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 Identifying Common Genetic Variants by High-Resolution Melting

2007 ◽  
Vol 53 (7) ◽  
pp. 1191-1198 ◽  
Author(s):  
Joshua G Vandersteen ◽  
Pinar Bayrak-Toydemir ◽  
Robert A Palais ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
False Negative ◽  
Melting Curve ◽  
High Resolution Melting Analysis ◽  
Common Variants ◽  
Difference Analysis ◽  
Common Genetic Variants ◽  
Blinded Study ◽  
Melting Analysis

Abstract Background: Heteroduplex scanning techniques usually detect all heterozygotes, including common variants not of clinical interest. Methods: We conducted high-resolution melting analysis on the 24 exons of the ACVRL1 and ENG genes implicated in hereditary hemorrhagic telangiectasia (HHT). DNA in samples from 13 controls and 19 patients was PCR amplified in the presence of LCGreen® I, and all 768 exons melted in an HR-1® instrument. We used 10 wild-type controls to identify common variants, and the remaining samples were blinded, amplified, and analyzed by melting curve normalization and overlay. Unlabeled probes characterized the sequence of common variants. Results: Eleven common variants were associated with 8 of the 24 HHT exons, and 96% of normal samples contained at least 1 variant. As a result, the positive predictive value (PPV) of a heterozygous exon was low (31%), even in a population of predominantly HHT patients. However, all common variants produced unique amplicon melting curves that, when considered and eliminated, resulted in a PPV of 100%. In our blinded study, 3 of 19 heterozygous disease-causing variants were missed; however, 2 were clerical errors, and the remaining false negative would have been identified by difference analysis. Conclusions: High-resolution melting analysis is a highly accurate heteroduplex scanning technique. With many exons, however, use of single-sample instruments may lead to clerical errors, and routine use of difference analysis is recommended. Common variants can be identified by their melting curve profiles and genotyped with unlabeled probes, greatly reducing the false-positive results common with scanning techniques.

scholarly journals High-Resolution Melting Analysis (HRMA)-More than just sequence variant screening

2009 ◽  
Vol 30 (6) ◽  
pp. 860-866 ◽  
Author(s):  
Rolf H.A.M. Vossen ◽  
Emmelien Aten ◽  
Anja Roos ◽  
Johan T. den Dunnen
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Sequence Variant ◽  
Melting Analysis

scholarly journals Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting

2008 ◽  
Vol 54 (1) ◽  
pp. 108-115 ◽  
Author(s):  
Michael T Seipp ◽  
David Pattison ◽  
Jacob D Durtschi ◽  
Mohamed Jama ◽  
Karl V Voelkerding ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Coagulation Factor ◽  
Temperature Correction ◽  
High Resolution Melting Analysis ◽  
Sequence Variant ◽  
Factor V ◽  
Real Time Analysis ◽  
Closed Tube ◽  
Melting Analysis

Abstract Background: Multiplexed amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, asymmetric PCR, or allele-specific PCR; however, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) analysis requires high-resolution melting analysis and controlled reaction conditions. Methods: We designed 4 amplicons bracketing the F5 [coagulation factor V (proaccelerin, labile factor)] 1691G>A, MTHFR (NADPH) 1298A>C, MTHFR 677C>T, and F2 [coagulation factor II (thrombin)] 20210G>A gene variants to melt at different temperatures by varying amplicon length and adding GC- or AT-rich 5′ tails to selected primers. We used rapid-cycle PCRs with cycles of 19–23 s in the presence of a saturating DNA dye and temperature-correction controls and then conducted a high-resolution melting analysis. Heterozygotes were identified at each locus by curve shape, and homozygous genotypes were assigned by Tm. We blinded samples previously genotyped by other methods before analysis with the multiplex melting assay (n = 110). Results: All samples were correctly genotyped with the exception of 7 MTHFR 1298 samples with atypical melting profiles that could not be assigned. Sequencing revealed that these 5 heterozygotes and 2 homozygotes contained the unexpected sequence variant MTHFR 1317T>C. The use of temperature-correction controls decreased the Tm SD within homozygotes by a mean of 38%. Conclusion: Rapid-cycle PCR with high-resolution melting analysis allows simple and accurate multiplex genotyping to at least a factor of 4.

scholarly journals Rapid Detection of Human Torque Teno Viruses Using High-Resolution Melting Analysis

