<meta charset="utf8"></head><body>使用野生型封闭 PCR 优化低频突变检测

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Y., 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=A+recurrent+inactivating+mutation+in+RHOA+GTPase+in+angioimmunoblastic+T+cell+lymphoma.">A recurrent inactivating mutation in RHOA GTPase in angioimmunoblastic T cell lymphoma.</a> Nat Genet. 46 (4), 371-375 (2014).</li><li>Lee, P. H., 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=RHOA+G17V+mutation+in+angioimmunoblastic+T-cell+lymphoma:A+potential+biomarker+for+cytological+assessment.">RHOA G17V mutation in angioimmunoblastic T-cell lymphoma:A potential biomarker for cytological assessment.</a> Exp Mol Pathol. 110, 104294 (2019).</li><li>Mamiko, S. Y., 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=Somatic+RHOA+mutation+in+angioimmunoblastic+T+cell+lymphoma.">Somatic RHOA mutation in angioimmunoblastic T cell lymphoma.</a> Nat Genet. 46 (2), 171-175 (2014).</li><li>Palomero, T., 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=Recurrent+mutations+in+epigenetic+regulators,+RHOA+and+FYN+kinase+in+peripheral+T+cell+lymphomas.">Recurrent mutations in epigenetic regulators, RHOA and FYN kinase in peripheral T cell lymphomas.</a> Nat Genet. 46 (2), 166-170 (2014).</li><li>Fujisawa, 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=Activation+of+RHOA-VAV1+signaling+in+angioimmunoblastic+T-cell+lymphoma.">Activation of RHOA-VAV1 signaling in angioimmunoblastic T-cell lymphoma.</a> Leukemia. 32 (3), 694-702 (2018).</li><li>Voena, C., 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=RHO+family+GTPases+in+the+biology+of+lymphoma.">RHO family GTPases in the biology of lymphoma.</a> Cells. 8 (7), 646 (2019).</li><li>Shendure, 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=DNA+sequencing+an+40:+past,+present+and+future.">DNA sequencing an 40: past, present and future.</a> Nature. 550 (7676), 345-353 (2017).</li><li>Athanasios, C. T., 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=Comparison+of+Sanger+sequencing,+pyrosequencing,+and+melting+curve+analysis+for+the+detection+of+KRAS+mutations:+diagnostic+and+clinical+implications.">Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.</a> J Mol Diagn. 12 (4), 425-432 (2010).</li><li>Wu, L. R., 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=Multiplexed+enrichment+of+rare+DNA+variants+via+sequence-selective+and+temperature-robust+amplification.">Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.</a> Nat Biomed Eng. 1, 714-723 (2017).</li><li>John, S. 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W., 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=General+concepts+for+PCR+primer+design.">General concepts for PCR primer design.</a> PCR Methods Appl. 3 (3), S30-S37 (1993).</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=User+Guide+for+Applied+Biosystems.">User Guide for Applied Biosystems.</a> 3730/3730xl DNA Analyzer. , (2014).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automatic DNA extractor</td>
			<td>9001301</td>
			<td>Qiagen</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA nucleic acid detector</td>
			<td>Q32854</td>
			<td>Thermo fisher</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR amplification kit</td>
			<td>P4600</td>
			<td>merck</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR instrument</td>
			<td>C1000 Touch</td>
			<td>Biorad</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase K solution</td>
			<td>D3001-2-A</td>
			<td>zymo research</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase K storage buffer</td>
			<td>D3001-2-C</td>
			<td>zymo research</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing amplification enzyme kit</td>
			<td>P7670-FIN</td>
			<td>Qiagen</td>
			<td></td>
		</tr>
	</tbody>
</table>

wild-type blocking pcr combined with sanger sequencing for detection of low-frequency somatic mutation

