Name: genetic variant detection in the calr gene using high resolution melting analysis
Uploaded: 2020-08-26T13:00:00
Description: Watch this Scientific Journal Video about Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis at JoVE.com

著者らは、大学医療センターリュブリャナの内科、血液学部門、専門血液学研究所の学術専門家と従業員全員に感謝したいと考えています。

この研究は、スロベニア共和国の医療倫理委員会によって承認されました。すべての手順はヘルシンキ宣言に従っていました。
1. 蛍光ベースの定量リアルタイム PCR (qPCR) および HRM による qPCR 後分析
<ol><li>材料表に記載されているプライマーを無菌、RNaseおよびDNaseフリーH2Oで100 μMに再懸濁します(材料表を参照)。10 μMの作業濃度プライ...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+melting+analysis+for+gene+scanning.">High resolution melting analysis for gene scanning.</a> Methods. 50, 250-261 (2010).</li><li>Bilbao-Sieyro, 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=High+Resolution+Melting+Analysis:+A+Rapid+and+Accurate+Method+to+Detect+CALR+Mutations.">High Resolution Melting Analysis: A Rapid and Accurate Method to Detect CALR Mutations.</a> PLoS One. 9, 103511 (2014).</li><li>S&#322;omka, M., Sobalska-Kwapis, M., Wachulec, M., Bartosz, G., Strapagiel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+melting+(HRM)+for+high-throughput+genotyping-limitations+and+caveats+in+practical+case+studies.">High resolution melting (HRM) for high-throughput genotyping-limitations and caveats in practical case studies.</a> International Journal of Molecular Sciences. 18, 2316 (2017).</li><li>Li, X., 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+three+common+DNA+concentration+measurement+methods.">Comparison of three common DNA concentration measurement methods.</a> Analytical Biochemistry. 451, 18-24 (2014).</li><li>Voytas, D. <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.">Agarose Gel Electrophoresis.</a> Current Protocols in Immunology. 2, (1992).</li><li>Lee, P. 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DNAの高解像度融解は、遺伝子型入力および遺伝的変異体走査14のための簡単なソリューションである。それは、融解曲線の形状を変化させるヘテロ二重鎖をもたらすシーケンスの違いに依存する。多様な頻度を有する異なる種類の遺伝的変異体は、癌1、2、15、16、10の特定のグループに特異的な遺伝子で観?...

カレチクリン遺伝子の体細胞遺伝学的変異体(CALR)は、2013年に骨髄増殖性新生物(MPN)の患者(必須血小板血症および原発性骨髄線維症1,2)で認識された。それ以来、CALR遺伝子の50以上の遺伝的変異体が発見され、+1(−1+2)フレームシフト3を誘導している。2つの最も頻繁なCALR遺伝的変異体は、タイプ1突然変異とも呼ばれる52bp欠失(NM_004343.3(CALR):c.1099_1150del52 p.(leu367Thrfs*46))、および5bp挿入(NM_004343.3(CALR):c.1154_1155insTTGTC、p.(Lys385Asnfs*47))、タイプ2突然変異とも呼ばれる。これら2つの遺伝的変異体は、全CALR遺伝的変異体の80%を表す。他のものは、野生型CALR4に近いαらせんの保存に基づくアルゴリズムを使用して、タイプ1-likeまたはタイプ2に分類されています。ここでは、CALR遺伝子変異体検出のための高感度かつ迅速な方法の1つ、高分解能融解解析法(HRM)を紹介する。この方法は、CALR突然変異5の大部分を表す1型および2型遺伝的変異体の迅速な検出を可能にする。HRMは、因子Vライデン6の変異を検出するためのツールとして1997年にリアルタイム»ポリメラーゼ連鎖反応«(qPCR)と組み合わせて導入されました。黄金の標準技術を表すサンガーシーケンシングと比較して、HRMはより敏感で、より特異的でない方法5です。HRM法は、多くのサンプルを迅速に分析し、費用便益5を実現する優れたスクリーニング方法である。蛍光色素の存在下で行う簡単なPCR法であり、特定のスキルを必要としません。もう一つの利点は、手順自体がHRM手順7の後に電気泳動またはサンガーシーケンシングのためにサンプルを再利用することを可能にする分析されたサンプルを損傷または破壊しないことである。唯一の欠点は、結果を解釈することが困難な場合もあるということです。さらに、HRMは、非タイプ1またはタイプ2突然変異8を有する患者において正確な突然変異を検出しない。これらの患者では、サンガーシーケンシングを行う必要があります(図1)。
HRMは、飽和DNA蛍光色素の存在下で特異的DNA領域の増幅に基づいており、これは二本鎖DNA(dsDNA)に組み込まれる。蛍光色素は、dsDNAに組み込まれると発光します。徐々に温度が上昇した後、dsDNAは一本鎖DNAに分解し、蛍光強度の急激な低下として融解曲線上で検出することができる。融解曲線の形状は、変異を検出するために使用されるDNA配列に依存する。試料の融解曲線は既知の突然変異または野生型CALRの融解曲線と比較される。明確な融解曲線は、タイプ 1 またはタイプ 29以外の異なる突然変異を表します。
HRMによる CALR 遺伝子における体細胞遺伝学的変異体検出のためのアルゴリズムとしては、アガロースゲル電気泳動およびシーケンシング法(図1)が使用され、10以前に発表された遡及研究で検証された。

