosystems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> User Guide for SeqScape Software 3. , (2012).</li><li>Mouritzen, P., Nielsen, A. T., Pfundheller, H. M., Choleva, Y., Kongsbak, L., M&#248;ller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphism+genotyping+using+locked+nucleic+acid+(LNA).">Single nucleotide polymorphism genotyping using locked nucleic acid (LNA).</a> Expert Rev Mol Diagn. 3 (1), 27-38 (2003).</li><li>Quach, N., Goodman, M. F., Shibata, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+mutation+artifacts+after+formalin+fixation+and+error+prone+translesion+synthesis+during+PCR.">In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR.</a> BMC Clin Pathol. 4 (1), (2004).</li><li>Gallegos Ruiz, M. I., Floor, K., Rijmen, F., Gr&#252;nberg, K., Rodriguez, J. A., Giaccone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+and+K-ras+mutation+analysis+in+non-small+cell+lung+cancer:+comparison+of+paraffin+embedded+versus+frozen+specimens.">EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen specimens.</a> Anal Cell Pathol. 29 (3), 257-264 (2007).</li><li>Solassol, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KRAS+mutation+detection+in+paired+frozen+and+formalin-fixed+paraffin-embedded+(FFPE)+colorectal+cancer+tissues.">KRAS mutation detection in paired frozen and formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues.</a> Int J Mol Sci. 12 (5), 3191-3204 (2011).</li><li>Yost, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+high-confidence+somatic+mutations+in+whole+genome+sequence+of+formalin-fixed+breast+cancer+specimens.">Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens.</a> Nucleic Acids Res. 40 (14), e107-e107 (2012).</li><li>Do, H., Wong, S. Q., Li, J., Dobrovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+sequence+artifacts+in+amplicon-based+massively+parallel+sequencing+of+formalin-fixed+paraffin-embedded+DNA+by+enzymatic+depletion+of+uracil-containing+templates.">Reducing sequence artifacts in amplicon-based massively parallel sequencing of formalin-fixed paraffin-embedded DNA by enzymatic depletion of uracil-containing templates.</a> Clin Chem. 59 (9), 1376-1383 (2013).</li><li>Oldenburg, R. P., Liu, M. S., Kolodney, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+amplification+of+rare+mutations+using+locked+nucleic+acid+oligonucleotides+that+competitively+inhibit+primer+binding+to+wild-type+DNA.">Selective amplification of rare mutations using locked nucleic acid oligonucleotides that competitively inhibit primer binding to wild-type DNA.</a> J Invest Dermatol. 128 (2), 398-402 (2008).</li><li>Mancini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+novel+methods+for+rapid+detection+and+quantification+of+DNMT3A+R882+mutations+in+acute+myeloid+leukemia.">Two novel methods for rapid detection and quantification of DNMT3A R882 mutations in acute myeloid leukemia.</a> J Mol Diagn. 17 (2), 179-184 (2015).</li><li>Parsons, B. L., Heflich, R. 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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...