2013 ◽  
Vol 16 (1) ◽  
pp. 55-62 ◽  
Author(s):  
S Spandole ◽  
D Cimponeriu ◽  
M Toma ◽  
I Radu ◽  
D.A. Ion
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Fluorescent Dyes ◽  
Melting Curve ◽  
High Resolution Melting Analysis ◽  
Dna Viruses ◽  
Mutation Scanning ◽  
Method Optimization ◽  
Melting Analysis ◽  
Curve Patterns

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.

scholarly journals Duplex high resolution melting analysis (dHRMA) to detect two hot spot CYP24A1 pathogenic variants (PVs) associated to idiopathic infantile hypercalcemia (IIH)

2021 ◽  
Author(s):  
Maria De Bonis ◽  
Elisa De Paolis ◽  
Maria Elisabetta Onori ◽  
Giorgia Mazzuccato ◽  
Antonio Gatto ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Hot Spot ◽  
Melting Curve ◽  
High Resolution Melting Analysis ◽  
Pathogenic Variants ◽  
Melting Analysis ◽  
Idiopathic Infantile Hypercalcemia ◽  
Set Up ◽  
Peculiar Case

AbstractPathogenic variants (PVs) in CYP24A1 gene are associated with Idiopathic Infantile Hypercalcemia disease (IIH). The identification of CYP24A1 PVs can be a useful tool for the improvement of target therapeutic strategies. Aim of this study is to set up a rapid and inexpensive High Resolution Melting Analysis (HRMA)-based method for the simultaneous genotyping of two hot spot PVs in CYP24A1 gene, involved in IIH. A duplex-HRMA (dHRMA) was designed in order to detect simultaneously CYP24A1 c.428_430delAAG, p.(Glu143del) (rs777676129) and c.1186C > T, p.(Arg396Trp) (rs114368325), in peculiar cases addressed to our Laboratory. dHRMA was able to identify clearly and simultaneously both hot spot CYP24A1 PVs evaluating melting curve shape and melting temperature (Tm). This is the first dHRMA approach to rapidly screen the two most frequent CYP24A1 PVs in peculiar case, providing useful information for diagnosis and patient management in IIH disease.

scholarly journals Parental mosaicism in Marfan and Ehlers–Danlos syndromes and related disorders

2021 ◽  
Author(s):  
Bertrand Chesneau ◽  
Aurélie Plancke ◽  
Guillaume Rolland ◽  
Nicolas Chassaing ◽  
Christine Coubes ◽  
...  
Keyword(s):  
High Resolution ◽  
Marfan Syndrome ◽  
High Resolution Melting ◽  
De Novo ◽  
Direct Sequencing ◽  
Causal Variant ◽  
Connective Tissue Disorder ◽  
High Resolution Melting Analysis ◽  
Aortic Diseases ◽  
Melting Analysis

AbstractMarfan syndrome (MFS) is a heritable connective tissue disorder (HCTD) caused by pathogenic variants in FBN1 that frequently occur de novo. Although individuals with somatogonadal mosaicisms have been reported with respect to MFS and other HCTD, the overall frequency of parental mosaicism in this pathology is unknown. In an attempt to estimate this frequency, we reviewed all the 333 patients with a disease-causing variant in FBN1. We then used direct sequencing, combined with High Resolution Melting Analysis, to detect mosaicism in their parents, complemented by NGS when a mosaicism was objectivized. We found that (1) the number of apparently de novo events is much higher than the classically admitted number (around 50% of patients and not 25% as expected for FBN1) and (2) around 5% of the FBN1 disease-causing variants were not actually de novo as anticipated, but inherited in a context of somatogonadal mosaicisms revealed in parents from three families. High Resolution Melting Analysis and NGS were more efficient at detecting and evaluating the level of mosaicism compared to direct Sanger sequencing. We also investigated individuals with a causal variant in another gene identified through our “aortic diseases genes” NGS panel and report, for the first time, on an individual with a somatogonadal mosaicism in COL5A1. Our study shows that parental mosaicism is not that rare in Marfan syndrome and should be investigated with appropriate methods given its implications in patient’s management.

scholarly journals Rapid detection of fungal pathogens in bronchoalveolar lavage samples using panfungal PCR combined with high resolution melting analysis

2016 ◽  
Vol 54 (7) ◽  
pp. 714-724 ◽  
Author(s):  
Matej Bezdicek ◽  
Martina Lengerova ◽  
Dita Ricna ◽  
Barbora Weinbergerova ◽  
Iva Kocmanova ◽  
...  
Keyword(s):  
High Resolution ◽  
Bronchoalveolar Lavage ◽  
Rapid Detection ◽  
Fungal Pathogens ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Melting Analysis ◽  
Panfungal Pcr
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