<table>
	<tbody>
		<tr>
			<td>E-Gel EX 4% Agarose</td>
			<td>Invitrogen, Thermo Fischer Scientific</td>
			<td>G401004</td>
			<td></td>
		</tr>
		<tr>
			<td>Fuorometer 3.0 QUBIT</td>
			<td>Invitrogen, Thermo Fischer Scientific</td>
			<td>Q33216</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen E-Gel iBase and E-Gel Safe Imager Combo Kit</td>
			<td>Invitrogen, Thermo Fischer Scientific</td>
			<td>G6465EU</td>
			<td></td>
		</tr>
		<tr>
			<td>MeltDoctor HRM MasterMix 2X</td>
			<td>Applied Biosystem, Thermo Fischer Scientific</td>
			<td>4415440</td>
			<td>Components: AmpliTaqGold 360 DNA Polymerase, MeltDoctor trade HRM dye, dNTP blend including dUTP, Magnesium salts and other buffer components, precisely formulated to obtain optimal HRM results</td>
		</tr>
		<tr>
			<td>MicroAmp Fast 96-well Reaction Plate (0.1 mL)</td>
			<td>Applied Biosystems, Thermo Fischer Scientific</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Optical adhesive film</td>
			<td>Applied Biosystems, Thermo Fischer Scientific</td>
			<td>4311971</td>
			<td></td>
		</tr>
		<tr>
			<td>NuGenius</td>
			<td>Syngene</td>
			<td>NG-1045</td>
			<td>Gel documentation systems</td>
		</tr>
		<tr>
			<td>Primer CALRex9 Forward</td>
			<td>Eurofins Genomics</td>
			<td></td>
			<td>Sequence: 5&#39;GGCAAGGCCCTGAGGTGT&#39;3 (High-Purity, Salt-Free)</td>
		</tr>
		<tr>
			<td>Primer CALRex9 Reverse</td>
			<td>Eurofins Genomics</td>
			<td></td>
			<td>Sequence: 5&#39;GGCCTCAGTCCAGCCCTG&#39;3 (High-Purity, Salt-Free)</td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>QIAGEN</td>
			<td>51306</td>
			<td>DNA isolation kit with the buffer for DNA dilution.</td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Invitrogen, Thermo Fischer Scientific</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>QUBIT dsDNA HS assay</td>
			<td>Invitrogen, Thermo Fischer Scientific</td>
			<td>Q32854</td>
			<td></td>
		</tr>
		<tr>
			<td>Trackit 100bp DNA Ladder</td>
			<td>Invitrogen, Thermo Fischer Scientific</td>
			<td>10488058</td>
			<td>Ladder consists of 13 individual fragments with the reference bands at 2000, 1500, and 600 bp.</td>
		</tr>
		<tr>
			<td>ViiA7 Real-Time PCR System</td>
			<td>Applied Biosystems, Thermo Fischer Scientific</td>
			<td>4453534</td>
			<td></td>
		</tr>
		<tr>
			<td>Water nuclease free</td>
			<td>VWR, Life Science</td>
			<td>436912C</td>
			<td>RNase, DNase and Protease free water</td>
		</tr>
	</tbody>
</table>

genetic variant detection in the calr gene using high resolution melting analysis