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&gt;。 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&gt; 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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 or 2 ml Safe-Lock microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>05-402-25</td>
			<td></td>
		</tr>
		<tr>
			<td>100% alcohol</td>
			<td>VWR</td>
			<td>89370-084</td>
			<td>Histology grade; 91.5% Ethanol, 5% Isopropyl alcohol, 4.5% Methyl alcohol</td>
		</tr>
		<tr>
			<td>3730XL sequencer</td>
			<td>ABI</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>For magnetic bead PCR purification</td>
		</tr>
		<tr>
			<td>Aluminum sealing foils</td>
			<td>GeneMate</td>
			<td>T-2451-1</td>
			<td>For PCR and cold storage</td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1 Cycle sequencing kit</td>
			<td>Life Technologies</td>
			<td>4337455</td>
			<td>For bi-directional sequencing. With 5X Sequencing Buffer</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 Series</td>
			<td>Eppendorf</td>
			<td></td>
			<td>A-2-DWP rotor (for PCR plate)</td>
		</tr>
		<tr>
			<td>Cold plate for 96 well plates</td>
			<td>Eppendorf</td>
			<td>Z606634</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse, RNAse-free, ultra-pure water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (100mM)</td>
			<td>Invitrogen</td>
			<td>10297-117</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-96 Side-Skirted Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td>12027</td>
			<td>For use in PCR Purification.</td>
		</tr>
		<tr>
			<td>Ethanol Absolute&#160;</td>
			<td>Sigma</td>
			<td>E7023</td>
			<td>200 proof, for molecular biology</td>
		</tr>
		<tr>
			<td>Exiqon website Oligo Tools</td>
			<td></td>
			<td></td>
			<td><a href="http://www.exiqon.com/oligo-tools">www.exiqon.com/oligo-tools</a></td>
		</tr>
		<tr>
			<td>FastStart Taq DNA polymerase (5 U/ul)</td>
			<td>Roche</td>
			<td>12032937001</td>
			<td>With10X concentrated PCR reaction buffer, with 20 mM MgCl2&#160;</td>
		</tr>
		<tr>
			<td>Gel electrophoresis apparatus</td>
			<td></td>
			<td></td>
			<td>2% agarose gel</td>
		</tr>
		<tr>
			<td>GeneRead DNA FFPE extraction Kit&#160;</td>
			<td>Qiagen</td>
			<td>180134</td>
			<td>Contains uracil DNA glycosylase necessary for reducing sequencing artifacts</td>
		</tr>
		<tr>
			<td>Hi-Di Formamide</td>
			<td>ABI</td>
			<td>4311320</td>
			<td>For sequencing.</td>
		</tr>
		<tr>
			<td>LNA oligonucleotide</td>
			<td>Exiqon</td>
			<td>500100</td>
			<td>5&#39;-TCAGA+AG+C+G+A+C+T+G+A+T+CC/invdT/ (+N = LNA bases)</td>
		</tr>
		<tr>
			<td>M13-F Sequencing Primer</td>
			<td>ABI</td>
			<td></td>
			<td>5&#39;-tgt aaa acg acg gcc agt</td>
		</tr>
		<tr>
			<td>M13-R Sequencing Primer</td>
			<td>ABI</td>
			<td></td>
			<td>5&#39;-cag gaa aca gct atg acc</td>
		</tr>
		<tr>
			<td>Mastercycler Pro S Thermocycler</td>
			<td>Eppendorf</td>
			<td>E950030020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Model 5430</td>
			<td>Eppendorf</td>
			<td></td>
			<td>FA-45-30-11 rotor (for 1.5/2 ml microcentrifuge tubes)</td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR forward primer</td>
			<td>IDT</td>
			<td></td>
			<td>5&#39;-tgt aaa acg acg gcc agt TGC CAG GGG TAC TTA GAT GG</td>
		</tr>
		<tr>
			<td>PCR reverse primer</td>
			<td>IDT</td>
			<td></td>
			<td>5&#39;-cag gaa aca gct atg acc GGT TGG TGT AGT CGC AGA CA</td>
		</tr>
		<tr>
			<td>PCR plates</td>
			<td>GeneMate</td>
			<td>T-3107-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors</td>
			<td></td>
			<td></td>
			<td>20, 200, 1000 &#181;l</td>
		</tr>
		<tr>
			<td>Plate septa, 96 well</td>
			<td>ABI</td>
			<td>4315933</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>Qiagen</td>
			<td>51304</td>
			<td>For BM aspirate and peripheral blood</td>
		</tr>
		<tr>
			<td>SeqScape Sortware v3.0</td>
			<td>ABI</td>
			<td>4474978</td>
			<td>For sequencing analysis</td>
		</tr>
		<tr>
			<td>Slide basket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate (3M, pH 5.2)&#160;</td>
			<td>Sigma</td>
			<td>S7899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile filtered pipette tips&#160;</td>
			<td></td>
			<td></td>
			<td>20, 200, 1000 &#181;l</td>
		</tr>
		<tr>
			<td>Thermomixer C&#160;</td>
			<td>Eppendorf</td>
			<td>5382000023</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex genie</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash reservoir</td>
			<td></td>
			<td></td>
			<td>~1000 ml</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>89370-088</td>
			<td>Histology grade</td>
		</tr>
	</tbody>
</table>

wild-type blocking pcr combined with direct sequencing as a highly sensitive method for detection of low-frequency somatic mutations

