scholarly journals Microsatellite Instability Detection by High-Resolution Melting Analysis

2010 ◽  
Vol 56 (11) ◽  
pp. 1750-1757 ◽  
Author(s):  
Ramunas Janavicius ◽  
Dovile Matiukaite ◽  
Arturas Jakubauskas ◽  
Laimonas Griskevicius
Keyword(s):  
Colorectal Cancer ◽  
Capillary Electrophoresis ◽  
High Resolution ◽  
Microsatellite Instability ◽  
High Resolution Melting ◽  
Sporadic Colorectal Cancer ◽  
Analytical Sensitivity ◽  
Analysis Method ◽  
Detection Techniques ◽  
Hrm Analysis

BACKGROUND Microsatellite instability (MSI) is an important marker for screening for hereditary nonpolyposis colorectal cancer (Lynch syndrome) as well as a prognostic and predictive marker for sporadic colorectal cancer (CRC). The mononucleotide microsatellite marker panel is a well-established and superior alternative to the traditional Bethesda MSI analysis panel, and does not require testing for corresponding normal DNA. The most common MSI detection techniques—fluorescent capillary electrophoresis and denaturing HPLC (DHPLC)—both have advantages and drawbacks. A new high-resolution melting (HRM) analysis method enables rapid identification of heteroduplexes in amplicons by their lower thermal stability, a technique that overcomes the main shortcomings of capillary electrophoresis and DHPLC. METHODS We investigated the straightforward application of HRM for the detection of MSI in 70 archival CRC samples. HRM analysis for 2 MSI markers (BAT25 and BAT26) was evaluated, and 2 different HRM-enabled instruments were compared—the LightCycler® 480 (Roche Diagnostics) and the LightScannerTM (Idaho Technology). We also determined the analytical sensitivity and specificity of the HRM assay on both instruments using 11 known MSI-positive and 54 microsatellite-stable CRC samples. RESULTS All MSI-positive samples were detected on both instruments (100% analytical sensitivity). The LightScanner performed better for analytical specificity, giving a combined specificity value of 99.1% compared with 92.3% on the LightCycler 480. CONCLUSIONS We expanded the application of the HRM analysis method as an effective MSI detection technique for clinical samples.

scholarly journals CAT25 defines microsatellite instability in colorectal cancer by high-resolution melting PCR

2020 ◽  
Vol 77 (3) ◽  
pp. 105-111 ◽  
Author(s):  
AG Sánchez ◽  
I Juaneda ◽  
H Eynard ◽  
AL Basquiera ◽  
E Palazzo ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
High Resolution ◽  
Microsatellite Instability ◽  
High Resolution Melting

Detection of KRAS mutations in colorectal cancer by high-resolution melting analysis

2009 ◽  
Vol 62 (10) ◽  
pp. 886-891 ◽  
Author(s):  
E S K Ma ◽  
C L P Wong ◽  
F B F Law ◽  
W-K Chan ◽  
D Siu
Keyword(s):  
Colorectal Cancer ◽  
High Resolution ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Tumour Tissue ◽  
Chinese Patients ◽  
Molecular Diagnostic ◽  
Service Utilisation ◽  
Exon 2 ◽  
Hrm Analysis

Aims:Mutation of the KRAS gene predicts the clinical response to the monoclonal antibody cetuximab in patients with advanced colorectal cancer (CRC). This study aimed to perform KRAS mutation detection on formalin-fixed paraffin-embedded (FFPE) tumour tissue by two different methods for comparison.Methods:The FFPE sample was microdissected to enrich for tumour cells. KRAS exon 2 mutations were performed on 100 Chinese patients with CRC by direct nucleotide sequencing and high-resolution melting (HRM) analysis.Results:KRAS exon 2 mutations were detected in a total of 62 patients with the two methods combined, comprising 11 different mutant alleles. Three common mutations p.Gly12Asp, p.Gly12Val and p.Gly13Asp accounted for approximately 70% of all cases. The concordant rate between the two methods was 95%. Four mutations not initially detected by direct sequencing were identified by HRM and confirmed by sequencing of the HRM amplicons. One mutation detected by direct sequencing was inadvertently grouped as a wild-type allele by HRM software, but this was readily rectified through manual review.Conclusion:HRM analysis is a sensitive method of detecting KRAS mutation on FFPE tumour tissue to guide cetuximab treatment and is applicable to routine molecular diagnostic service. Utilisation of HRM to screen for mutations upfront economises the resource used in the sequencing reaction.

scholarly journals Differentiation of Mitragyna speciosa, a narcotic plant, from allied Mitragyna species using DNA barcoding-high-resolution melting (Bar-HRM) analysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chayapol Tungphatthong ◽  
Santhosh Kumar J. Urumarudappa ◽  
Supita Awachai ◽  
Thongchai Sooksawate ◽  
Suchada Sukrong
Keyword(s):  
High Resolution ◽  
Dna Barcoding ◽  
High Resolution Melting ◽  
Dna Barcode ◽  
Herbal Medicines ◽  
Efficient Tool ◽  
Hrm Analysis ◽  
Mitragyna Speciosa ◽  
Leaf Powder

