This research was completed with the financial support of Kindstar Global Corporation and the help of the leaders of the Molecular Biology Laboratory and related colleagues. Thanks to the company, leaders, and relevant colleagues for their support and help. This article is only used for scientific research and does not constitute any commercial activity.

This study was approved by the medical ethics review committee of Yongzhou Central Hospital (approval number: 2024022601). The participants provided informed consent.
1. Primer design
<ol>
	<li>Conventional primer design: Design primers according to the reported primer design rules20, primer design by NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). For the mutation site of RHOA G17V, all reference sequ...

Optimizing Low-Frequency Mutation Detection with Wild-Type Blocking PCR

<ol>
	<li>Penn Medicine. Testing for measurable/minimal residual disease (MRD) Available from: <a target="_blank" href="https://www.oncolink.org/cancer-treatment/procedures-diagnostic-tests/blood-tests-tumordiagnostic-tests/testing-for-measurable-minimal-residualdisease-mrd">https://www.oncolink.org/cancer-treatment/procedures-diagnostic-tests/blood-tests-tumordiagnostic-tests/testing-for-measurable-minimal-residualdisease-mrd</a> (2019)</li><li>Xie, Y., Elaine, S. 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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. 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=The+thermodynamics+of+DNA+structural+motifs.">The thermodynamics of DNA structural motifs.</a> Annu Rev Biophys Biomol Struct. 33, 415-440 (2004).</li><li>Coren, A. 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=PCR-based+methods+for+the+enrichment+of+minority+alleles+and+mutations.">PCR-based methods for the enrichment of minority alleles and mutations.</a> Clin Chem. 55 (4), 632-640 (2009).</li><li>Eliza, M. 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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=Detection+of+the+G17V+RHOA+mutation+in+angioimmunoblastic+T-cell+lymphoma+and+related+lymphomas+using+quantitative+allele-specific+PCR.">Detection of the G17V RHOA mutation in angioimmunoblastic T-cell lymphoma and related lymphomas using quantitative allele-specific PCR.</a> PLoS One. 9 (10), e109714 (2014).</li><li>Dieffenbach, C. 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>

The authors have nothing to disclose.

The WTB-PCR based on BDA technology, described in this article, introduces a mismatched primer complementary to the mutant type when designing conventional primers to compete with the wild type, suppress the wild type, and amplify the mutant product. Then, the WTB-PCR products were sequenced for mutation analysis. The utility of WTB-PCR/Sanger is its simplicity and high sensitivity. According to the detection scheme established in this paper, most existing Sanger-based assays can be added with suppressor primers via BDA,...

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.

<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

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues

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

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.

Compare the test sample&#39;s sequence with the reference sequence to obtain the test sample&#39;s mutation status. BDA-based WBT-PCR technology can detect the known RHOA G17V mutation and other low-frequency mutations in the amplification interval of upstream and downstream primers. See Figure 1. Two additional genes, namely IDH2 and JAK1, were also analyzed using this method, Figure 2 and Figure 3, respectively. ...

This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other 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

DNA Extraction from Cancer Tissues for the Detection of Low-Frequency Mutations Using Blocker Displacement Amplification Technology

To begin, prepare all the reagents required for tissue sample preparation and DNA extraction. Add 500 microliters of deep paraffin buffer, and 20 microliters of proteinase K solution to about 30 milligrams of tissue in a micro centrifuge tube. Vortex the micro centrifuge tube for at least 10 seconds and place it in a dry thermostat set at 55 degrees Celsius for 90 minutes.After incubation, transfer the sample including liquid and tissue to the spin column that is placed in a filter tube. Centrifuge at 16, 500 G for five minutes to separate the liquid into the filter tube. Then, transfer the liquid from the filter tube to a labeled sample tube, and perform DNA extraction according to the manufacturer's instructions.Measure the concentration and purity of the extracted DNA.

PCR Amplification for Sensitive Detection of Tumor Mutations in the Extracted Cancer DNA Samples

To begin extract the DNA from formal and fixed paraffin embedded tissue samples. Then centrifuge the primer dry powder at 5, 400 to 8, 400 G for two to three minutes, and add the appropriate volume of double distilled water along the wall of the primer tube. Add five microliters each of the forward and the reverse primer to 50 microliters of wild type primer stock, and 40 microliters of double distilled water.After mixing two to three times, vortex the tube and centrifuge it briefly. For the polymerase chain reaction or PCR procedure, add the required reagents to each reaction tube. Vortex to mix and centrifuge briefly.Place the PCR tube in the PCR instrument for amplification. Set the thermal cycle program and run it. Then perform gel electrophoresis on the PCR product using 2%Agarose to check whether the target fragment is amplified.To purify the PCR products, briefly centrifuge the purificase one and purificase two that have been brought to room temperature and prepare the purification reaction mixture. Place the PCR tube in the PCR instrument and run the machine with the preset parameters.

Blocker Displacement Amplification-Enhanced Sanger Sequencing for Low-Frequency Mutation Detection in Cancer Tissues

To begin, perform polymerase chain reaction, or PCR, to amplify the desired region of the tissue-extracted DNA. Bring the sequencing amplification enzyme master mix, buffer, and double-distilled water to room temperature and spin off instantly. After preparing the tubes for sequencing, place the PCR tube on the preset PCR instrument for amplification.Then remove the product from the PCR instrument and store the reaction products in the refrigerator, protected from light. Vortex the magnetic beads thoroughly. Add 10 microliters of magnetic beads and 40 microliters of 80%ethanol to the 96-well plate and seal the plate with a film.Place the 96-well plate on a vortex shaker for 10 seconds to fully suspend and mix the beads. Then secure the plate with a rubber band on a horizontal oscillator and vibrate at the maximum setting for three to five minutes. Next, place the PCR plate on a magnetic stand, ensuring it is firmly pressed.Keep the stand at four degrees Celsius for three minutes for static adsorption. Once the beads are adsorbed, remove the ceiling film and pour out the waste liquid. Rinse the beads twice with 40 microliters of 80%ethanol.After pouring off the waste liquid, place the plate on absorbent paper and gently pat it dry. Place the absorbent paper with the magnetic plate into a centrifuge and spin at 120 g briefly. After removing the alcohol, place the plate in an oven at 60 degrees Celsius for three to five minutes to dry the beads completely.Next, add 40 microliters of pure water to the plate and seal it with a film. Shake it on a vortex shaker for three to five seconds and place the plate on a horizontal shaker. Secure it and let the plate shake for three to five minutes to dissolve the purified sequencing product.Finally, spin the dissolved sequencing product at 180 g and use the product for genomic analysis using capillary electrophoresis. This method could detect the known RHOA G17V mutation and other low-frequency mutations in the amplification interval of upstream and downstream primers. Mutations in two additional genes, namely, IDH2 and JAK1, were also correctly analyzed using this method.

320 Views.  Kindstar Global Technology Inc..  This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.

Optimizing Low-Frequency Mutation Detection with Wild-Type Blocking PCR

Summary