저자는 전문 혈액학 연구소, 혈액학학과, 내과, 대학 의료 센터 류블랴나의 모든 학술 전문가와 직원에게 감사드립니다.

연구 결과는 슬로베니아 공화국의 의학 윤리위원회에 의해 승인되었습니다. 모든 절차는 헬싱키 선언에 따른 것입니다.
1. HRM에 의한 형광 기반 정량적 실시간 PCR(qPCR) 및 사후 qPCR 분석
<ol><li>재료 표에 나열된 프라이머를 멸균, RNase 및 DNase 무료 H2O로 100 μM로 재일시 중지합니다(재료 표참조). 10 μM 작업 농도 프라이머를 만듭니다. 프로토콜?...

<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=Sensitivity+and+specificity+of+single-nucleotide+polymorphism+scanning+by+high-resolution+melting+analysis.">Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis.</a> Clinical Chemistry. 50, 1748-1754 (2004).</li><li>Belcic Mikic, T., Pajic, T., Sever, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CALR+mutations+in+a+cohort+of+JAK2+V617F+negative+patients+with+suspected+myeloproliferative+neoplasms.">CALR mutations in a cohort of JAK2 V617F negative patients with suspected myeloproliferative neoplasms.</a> Scientific Reports. 9, 19838 (2019).</li><li>Invitrogen, Thermo Fischer Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QubitTM dsDNA HS Assay Kits. Manual (MAN0002326, MP32851, Revision: B.0). , (2015).</li><li>Applied Biosystems, Thermo Fisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High Resolution Melt Module for ViiA TM 7 Software v1. Getting Started Guide. Part Number 4443531 Rev. B. , (2011).</li><li>Applied Biosystems, Thermo Fischer Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High-Resolution Melt Analysis for Mutation Scanning Prior to Sequencing. Application Note. , (2010).</li><li>Reed, G. H., Kent, J. O., Wittwer, C. 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+DNA+melting+analysis+for+simple+and+efficient+molecular+diagnostics.">High-resolution DNA melting analysis for simple and efficient molecular diagnostics.</a> Pharmacogenomics. 8, 597-608 (2007).</li><li>Gonzalez-Bosquet, 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=Detection+of+somatic+mutations+by+high-resolution+DNA+melting+(HRM)+analysis+in+multiple+cancers.">Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.</a> PLoS One. 6, 14522 (2011).</li><li>Van Der Stoep, 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=Diagnostic+guidelines+for+high-resolution+melting+curve+(HRM)+analysis:+An+interlaboratory+validation+of+BRCA1+mutation+scanning+using+the+96-well+LightScanner.">Diagnostic guidelines for high-resolution melting curve (HRM) analysis: An interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner.</a> Human Mutation. 30, 899-909 (2009).</li><li>Singdong, 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=Characterization+and+Prognosis+Significance+of+JAK2+(V617F),+MPL,+and+CALR+Mutations+in+Philadelphia-Negative+Myeloproliferative+Neoplasms.">Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms.</a> Asian Pacific Journal of Cancer Prevention. 17, 4647-4653 (2016).</li><li>Erali, M., Wittwer, C. 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. 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> Journal of Visualized Experiments. , e3923 (2012).</li><li>Helling, R. B., Goodman, H. M., Boyer, H. W. <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+Endonuclease+R&#183;EcoRI+Fragments+of+DNA+from+Lambdoid+Bacteriophages+and+Other+Viruses+by+Agarose-Gel+Electrophoresis.">Analysis of Endonuclease R&#183;EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis.</a> Journal of Virology. , 1235-1244 (1974).</li></ol>

DNA의 고해상도 용융은 유전화 및 유전 적 변이체 스캐닝14에대한 간단한 솔루션이다. 그것은 용융 곡선의 모양을 변경 하는 heteroduplexes 귀착되는 시퀀스 차이에 따라 달라 집니다. 다양한 주파수를 가진 유전이체의 상이한유형은 암1, 2,15,16,10을가진 환자의 특정 집단에 대한 ...