高分解能溶融解析(HRM)は、遺伝子型入力や遺伝子変異スキャンのための強力な方法です。ほとんどのHRMアプリケーションは、配列の違いを検出する飽和DNA色素と、融解曲線の形状を変化させるヘテロ二重鎖に依存しています。優れた装置分解能および特殊なデータ解析ソフトウェアは、変異体または遺伝子型を識別する小さな融解曲線の違いを識別するために必要とされます。さまざまな頻度を有する異なる種類の遺伝的変異体は、特定の疾患を有する患者、特に癌、およびフィラデルフィア染色体-陰性骨髄増殖性新生物の患者における CALR 遺伝子に特異的な遺伝子で観察することができる。関心のある遺伝子における単一ヌクレオチドの変化、挿入および/または欠失(インデル)は、HRM分析によって検出され得る。異なる種類の遺伝的変異体の同定は、主にqPCR HRMアッセイで使用されるコントロールに基づいています。しかし、製品の長さが長くなるにつれて、野生型とヘテロ接合曲線の差が小さくなり、遺伝的変異体の種類を特定することがより困難になります。したがって、インデルが目的の遺伝子に期待される一般的な遺伝的変異体である場合、アガロースゲル電気泳動などの追加の方法をHRM結果の解明に使用することができる。場合によっては、決定的な結果を、標準のサンガーシーケンシングによって再チェック/再診断する必要があります。本回顧研究では、MPNを有する JAK2 V617F陰性患者にこの方法を適用した。

循環15と35の間の閾値を超える蛍光の指数関数的増加と、すべての複製されたサンプルおよびコントロールにおける定量サイクルの非常に狭い値(Cq)を有する目的のDNA領域の増幅に成功した(図2)は、HRM分析による遺伝的変異体の確実な同定のための前提条件である。これは、qPCR HRM実験で蛍光染色と同量のDNAを正確に測定することによって達成される(ステップ1.2参照)。 <st...

Watch this Scientific Journal Video about Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis at JoVE.com

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

高分解能溶融解析(HRM)は、遺伝的変異体検出のための敏感で迅速なソリューションです。それは、ヘテロ二重鎖が融解曲線の形状を変化させる結果となるシーケンスの違いに依存する。HRMとアガロースゲル電気泳動を組み合わせて、インデルなどの異なる種類の遺伝的変異体を同定することができる。

高分解能融解解析を用いた CALR 遺伝子の遺伝的変異体検出

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA ...