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。

延伸期间WTB-PCR的概念的概述示于图1。因为在阻滞剂-DNA杂交的单个核苷酸错配大幅降低其熔化温度（ΔTM = 20 - 30℃）中，WT等位基因的扩增而突变体的模板DNA是免费完成延伸部17被阻断。以这种方式，而WT DNA线性扩增的突变的DNA指数扩增。 由WTB-PCR获得的突变体富集的示范显示在图2。...

Watch this Scientific Journal Video about Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations at JoVE.com

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

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.

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

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

The overall goal of this procedure is to detect low frequency somatic mutations in a variety of sample types. This methodology can help answer key questions in somatic mutation testing by facilitating the detection of very low frequency mutations. Here we will look for mutations in the myd88 gene in bone marrow samples.The main advantage of this technique is that it provides highly accurate and sensitive information about the presence of somatic mutations, even with very few neoplastic cells. This wild type blocking PCR based sequencing assay was developed to amplify part of exon five in the myd88 gene, ensuring coverage of the L265 hotspot. The forward and reverse primers were designed with a five prime M13 sequence to allow for a kneeling of complimentary sequencing primers.Design the blocking oligonucleotide to be approximately 10 to 15 bases in length and complimentary to the wild type template where mutant enrichment is desired. A shorter oligo will improve mismatch discrimination. To achieve high target specificity it's important not to use too much of the blocking nucleotides, as this will result in very sticky oligonucleotides.To design the blocking oligonucleotide begin by navigating to the Oligo Tools website. Select the Oligo TM prediction tool. A new window will open up.Paste the sequence of the wild type template to be blocked into the oligo sequence box. Add a plus sign in front of the blocker bases to mark them. Click on the calculate button to determine the approximate TM of the DNA blocker hybrid.The calculated melting temperatures will appear in the boxes below. Design the blocking oligo to have a melting temperature 10 to 15 degrees Celsius above the extension temperature during thermocycling. Here, the extension temperature is 72 degrees Celsius.To adjust the melting temperature, add, remove, or substitute blocking bases. Avoid long stretches of three to four blocking C or G bases. Next, to avoid secondary structure formation or self dimerization go back to the Oligo Tools website home screen and select the Oligo Optimizer tool.A new window will open. Paste the sequence of the wild type template to be blocked into the box. Add a plus sign to indicate blocking bases.Select the two boxes for secondary structure and self only and press the analyze button to see the scores for hybridization and secondary structure. These scores represent very rough estimates of the melting temperatures of the self dimers and secondary structures respectively. Lower scores are optimal and can be achieved by limiting blocker blocker pairing.Remove or reposition blocking nucleotides in order to achieve lower scores. The optimized blocking oligo nucleotide for myd88 shown here strikes a balance between the DNA blocker hybrid melting temperature and low enough hybridization and secondary structure scores. It was designed to cover amino acids Q262 to I266, and features a three prime inverted DT to inhibit both extension by DNA polymerase and degradation by three prime exonuclease.Once the primers have been designed set up the wild type blocking PCR and perform thermocycling as described in the accompanying document. Remove magnetic beads from storage at four degrees Celsius and bring them to room temperature. Transfer 10 microliters of the PCR product to a new PCR plate.Vortex the magnetic beads vigorously to fully resuspend the magnetic particles and then add 18 microliters of magnetic beads to each well on the new plate. Pipette up and down 10 times to mix. Then incubate the plate at room temperature for five minutes.Following the incubation, place the PCR plate onto the side skirted magnet plate for two minutes to separate the beads from the solution. Use a multi channel pipette to aspirate the supernatant. Take care to avoid the bead pellet.Next, dispense 150 microliters of 70%ethanol into each well and incubate the plate at room temperature for at least 30 seconds. Then