Minimal residual disease (MRD) is the small number of cancer cells that are still present in the body after treatment. Due to their small number, they do not lead to any physical signs or symptoms. They often go undetected by traditional methods, such as microscopic visualization and/or tracking abnormal serum proteins in the blood. An MRD positive test result indicates the presence of residual diseased cells. A negative result means that residual diseased cells are absent. Post-cancer treatment, the remaining cancer cells in the body can become active and start to multiply, causing a disease relapse. Detecting MRD is indicative that either the treatment was not completely effective or that the treatment was incomplete. Another reason for MRD-positive results after treatment might be that not all the cancer cells responded to the therapy or because the cancer cells became resistant to the medications used1.
Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphoma (PTCL) derived from T follicular helper cells2; it is the most common type of T-cell lymphoma, accounting for about 15%-20% of PTCL3. It is a group of related malignancies that affect the lymphatic system. The cell of origin is the follicular T helper cell. The 2016 WHO classification categorizes it as Angioimmunoblastic T-cell Lymphoma4. In 2022, WHO renamed it as Nodal T-follicular helper cell lymphoma, angioimmunoblastic-type (nTFHL-AI), together with Nodal T-follicular helper cell lymphoma, follicular-type (nTFHL-F) and Nodal T-follicular helper cell lymphoma, not otherwise specified (nTFHL-NOS), collectively referred to as nodular follicular helper T cell lymphoma (nTFHL). This was done to identify its important clinical and immunophenotypic features and similar T follicular helper (TFH) gene expression signatures and mutants. Genetically, nTFHL-AI is characterized by the progressive acquisition of somatic mutations in early hematopoietic stem cells through TET2 and DNMT3A mutations, while RHOA and IDH2 mutations are also present in TFH tumor cells5. Several studies have shown that RHOA G17V mutation occurs in 50%-80% of AITL patients6,7,8,9. The RHOA protein encoded by the RHOA gene is activated by guanosine triphosphate (GTP) binding and inactivated by guanosine diphosphate (GDP) binding. When activated, it can bind to a variety of effector proteins and regulate a variety of biological processes. Physiologically, RHOA mediates T cell migration and polarity, plays a role in thymocyte development, and mediates activation of pre-T cell receptor (pre-TCR) signaling10. The RHOA G17V mutation is a loss-of-function mutation that plays a driving role in lymphoma pathogenesis11. The detection of its low-frequency mutation is helpful for the MRD monitoring of AITL.
Sanger sequencing has been used for more than 40 years as the gold standard for the detection of known and unknown mutations. However, its detection limit is only 5%-20%, which limits its application for low-frequency mutation detection12,13. In Sanger sequencing, the detection sensitivity can be decreased to 0.1% by replacing the traditional PCR with BDA, a wild-blocking technology14. BDA technology mainly adds a mismatched primer complementary to the mutant type when designing conventional primers to compete with the wild type so as to achieve the purpose of amplifying the mutant type. The key to primer design is the mismatch primer and the terminal modification. At the same time, according to the structural principle of DNA, the difference between the Gibbs free energy of the two primers is between 0.8 kcal/mol and 5 kcal/mol. Another key step in this technique is to suppress wild-type amplification by adjusting the ratio of wild-type and blocking primers14,15.
At present, common low-frequency somatic mutation detection techniques include PCR-based allele-specific PCR (allele-specific polymerase chain reaction, ASPCR), amplification-refractory mutation system PCR (amplification-refractory mutation system-PCR, ARMS-PCR) used for genotyping SNP with the help of refractory primers. Designing primers for the mutant and normal alleles allows selective amplification and digital PCR (Droplet Digital PCR, ddPCR), a method for performing digital PCR that is based on water-oil emulsion droplet technology16. A sample is fractionated into 20,000 droplets, and for each droplet, a PCR amplification of the template molecules is done with a sensitivity of 1 x 10-5; blocker displacement amplification (BDA) is also a PCR-based rare allele enrichment method used for accurate detection and quantitation of SNVs and indels down to 0.01% VAF in a highly multiplexed environment; locked nucleic acid technology (non-extendable locked nucleic acid, LNA), is a class of high-affinity RNA analogs in which the ribose ring is locked in the ideal conformation for Watson-Crick binding; hotspot-specific probes (Hot-Spot-Specific Probe, HSSP), overlap the target primer sequence, include a single mutation, and are modified with a C3 spacer at the C3&#39; end to prevent amplification by qPCR14,16,17,18. When a mutation in the sequence exists, the HSSP competitively attaches to the target mutation and prevents the primer from binding to the target mutant sequence which stops sequence amplification; NGS (next-generation sequencing) - based immunoglobulin high-throughput sequencing (igHTS) Cancer Personalized Profiling by Deep Sequencing (CAAP-seq), it is a next-generation sequencing-based method used to quantify circulating DNA in cancer cells (sensitivity is 1 x 10-4); etc. Among them, most methods are based on PCR and can only detect a small number of mutation sites, and the NGS-based methods can detect multiple sites, but the cost is high, and the process is complicated14,16,17,18. There have been reports on the detection of RHOA G17V low-frequency mutations based on the qPCR, but the detection limit can only reach about 2%19. There is no report on the detection of RHOA G17V low-frequency mutation based on Sanger sequencing. Here, we demonstrate the increase in sensitivity achieved by BDA, a wild-type block PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%. Additional data for IDH2 and JAK1 is also provided.
This article provides a detailed protocol of RHOA G17V low-frequency detection scheme by Sanger sequencing and provides a reference for the development of more low-frequency mutation detection based on the Sanger sequencing platform. This method can be used to detect and monitor possible drug-resistance mutations and minimal residues in tumors.

<meta charset="utf8"></head><body>作者聚焦：推进癌症组织中低频突变的检测

野生型封闭 PCR 联合 Sanger 测序检测低频体细胞突变

We are interested in understanding low frequency somatic mutation in cancer tissues. This article introduces the application for low frequency mutation detection method based on our Sanger sequencing in angioimmunoblastic lymphoma test, providing a basis for applying this method to other disease tests. Currently there are low frequency mutation detection methods such as quantitative PCR, digital PCR, and NGS.But there's no report on detection of IGHV7-3 low frequency mutation based on Sanger sequencing, of which detection limit is only 5%to 20%Generally due to technical limitations, low frequency detection based on Sanger sequencing cannot be accurately quantified. Our technique offers high sensitivity, low cost, and strong flexibility, making it an ideal choice for low frequency mutation detection. We are planning to design pseudogene primers as well as multiplex GCR primers to explore their application in blood diseases.

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting ...

wild-type-blocking-pcr-combined-with-sanger-sequencing-for-detection

Research

JoVE Journal

Genetics

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

391 次观看.  Kindstar Global Technology Inc.. 我们对了解癌组织中的低频体细胞突变感兴趣。本文介绍了基于我们的 Sanger 测序的低频突变检测方法在血管免疫母细胞淋巴瘤检测中的应用，为该方法应用于其他疾病检测提供了依据。目前有定量 PCR、数字 PCR 和 NGS 等低频突变检测方法。但尚无基于 Sanger 测序检测 IGHV7-3 低频突变的报道，其中检出限仅为 5%-20%一般由于技术限制，基于 Sanger 测...