AbstractMitragyna speciosa (Korth.) Havil. [MS], or “kratom” in Thai, is the only narcotic species among the four species of Mitragyna in Thailand, which also include Mitragyna diversifolia (Wall. ex G. Don) Havil. [MD], Mitragyna hirsuta Havil. [MH], and Mitragyna rotundifolia (Roxb.) O. Kuntze [MR]. M. speciosa is a tropical tree belonging to the Rubiaceae family and has been prohibited by law in Thailand. However, it has been extensively covered in national and international news, as its abuse has become more popular. M. speciosa is a narcotic plant and has been used as an opium substitute and traditionally used for the treatment of chronic pain and various illnesses. Due to morphological disparities in the genus, the identification of plants in various forms, including fresh leaves, dried leaf powder, and finished products, is difficult. In this study, DNA barcoding combined with high-resolution melting (Bar-HRM) analysis was performed to differentiate M. speciosa from allied Mitragyna and to assess the capability of Bar-HRM assays to identify M. speciosa in suspected kratom or M. speciosa-containing samples. Bar-HRM analysis of PCR amplicons was based on the ITS2, rbcL, trnH-psbA, and matK DNA barcode regions. The melting profiles of ITS2 amplicons were clearly distinct, which enabled the authentication and differentiation of Mitragyna species from allied species. This study reveals that DNA barcoding coupled with HRM is an efficient tool with which to identify M. speciosa and M. speciosa-containing samples and ensure the safety and quality of traditional Thai herbal medicines.

scholarly journals A Forensic Detection Method for Hallucinogenic Mushrooms via High-Resolution Melting (HRM) Analysis

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 199
Author(s):  
Xiaochun Zhang ◽  
Huan Yu ◽  
Qi Yang ◽  
Ziwei Wang ◽  
Ruocheng Xia ◽  
...  
Keyword(s):  
High Resolution ◽  
Dna Barcoding ◽  
High Resolution Melting ◽  
Its Region ◽  
Its Sequencing ◽  
Melting Temperatures ◽  
Hrm Analysis ◽  
National Drug ◽  
Species Specific ◽  
Its1 And Its2

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

scholarly journals Genotyping of GATA4 Gene Variant (G296S) in Malaysian Congenital Heart Disease Subjects by Real-Time PCR High Resolution Melting Analysis

2013 ◽  
Vol 32 (2) ◽  
pp. 152-157
Author(s):  
Nora Fawzi ◽  
Ramachandran Vasudevan ◽  
Patimah Ismail ◽  
Mazeni Alwi ◽  
Ahmad Fazli Abdul Aziz ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
High Resolution ◽  
Congenital Heart ◽  
High Resolution Melting ◽  
New Technology ◽  
Cost Effective ◽  
Study Cohort ◽  
Hrm Analysis ◽  
Melting Analysis

Summary Background: Congenital heart disease (CHD) is the most common birth defect; however, the underlying etiology is unrecognized in the majority of cases. GATA-binding protein 4 (GATA4), a cardiac transcription factor gene, has a crucial role in the cardiogenesis process; hence, a number of heterozygote sequence variations were identified as a cause of CHD. G296S heterozygote variant is the most frequently reported GATA4 gene sequence alteration. This study aims to investigate the role of G296S variant of the GATA4 gene in Malaysian CHD subjects. Methods: We have investigated 86 Malaysian CHD subjects with cardiac septation defects for the presence of the GATA4 gene heterozygote variant (G296S) by the new technology of high resolution melting (HRM) analysis. Results: Genotyping of G296S (c.886G>A) by HRM analysis shows that all the sample genotypes were of the wild GG type genotype and the heterozygote mutant GA genotype was totally absent from this study cohort. Conclusions: The results of our study showed that the G296S variant of the GATA4 gene was not associated with the development of CHD in Malaysian subjects. The use of HRM analysis proved a cost-effective, high-throughput, specific and sensitive genotyping technique which eliminates the need for unnecessary sequencing.

scholarly journals Distinct BRAF (V600E) and KRAS Mutations in High Microsatellite Instability Sporadic Colorectal Cancer in African Americans

2009 ◽  
Vol 15 (4) ◽  
pp. 1155-1161 ◽  
Author(s):  
Krishan Kumar ◽  
Hassan Brim ◽  
Francis Giardiello ◽  
Duane T. Smoot ◽  
Mehdi Nouraie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
African Americans ◽  
Microsatellite Instability ◽  
Braf V600e ◽  
Sporadic Colorectal Cancer ◽  
Kras Mutations

scholarly journals Extreme PCR: Efficient and Specific DNA Amplification in 15–60 Seconds

2015 ◽  
Vol 61 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Jared S Farrar ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
High Resolution Melting ◽  
Dna Amplification ◽  
Temperature Cycling ◽  
High Yield ◽  
Temperature Cycle ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Agarose Gels

Abstract BACKGROUND PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown. METHODS We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4–2.0 s at annealing/extension temperatures of 62 °C–76 °C and denaturation temperatures of 85 °C–92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring. RESULTS Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15–60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any “hot start” technique. Amplification efficiencies were 91.7%–95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles. CONCLUSIONS The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4–2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.

High-Resolution Melting Analyses for Gene Scanning of APC, MLH1, MSH2, and MSH6 Associated with Hereditary Colorectal Cancer

2012 ◽  
Vol 16 (5) ◽  
pp. 406-411 ◽  
Author(s):  
Jurat Obul ◽  
Sakae Itoga ◽  
Maynur Abliz ◽  
Kenichi Sato ◽  
Takayuki Ishige ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
High Resolution ◽  
High Resolution Melting ◽  
Hereditary Colorectal Cancer ◽  
Gene Scanning
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