Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the MGMT Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using a Single Primer Set

Due to its cost-efficiency, high resolution melting (HRM) analysis plays an important role in genotyping of candidate single nucleotide polymorphisms (SNPs). Studies indicate that HRM analysis is not only suitable for genotyping individual SNPs, but also allows genotyping of multiple SNPs in one and the same amplicon, although with limited discrimination power. By targeting the three C>T SNPs rs527559815, rs547832288, and rs16906252, located in the promoter of the O6-methylguanine-DNA methyltransferase (MGMT) gene within a distance of 45 bp, we investigated whether the discrimination power can be increased by coupling HRM analysis with pyrosequencing (PSQ). After optimizing polymerase chain reaction (PCR) conditions, PCR products subjected to HRM analysis could directly be used for PSQ. By analyzing oligodeoxynucleotide controls, representing the 36 theoretically possible variant combinations for diploid human cells (8 triple-homozygous, 12 double-homozygous, 12 double-heterozygous and 4 triple-heterozygous combinations), 34 out of the 36 variant combinations could be genotyped unambiguously by combined analysis of HRM and PSQ data, compared to 22 variant combinations by HRM analysis and 16 variant combinations by PSQ. Our approach was successfully applied to genotype stable cell lines of different origin, primary human tumor cell lines from glioma patients, and breast tissue samples.

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High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

Transfusion Medicine and Hemotherapy ◽

10.1159/000520713 ◽

2021 ◽

pp. 1-8

Author(s):

Oytip Nathalang ◽

Kamphon Intharanut ◽

Sarisa Chidtrakoon

Keyword(s):

High Resolution ◽

Blood Group ◽

High Resolution Melting ◽

Melting Curve ◽

Melting Curve Analysis ◽

Blood Group Antigens ◽

Red Cell ◽

Nucleotide Polymorphisms ◽

Hrm Analysis ◽

Antibody Specificities

Background: High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. Methods: Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of KEL*01 and KEL*02, JK*01 and JK*02, FY*01, FY*02, and FY*02 N.01, DI*01 and DI*02, GYPB*03 and GYPB*04, RHCE*E and RHCE*e, and DO*01 and DO*02. Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. Results: The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. Conclusions: We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

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Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis

Frontiers in Plant Science ◽

10.3389/fpls.2015.01068 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 8

Author(s):

Yanbin Hong ◽

Manish K. Pandey ◽

Ying Liu ◽

Xiaoping Chen ◽

Hong Liu ◽

...

Keyword(s):

High Resolution ◽

Single Nucleotide Polymorphisms ◽

Arachis Hypogaea ◽

High Resolution Melting ◽

Amplicon Sequencing ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Hrm Analysis ◽

Arachis Hypogaea L

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High-resolution melting (HRM) analysis for the detection of single nucleotide polymorphisms in microRNA target sites

Clinica Chimica Acta ◽

10.1016/j.cca.2012.03.007 ◽

2012 ◽

Vol 413 (13-14) ◽

pp. 1092-1097 ◽

Cited By ~ 6

Author(s):

Pei-Chin Lin ◽

Ta-Chih Liu ◽

Chun-Chi Chang ◽

Yung-Hsiu Chen ◽

Jan-Gowth Chang

Keyword(s):

High Resolution ◽

Single Nucleotide Polymorphisms ◽

High Resolution Melting ◽

Nucleotide Polymorphisms ◽

Microrna Target ◽

Single Nucleotide ◽

Hrm Analysis ◽

Target Sites

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Application of High-Resolution Melting to Large-Scale, High-Throughput SNP Genotyping

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110365900 ◽

2010 ◽

Vol 15 (6) ◽

pp. 623-629 ◽

Cited By ~ 40

Author(s):

Alessandro Martino ◽

Tommaso Mancuso ◽

Anna Maria Rossi

Keyword(s):

High Resolution ◽

High Throughput ◽

Large Scale ◽

High Resolution Melting ◽

Fluorescent Dyes ◽

Gc Content ◽

Snp Genotyping ◽

Nucleotide Polymorphisms ◽

Experimental Conditions ◽

Discrimination Power

Because of the wide use of single-nucleotide polymorphisms (SNPs) as markers of genetic variation, several high-throughput genotyping methods have been developed and applied during the past decades. High-resolution melting (HRM) is a very attractive, advanced, fast, and cost-effective SNP genotyping technology based on the analysis of the melting profile of PCR products, using intercalating fluorescent dyes to monitor the transition from unmelted to melted DNA. The authors used HRM for genotyping 215 human DNA samples for SNPs in the ABCB1, NQO1, and SLC19A1 genes and 96 samples for SNPs in the IL1A and IL12B genes with the aim of assessing HRM sensitivity and accuracy in comparisons with the TaqMan® assay in view of large-scale, high-throughput SNP-typing applications. The potential effect of PCR product size, TM, GC content, and SNP position on HRM performances was explored with amplicons that were heterogeneous for these factors. Discrimination power ranged from 91.4% to 98.4%, being significantly lower only when the number of rare homozygotes dropped to 1 or few units. The availability of specific and validated assays, in addition to a better standardization of HRM experimental conditions, can considerably reduce time and costs of large-scale genotyping studies with a negligible risk of failure or misclassification.

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Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment

Applied and Environmental Microbiology ◽

10.1128/aem.01164-09 ◽

2009 ◽

Vol 76 (2) ◽

pp. 493-499 ◽

Cited By ~ 26

Author(s):

Shreema Merchant-Patel ◽

Patrick J. Blackall ◽

Jillian Templeton ◽

Erin P. Price ◽

Steven Y. C. Tong ◽

...

Keyword(s):

High Resolution ◽

Campylobacter Jejuni ◽

High Resolution Melting ◽

Resolving Power ◽

Campylobacter Coli ◽

Sequence Variants ◽

Nucleotide Polymorphisms ◽

Hrm Analysis ◽

Pcr Product ◽

Binary Markers

ABSTRACT The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP. flaA HRM analysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.

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

Scientific Reports ◽

10.1038/s41598-021-86228-9 ◽

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.

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

Genes ◽

10.3390/genes12020199 ◽

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.

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

Journal of Medical Biochemistry ◽

10.2478/jomb-2013-0006 ◽

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.

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