scholarly journals Snapback Primer Genotyping of the Gilbert Syndrome UGT1A1 (TA)n Promoter Polymorphism by High-Resolution Melting

2011 ◽  
Vol 57 (9) ◽  
pp. 1303-1310 ◽  
Author(s):  
Jared S Farrar ◽  
Robert A Palais ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Pcr Primers ◽  
Promoter Polymorphism ◽  
Fragment Analysis ◽  
Gilbert Syndrome ◽  
Blinded Study ◽  
Thymine Adenine ◽  
Local Deviation ◽  
Genotype Clustering

BACKGROUND Gilbert syndrome, a chronic nonhemolytic unconjugated hyperbilirubinemia, is associated with thymine–adenine (TA) insertions in the UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1) promoter. The UGT1A1 promoter genotype also correlates with toxicity induced by the chemotherapeutic drug irinotecan. Current closed-tube assays for genotyping the UGT1A1 (TA)n promoter polymorphism require multiple labeled probes and/or have difficulty classifying the (TA)5 and (TA)8 alleles. METHODS An unlabeled 5′ extension on one primer that creates a hairpin after asymmetric PCR was used to develop a snapback primer high-resolution melting assay for the (TA)n polymorphism. A new method that plots the local deviation from exponential decay to improve genotype clustering was used to remove background fluorescence and to analyze the data. The snapback assay was compared with small-amplicon melting and fragment length analyses in a blinded study of DNA samples from 100 African Americans. RESULTS Genotyping results obtained by small-amplicon melting and snapback primer melting were 83% and 99% concordant, respectively, with results obtained by fragment analysis. Reanalysis of the single discordant sample in the results of the snapback genotyping assay and the fragment analysis revealed an error in the fragment analysis. High-resolution melting was required for accurate snapback genotyping of the UGT1A1 (TA)n polymorphism. The 100% accuracy obtained with a capillary-based instrument fell to ≤81% with plate-based instruments. CONCLUSIONS In contrast to small-amplicon genotyping, snapback primer genotyping can distinguish all UGT1A1 promoter genotypes. Rapid-cycle PCR combined with snapback primer analysis with only 2 unlabeled PCR primers (one with a 5′ extension) and a saturating DNA dye can genotype loci with several alleles in <30 min.

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 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.

High Resolution Melting Analysis (HRMA) for the identification of a rare UGT1A1 promoter polymorphism

2011 ◽  
Vol 44 (16) ◽  
pp. 1359-1360 ◽  
Author(s):  
Angelo Minucci ◽  
Enrica Mello ◽  
Domenico Tripodi ◽  
Paola Concolino ◽  
Cecilia Zuppi ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Promoter Polymorphism ◽  
High Resolution Melting Analysis ◽  
Melting Analysis

scholarly journals A Combination of Real-Time PCR and High-Resolution Melting Analysis to Detect and Identify CpGV Genotypes Involved in Type I Resistance

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 723 ◽  
Author(s):  
Aurélie Hinsberger ◽  
Stéphane Theulier Saint Germain ◽  
Patrice Guerrero ◽  
Christine Blachère-López ◽  
Miguel López-Ferber ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Cydia Pomonella ◽  
Relative Proportion ◽  
Variable Region ◽  
Codling Moth ◽  
Pcr Primers ◽  
Type I ◽  
Occlusion Bodies ◽  
Type I Resistance

Cydia pomonella granulovirus, in particular CpGV-M isolate, is used as a biological control against the codling moth (CM), Cydia pomonella. As a result of intensive control over the years, codling moth populations have developed resistance against this isolate. This resistance is now called type I resistance. Isolates, among them, CpGV-R5, have been found that are able to overcome type I resistance. Both CpGV-M and CpGV-R5 are used in orchards to control the codling moth. High resolution melting (HRM) has been adapted to differentiate between CpGV-M and CpGV-R5 isolates. Specific PCR primers have been designed for the CpGV p38 gene, encompassing the variable region responsible for the ability to overcome resistance. Because each amplicon has a specific melting point, it is possible to identify the CpGV-M and CpGV-R5 genotypes and to quantify their relative proportion. This method has been validated using mixtures of occlusion bodies of each isolate at various proportions. Then, the HRM has been used to estimate the proportion of each genotype in infected larvae or in occlusion bodies (OBs) extracted from dead larvae. This method allows a rapid detection of genotype replication and enables the assessment of either success or failure of the infection in field conditions.

scholarly journals Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM)

2021 ◽  
pp. 1-8
Author(s):  
Xiaochun Zhang ◽  
Huan Yu ◽  
Ziwei Wang ◽  
Qi Yang ◽  
Ruocheng Xia ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting

High-resolution melting PCR assay as a powerful tool for the epidemiological surveillance of tularemia in Western Europe

2021 ◽  
Vol 90 ◽  
pp. 104741
Author(s):  
Maëllys Kevin ◽  
Guillaume Girault ◽  
Yvan Caspar ◽  
Moulay Ali Cherfa ◽  
Christiane Mendy ◽  
...  
Keyword(s):  
High Resolution ◽  
Western Europe ◽  
High Resolution Melting ◽  
Epidemiological Surveillance ◽  
Pcr Assay

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

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.

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

Genes ◽  
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.

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.

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