This method accurately defines calreticulin mutations in patients with suspected hematologic condition, especially where other diagnostic options are limited or there is an overlap with non-hematologic conditions. The main advantage of the high resolution mirror technique is detecting the common somatic genetic variants in the hotspot region of the calreticulin gene at the acceptable limit of detection and affordable cost. This technique enables rapid and noninvasive diagnosis of myeloproliferative disorders, especially essential thrombocythemia and primary myelofibrosis.Currently it has no effect on the choice of therapy in these patients. However, this may change in the future. Begin by preparing the qPCR HRM master mix as described in the text manuscript, making enough for three replicates of each DNA sample and control.Mix the reaction contents by gently tapping and inverting the tube and vortexing for two to three seconds, then briefly spin the tube to collect the scattered droplets. Transfer 19 microliters of the qPCR HRM master mix to each well of the 96 Well Optical Reaction Plate. Pipette one microliter of the negative controls, positive controls and samples into the appropriate wells of the plate for the no template control or NTC at one microliter of sterile RNAs and DNA's free water used for preparing the qPCR HRM master mix instead of DNA.Seal the reaction plate firmly with the optical adhesive film to prevent evaporation during the run. Then spin it at 780 times G at room temperature for one minute. Check that the liquid is at the bottom of the wells in the reaction plate and run the assay.Assign the controls and samples to the appropriate wells in the instrument system software and change the default instrument amplification protocol. Program the instrument as described in the text manuscript to perform the dissociation analysis immediately after qPCR. when the run end review and verify the amplification plot.In the experiment menu of the instrument system software select the amplification plot option. If no data are displayed, click the green analyze button. In the Amplification Plot tab from the plot type dropdown menu, select the plot that displays the amplification data as the fluorescence readings, normalized to the fluorescence from the passive reference as a function of cycle number, In the plot color dropdown menu, select sample.From the graph type dropdown menu, select the linear amplification graph type, verify that the baseline start and end cycle is set correctly. The end cycle should be set a few cycles before the cycle number where a significant fluorescent signal is detected. From the graph type dropdown menu, select the log 10 amplification graph type show the threshold line on the graph by selecting the threshold option and adjust the threshold.The threshold line should cross the exponential phase at the qPCR curves. Verify that all sample and positive control Wells have normal amplification curves and that there is no amplification in the NTC Wells. A normal amplification plot shows an exponential increase in florescence that exceeds the threshold between cycles 15 and 35.In the derivative melt curves review the pre and post melt regions or temperature lines. The pre and post melt regions should be within a flat area where there are no large peaks or slopes in the fluorescent levels. If needed set the start and stop of the pre and post melt temperature lines, approximately 0.5 degrees Celsius apart from each other.Restart the analysis if the parameters are adjusted by clicking on the analyze button. In the plot settings tab of the difference pot tab, choose one of the wild type control replicates as the reference DNA and restart the analysis by clicking on the analyze button. In the aligned Melt curves tab, confirm that all positive controls have the correct genotype and that the NTC failed to amplify.From the well table, select a control well to highlight the corresponding melt curve in the analysis plots and confirm that the color of the line corresponds to the correct genotype. In the aligned melt curves tab carefully review the plot displays for the unknown samples and compare them to the plot displays for controls. Select the Wells containing the unknown sample replicates and align the melt curves with the controls in an ordered sequence by selecting the wells containing controls one by one.Analyze the result for the unknown sample in the Difference Plot tab to verify that the results obtained are consistent. Select the wells containing the unknown sample replicates and align the melt curves with the controls in an ordered sequence, selecting the Wells containing controls one by one. Run qPCR HRM products on a 4%agarose precast gel containing a fluorescent nucleic acid strain.Run only one positive negative NTC and sample qPCR HRM replicates. Remove the precast gel and the cassette from the package then remove the comb and insert the gel into the apparatus according to the manufacturer's instructions. Dilute a 10 microliter sample to 20 microliters with sterile RNA and DNA free water and load each well with 20 microliters of the diluted sample.Dilute three microliters of DNA size standard solution to 20 microliters and load it into the marker well. Fill any empty wells with 20 microliters of sterile RNA and DNA's free water. Immediately select the program according to the percentage of the gel being run and set the runtime on the apparatus to 20 minutes.Start the electrophoresis within one minute of loading the samples. When the electrophoresis is completed, visualize the DNA and the gel by using blue light or UV transillumination. And successfully amplified DNA region of interest with an exponential increase in fluorescents that exceeds the threshold between cycles 15 and 35 and very narrow values of the cycle of quantification in all replicates and controls is a prerequisite for the reliable identification of genetic variance by high resolution melting analysis.HRM analysis is performed immediately after qPCR, the active melt regions of the samples, the controls and the NTC are used to create their aligned melt curve plots. Correctly set pre and post melt regions are important for properly visualizing and identifying genetic variance in the samples. Aligned melt curves and different pots are shown here.The unknown samples are tightly aligned with one of the positive controls. Incorrectly set pre and post melt regions or temperature lines result in aligned melt curves and different plots where correct identification of the genetic variance is more difficult. The genetic variant in the sample can be identified by comparing the band pattern of the sample to the controls.And by combining the HRM and agarose gel electrophoresis. Correct genetic variant identification can only be done for the samples that contain the same genetic variant as one of the controls used in the HRM assay. Samples containing rare calreticulin genetic variants differ in the HRM result and electrophoresis band pattern.When attempting this protocol, it is important to carefully compare the aligned melt curve plot displays of the known and control samples.

Research

JoVE Journal

Genetics

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

Detection of Copy Number Alterations Using Single Cell Sequencing

単一細胞のシーケンシングを使用したコピー数変化の検出

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

遺伝学

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

補体受容体1長多型のための高分解能Melting PCR Genotyping：アルツハイマー病の遺伝子感受性評価のための革新的なツール

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

ショウジョウバエの胚とパスチラミドシグナル信号増幅を利用した生体表面の高解像度蛍光In Situハイブリダイゼーション

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

プロモーター キャプチャこんにちは-c: 高解像度, ゲノム プロモーターの相互作用のプロファイリング

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

細菌の活用頻度の高解像度の比較

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

円の染色体構造の次世代シーケンシングによる遺伝子制御配列の高スループット識別キャプチャ (4 C seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation

遺伝的変異のアミノ酸レベル信号対雑音解析によるバリアント病原性の可能性を決定します。

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

植物におけるマイクロRNA転写物を正確かつ効率的に分析するバイオインフォマティクスパイプライン

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

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

米の耕作集団における高解像度溶融による変異の同定

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

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

細菌性ウィルト病の簡単な遺伝子解析のためのラストニア・ソラナセアラムによるトマト根形質転換

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...