High resolution melting analysis for gene scanning

Erali, Maria; Wittwer, Carl T.

doi:10.1016/j.ymeth.2010.01.013

Cited by 137 publications

(90 citation statements)

References 37 publications

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“…Several reports about HRM analysis have discussed the optimization of HRM reaction conditions, and most of them focused on the choice of proper annealing temperatures (Erali and Wittwer 2010;Mackay et al 2008). Except for proper annealing temperature, DNA quality is also an important factor for HRM analysis, and we therefore confirmed the DNA quality of all samples before further tests.…”

Section: Optimization Of Hrm Reaction Conditionssupporting

confidence: 63%

Evaluation of High-Resolution Melting for Gene Mapping in Rice

Wang

Dong

et al. 2011

Plant Mol Biol Rep

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In this study, high-resolution melting (HRM) analysis was evaluated for gene mapping in rice with sequence-tagged site (STS) and simple sequence repeat (SSR) markers. A total of 103 out of 353 normal STS and SSR markers revealed polymorphic melting curves among the parental genotypes, and 12 of these were successfully used to genotype the F 2 mapping population for HRM analysis. Additional electrophoresis findings demonstrated that HRM genotyping matched with traditional electrophoresis results. To optimize the HRM-marker screening efficiency, different HRM reaction conditions were evaluated. A 5-μl touchdown-polymerase chain reaction (PCR) system provided no significant improvement in screening efficiency but in a 10-μl touchdown-PCR system, the marker screening efficiency increased by 75%. Twenty-one markers were obtained for mapping purposes under the optimized reaction conditions. This study indicates that HRM analysis can speed up the gene mapping progress in rice, while saving a lot of manpower.

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Section: Optimization Of Hrm Reaction Conditionssupporting

confidence: 63%

Evaluation of High-Resolution Melting for Gene Mapping in Rice

Wang

Dong

et al. 2011

Plant Mol Biol Rep

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“…for the wild type (10). Only two primers and one PCR per target and a melting instrument are required.…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis

et al. 2011

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A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis by targeting resistance-associated mutations in the katG, mabA-inhA promoter, rpoB, and gyrA genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical M. tuberculosis isolates was selected for development and evaluation of HRMA. PCR amplicons from the katG, mabA-inhA promoter, rpoB, and gyrA genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified katG and/or mabA-inhA promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates, rpoB mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and gyrA mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in M. tuberculosis which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.One-third of the world's population is reportedly infected with Mycobacterium tuberculosis, causing high mortality and morbidity. Eight million to nine million new tuberculosis (TB) cases and about 2 million deaths are reported each year (9, 18, 28). Multidrug-resistant TB (MDR-TB) is defined as resistance of the isolate to at least isoniazid (INH) and rifampin (RIF); extensively drug-resistant TB (XDR-TB) is MDR-TB in which the isolate has additional resistance to any of fluoroquinolones and at least one of three injectable second-line antituberculosis drugs used in TB treatment, namely, capreomycin, kanamycin, and amikacin (14, 21). MDR-TB and XDR-TB are major obstacles to the control of TB worldwide. More than 2.5 million patients were diagnosed with active TB in 116 countries and regions in 2006; of them, 4.8% had MDR-TB (3). About one in six (15%) patients with MDR-TB had previously received treatment for TB for at least 1 month (18), and at least 8.0% had XDR-TB, according to incomplete data collected from 37 countries between 2002 and 2007 (41).Drug susceptibility testing using molecular techniques can enhance the identification of drug-resistant M. tuberculosis. Several scanning methods are available for detection of drug resistance mutations, including denaturing gradient gel electrophoresis, conformation-sensitive gel electrophoresis, temperature gradient capillary electrophoresis, denaturing high-performance liquid chromatography (dHPLC), and high-density oligonucleotide arrays (15,20,22,30,31,33). These methods vary in sensitivity and...

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“…The analytical sensitivity and specificity of HRM depends on the dye, instrument, and software used (30 ). In this study we evaluated and validated HRM analysis for MSI detection on 2 distinct instruments.…”

Section: Discussionmentioning

confidence: 99%

Microsatellite Instability Detection by High-Resolution Melting Analysis

et al. 2010

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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.

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High resolution melting analysis for gene scanning

Cited by 137 publications

References 37 publications

Evaluation of High-Resolution Melting for Gene Mapping in Rice

Evaluation of High-Resolution Melting for Gene Mapping in Rice

Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis

Microsatellite Instability Detection by High-Resolution Melting Analysis

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