Mutation scanning using high-resolution melting

2009 ◽  
Vol 37 (2) ◽  
pp. 433-437 ◽  
Author(s):  
Claire F. Taylor
Keyword(s):  
High Resolution ◽  
Prior Knowledge ◽  
Sensitivity And Specificity ◽  
Mutation Detection ◽  
High Resolution Melting ◽  
Sequence Variants ◽  
Precise Location ◽  
Mutation Scanning ◽  
Scanning Technique ◽  
The Status

Mutation scanning techniques are used to detect sequence variants without the need for prior knowledge of the identity or precise location of the variant, in contrast with genotyping techniques, which determine the status of a specific variant. High-resolution melting is a recently developed method that shows great potential as a mutation scanning technique. Sensitivity and specificity for mutation detection are extremely high and the technique also has advantages of cost and throughput. Practical considerations for successful mutation scanning by high-resolution melting are also discussed in this review.

scholarly journals Comparative analysis of pyrosequencing and QMC-PCR in conjunction with high resolution melting for KRAS/BRAF mutation detection

2010 ◽  
Vol 91 (6) ◽  
pp. 500-505 ◽  
Author(s):  
Salih Ibrahem ◽  
Rashmi Seth ◽  
Brendan O’Sullivan ◽  
Wakkas Fadhil ◽  
Philippe Taniere ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
High Resolution ◽  
Braf Mutation ◽  
Mutation Detection ◽  
High Resolution Melting

scholarly journals Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes

2003 ◽  
Vol 49 (3) ◽  
pp. 396-406 ◽  
Author(s):  
Cameron N Gundry ◽  
Joshua G Vandersteen ◽  
Gudrun H Reed ◽  
Robert J Pryor ◽  
Jian Chen ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Rapid Heating ◽  
Melting Curve ◽  
Pcr Primers ◽  
High Resolution Melting Analysis ◽  
Melting Transition ◽  
Sequence Variant ◽  
Sequence Variants ◽  
Melting Analysis

Abstract Background: Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR. Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (Tm). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required ∼1 min and no sample processing was needed after PCR. Results: Polymorphisms in the HTR2A (T102C), β-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 °C/s) of denatured products, followed by rapid heating during melting analysis (0.2–0.4 °C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in Tm by <0.2 °C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5′ tail of identical sequence was added to one of the two unlabeled primers. Conclusion: High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.

scholarly journals Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

2016 ◽  
Vol 54 (8) ◽  
pp. 2074-2081 ◽  
Author(s):  
Valentina Donà ◽  
Sara Kasraian ◽  
Agnese Lupo ◽  
Yuvia N. Guilarte ◽  
Christoph Hauser ◽  
...  
Keyword(s):  
High Resolution ◽  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Health Concern ◽  
Content Type ◽  
Resistance To Antibiotics

Resistance to antibiotics used againstNeisseria gonorrhoeaeinfections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences forN. gonorrhoeaeidentification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterizedN. gonorrhoeaestrains, 19 commensalNeisseriaspecies strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identifiedN. gonorrhoeaeand the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcalNeisseriaspecies, and the detection limit was 103to 104genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis

2010 ◽  
Vol 43 (1-2) ◽  
pp. 178-185 ◽  
Author(s):  
Panagiotis A. Vorkas ◽  
Konstantinos Christopoulos ◽  
Christos Kroupis ◽  
Evi S. Lianidou
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Brca1 Gene ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Mutation Scanning ◽  
High Resolution Melting Curve ◽  
Exon 20

scholarly journals High resolution melting for mutation scanning of TP53exons 5–8

BMC Cancer ◽  
2007 ◽  
Vol 7 (1) ◽  
Author(s):  
Michael Krypuy ◽  
◽  
Ahmed Ashour Ahmed ◽  
Dariush Etemadmoghadam ◽  
Sarah J Hyland ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Mutation Scanning
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