aspirate the ethanol with a multichannel pipette and discard the tips. Repeat this wash procedure once more.Using a 20 microliter multichannel pipette aspirate the remaining ethanol from each well and discard the tips. After allowing about 10 minutes for the wells of the plate to dry remove it from the magnet and add 40 microliters of nuclease free water to each well. Pipette up and down 15 times to mix.Then incubate at room temperature for two minutes. After incubating, place the PCR plate back onto the magnet plate for one minute to separate the beads from solution. Transfer 35 microliters of purified product to a new PCR plate, and perform bidirectional sequencing as described in the accompanying document.Once bidirectional sequencing has been performed to purify the sequencing products prepare a fresh one in 25 solution of three molar sodium acetate at PH 5.2 and 100%ethanol. Also prepare a fresh solution of 70%ethanol. To each well of both the forward and reverse sequencing plates add 30 microliters of sodium acetate in 100%ethanol and pipette up and down five times to mix.Then reseal the plate, and incubate in the dark at room temperature for 20 minutes. After 20 minutes have passed, centrifuge the plate at 2, 250 times G for 15 minutes. After the spin, remove the plate sealer, and invert the plate once over a waste container.If the plate is inverted multiple times the pellets may loosen from the well bottoms. Place the inverted plate on a clean paper towel, and centrifuge at 150 times G for one minute. Next, add 150 microliters of 70%ethanol to each well and reseal the plate.Spin at 2, 250 times G for five minutes. Then repeat the process of removing the plate sealer and inverting the plate. If the wells are not completely dry, allow them to air dry at room temperature.Make sure the samples are protected from light. Once the wells are completely dry add 10 microliters of formamide to each well and pipette up and down 10 times to mix. Reseal the plate.Denature using a thermo cycler at 95 degrees Celsius for three minutes followed by four degrees Celsius for five minutes. After denaturing, replace the plate sealer with a septa and sequence on a sequencing platform according to the manufacturer's instructions. Using sequence analysis software, visualize the traces, and align the sequences to the appropriate reference sequences.Align myd88 to NCBI reference sequence NM002468. Genomic DNA from patients with and without mutations underwent both conventional and wild type blocking PCR and the resulting PCR products were then sequenced. As can be seen here, the mutant allele was enriched when wild type blocking PCR was performed, and false positives were not seen in wild type DNA.A characteristic drop off in signal intensity as shown here is often seen if too high a concentration of blocker is used, or if post PCR purification failed to remove blocker prior to bidirectional sequencing. This occurs when enzymatic purification is performed in place of magnetic bead purification. Any PCR based assay that enriches for mutant alleles will detect low frequency artifacts.These traces show an increase in CG sequencing artifacts, relative to TA artifacts in FFPE tissue when cytosine or methylated cytosine are deaminated via formal infixation to uracil or thiamine respectively. Uracil DNA glycosylase or UDG can excise uracil prior to wild type blocking PCR, helping to reduce sequencing artifacts. However, thiamine resulting from deaminated five methyl cytosine which frequently occurs at CPG islands cannot be excised by UDG.Decreasing the concentration of blocker used in wild type blocking PCR may help to reduce the occurrence of sequencing artifacts that are not remedied by UDG treatment. After watching this video, you should have a good understanding of how to test somatic mutations with a high degree of accuracy and sensitivity, using the wild type blocking technique. The principle of this technology can be applied to the detection of mutations in small sub populations of cells.Therefore, it has utility in detecting minimal residual disease, monitoring patients, and predicting early relapse in patients with various neoplasms.

Research

JoVE Journal

Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

科研

遗传学

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

通过高分辨率熔融在水稻种群中识别突变

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

以乌苏图病毒为例，验证全基因组纳米孔测序

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for ...