calreticulin유전자(CALR)의체세포 유전 변이체는 2013년에 필수 혈전세포증 및 1차 골수혈증과같은 골수증성 신생물(MPN)을 가진 환자에서1,2를인식하였다. 그 이후로 CALR 유전자에 있는 50개 이상의 유전 변이체가 발견되어 +1(−1+2) 프레임 시프트3을유도합니다. 가장 빈번한 CALR 유전변이체 는 52bp 삭제(NM_004343.3(CALR):c.1099_1150del52, p.(Leu367Thrfs*46)) 및 타입-1 돌연변이라고도 하며, 5bp 삽입(NM_004343.3(CALR):c.1154_1155insTTGTC, p.(lys385Asnfs*47))라고도 합니다. 이 2개의 유전 이체는 모든 CALR 유전 이체의 80%를 나타냅니다. 다른 것들은 야생형 CALR4에가까운 α 나선의 보존에 기초한 알고리즘을 사용하여 유형 1-like 또는 유형 2-와 같은 유형으로 분류되었습니다. 여기서, 우리는 CALR 유전이체 검출을 위한 고민감하고 신속한 방법 중 하나인 고해상도 용융 분석 방법(HRM)을 제시한다. 이 방법은 CALR 돌연변이5의대다수를 나타내는 타입-1 및 타입-2 유전 이체의 급속한 검출을 가능하게 합니다. HRM은 1997년 V 라이덴6의돌연변이를 검출하는 도구로 실시간 »폴리머라제 연쇄 반응«(qPCR)과 함께 도입되었다. 황금 표준 기술을 나타내는 Sanger 시퀀싱과 비교하여 HRM은 보다 민감하고 덜 구체적인 방법5입니다. HRM 방법은 비용 이점5를가진 많은 수의 샘플을 신속하게 분석할 수 있는 좋은 스크리닝 방법입니다. 형광염의 존재에서 수행되는 간단한 PCR 방법이며 특정 기술이 필요하지 않습니다. 또 다른 이점은 절차 자체가 HRM 시술7후 전기전도 또는 Sanger 시퀀싱시퀀싱시료를 재사용할 수 있는 분석된 샘플을 손상시키거나 파괴하지 않는다는 것입니다. 유일한 단점은 때때로 결과를 해석하기 어렵다는 것입니다. 또한, HRM은 비형 1 또는 타입-2 돌연변이8을가진 환자에서 정확한 돌연변이를 검출하지 않습니다. 이러한 환자에서, Sanger 시퀀싱을 수행해야한다(도 1).
HRM은 이중 좌초 된 DNA (dsDNA)에 통합되는 포화 DNA 형광 염료의 존재에서 특정 DNA 영역의 증폭에 기초한다. 형광 염료는 dsDNA에 통합 될 때 빛을 방출합니다. 온도가 점진적으로 증가한 후 dsDNA는 단일 좌초 된 DNA로 분해되어 용융 곡선에서 형광 강도가 급격히 감소함에 따라 감지 될 수 있습니다. 용융 곡선의 모양은 돌연변이를 검출하는 데 사용되는 DNA 서열에 따라 달라집니다. 시료의 용융 곡선은 알려진 돌연변이 또는 야생 형 CALR의 용융 곡선과 비교됩니다. 뚜렷한 용융 곡선은 비타입 1 또는 유형 29인다른 돌연변이를 나타낸다.
HRM, 아가로즈 겔 전기포진 및 시퀀싱방법(도 1)에의한 CALR 유전자내체 유전적 이체 검출을 위한 알고리즘은10일전에 발표된 회고전 연구에서 사용되고 검증되었다.

<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

고해상도 용융 분석(HRM)은 유전화 및 유전적 변이 스캐닝을 위한 강력한 방법입니다. 대부분의 HRM 응용 프로그램은 서열 차이를 감지하는 DNA 염료를 포화시키고 용융 곡선의 모양을 변경하는 이테로두플렉스에 의존합니다. 우수한 계측기 해상도와 특수 데이터 분석 소프트웨어는 변형 또는 유전자형을 식별하는 작은 용융 곡선 차이를 식별하는 데 필요합니다. 다양한 주파수를 가진 유전 이체의 다른 모형은 특정 질병을 가진 환자를 위해 특정한 유전자에서 관찰될 수 있습니다, 특히 암 및 필라델피아 염색체-음성 골수증성 신생물을 가진 환자에 있는 CALR 유전자에서. 관심 유전자의 단일 뉴클레오티드 변화, 삽입 및/또는 삭제(indels)는 HRM 분석에 의해 검출될 수 있다. 유전 이체의 다른 모형의 확인은 주로 qPCR HRM 분석에서 사용된 통제에 근거를 두릅니다. 그러나 제품 길이가 증가함에 따라 야생형곡선과 이종조곡선의 차이가 작아지고 유전적 변이체의 유형이 결정되기 가 더 어려워진다. 따라서, 인델이 관심 있는 유전자에서 예상되는 널리 퍼진 유전적 이체인 경우, 아가로즈 겔 전기포진과 같은 추가 방법이 HRM 결과의 해명에 사용될 수 있다. 경우에 따라 표준 Sanger 시퀀싱에 의해 결정적인 결과를 다시 검사/다시 진단해야 합니다. 이 회고 연구에서, 우리는 MPN을 가진 JAK2 V617F 음성 환자에 방법을 적용했습니다.

모든 복제된 샘플 및 대조군(그림2)에서주기 15및 35 사이의 임계값과 매우 좁은 값사이의 임계값을 초과하는 형광의 기하급수적 증가와 함께 성공적으로 증폭된 DNA 영역은 HRM 분석에 의한 유전적 이체의 신뢰성 있는 식별을 위한 전제 조건이다. 이것은 형광 염색을 가진 DNA의 정확한 측정 및 qPCR HRM 실험에 있는 DNA의 동등한 양을 사용하여 달성됩니다 (단계 1.2 참조). <strong cla...

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

고해상도 용융 분석(HRM)은 유전적 이체 검출을 위한 민감하고 신속한 솔루션입니다. 그것은 이발 곡선의 모양을 변경 하는 heteroduplexes 귀착되는 시퀀스 차이에 따라 달라 집니다. HRM 및 아가로즈 젤 전기전도를 빗질함으로써, 인델과 같은 유전적 변이체의 상이한 유형을 확인할 수 있다.

고분해능 용융 분석을 이용한 CALR 유전자의 유전적 변이체 검출

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.

고분해능 용융 분석을 이용한 CALR 유전자의 유전적 변이체 검출

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