Comparison of High-Resolution Melting Analysis with Denaturing High-Performance Liquid Chromatography for Mutation Scanning in theABCA4Gene

Abstract Torque teno viruses (TTVs) are recently discovered DNA viruses, with heterogeneous genomes, highly prevalent in populations worldwide. The species that infect humans are Torque teno virus (TTV), Torque teno midi virus (TTMDV) and Torque teno mini virus (TTMV). High-resolution melting analysis (HRMA) is a sensitive and effective method for genotyping and mutation scanning. Up to now, HRMA has not been utilized for detection of TTVs. The aim of this study was to asses if HRMA is suitable for detecting TTVs variants. DNA was extracted from the blood and saliva of 13 healthy subjects for method optimization. Additionally, saliva samples from 100 healthy individuals were collected for estimating the TTVs’ prevalence. Viral DNA was amplified by heminested polymerase chain reaction (PCR). Second round amplicons were used for the HRMA. The samples were analyzed using two fluorescent dyes, SYBR® Green I and EvaGreen®. The prevalence values for TTV, TTMDV and TTMV were 71.0, 31.0 and 54.0%, respectively. The three major melting curve patterns corresponding to TTV, TTMDV and TTMV on HRMA can be easily distinguished regardless of kit used. Our results showed that HRMA is a rapid and efficient method of detecting human TTVs.

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KRAS mutation detection in Tunisian sporadic coloractal cancer patients with direct sequencing, high resolution melting and denaturating high performance liquid chromatography

Cancer Biomarkers ◽

10.3233/cbm-2011-0222 ◽

2011 ◽

Vol 8 (6) ◽

pp. 331-340 ◽

Cited By ~ 3

Author(s):

Bougatef Karim ◽

Coulet Florence ◽

Rouissi Kamel ◽

Kourda Nadia ◽

Omrane Ines ◽

...

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

High Resolution ◽

Cancer Patients ◽

Kras Mutation ◽

High Performance ◽

Mutation Detection ◽

High Resolution Melting ◽

Direct Sequencing

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High-resolution melting analysis, a simple and effective method for reliable mutation scanning and frequency studies in the ACADVL gene

Journal of Inherited Metabolic Disease ◽

10.1007/s10545-010-9101-y ◽

2010 ◽

Vol 33 (3) ◽

pp. 247-260 ◽

Cited By ~ 8

Author(s):

Rikke Katrine Jentoft Olsen ◽

Steven F. Dobrowolski ◽

Margrethe Kjeldsen ◽

David Hougaard ◽

Henrik Simonsen ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

High Resolution Melting Analysis ◽

Mutation Scanning ◽

Melting Analysis

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High-resolution melting analysis for mutation scanning in the non-coding control region of JC polyomavirus from patients with progressive multifocal leukoencephalopathy

Archives of Virology ◽

10.1007/s00705-014-1988-4 ◽

2014 ◽

Vol 159 (7) ◽

pp. 1687-1696 ◽

Cited By ~ 6

Author(s):

Kazuo Nakamichi ◽

Shigeru Tajima ◽

Chang-Kweng Lim ◽

Masayuki Saijo

Keyword(s):

High Resolution ◽

Progressive Multifocal Leukoencephalopathy ◽

Control Region ◽

High Resolution Melting ◽

High Resolution Melting Analysis ◽

Jc Polyomavirus ◽

Mutation Scanning ◽

Melting Analysis ◽

Coding Control

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Mutation Scanning the GJB1 Gene with High-Resolution Melting Analysis: Implications for Mutation Scanning of Genes for Charcot-Marie-Tooth Disease

Clinical Chemistry ◽

10.1373/clinchem.2006.080010 ◽

2007 ◽

Vol 53 (2) ◽

pp. 349-352 ◽

Cited By ~ 41

Author(s):

Marina L Kennerson ◽

Trent Warburton ◽

Eva Nelis ◽

Megan Brewer ◽

Patsie Polly ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Mutation Screening ◽

Gene Mutations ◽

High Resolution Melting Analysis ◽

Charcot Marie Tooth ◽

Charcot Marie Tooth Disease ◽

Mutation Scanning ◽

Junction Protein ◽

Melting Analysis

Abstract Background: X-linked Charcot-Marie-Tooth type 1 disease has been associated with 280 mutations in the GJB1 [gap junction protein, beta 1, 32kDa (connexin 32, Charcot-Marie-Tooth neuropathy, X-linked)] gene. High-resolution melting analysis with an automated instrument can be used to scan DNA for alterations, but its use in X-linked disorders has not been described. Methods: A 96-well LightScanner for high resolution melting analysis was used to scan amplicons of the GJB1 gene. All mutations reported in this study had been confirmed previously by sequence analysis. DNA samples were amplified with the double-stranded DNA-binding dye LC Green Plus. Melting curves were analyzed as fluorescence difference plots. The shift and curve shapes of melting profiles were used to distinguish controls from patient samples. Results: The method detected each of the 23 mutations used in this study. Eighteen known mutations provided validation of the high-resolution melting method and a further 5 mutations were identified in a blind study. Altered fluorescence difference curves for all the mutations were easily distinguished from the wild-type melting profile. Conclusion: High-resolution melting analysis is a simple, sensitive, and cost-efficient alternative method to scan for gene mutations in the GJB1 gene. The technology has the potential to reduce sequencing burden and would be suitable for mutation screening of exons of large multiexon genes that have been discovered to be associated with Charcot Marie Tooth neuropathy.

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