scholarly journals High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen

2003 ◽  
Vol 49 (6) ◽  
pp. 853-860 ◽  
Author(s):  
Carl T Wittwer ◽  
Gudrun H Reed ◽  
Cameron N Gundry ◽  
Joshua G Vandersteen ◽  
Robert J Pryor
Keyword(s):  
High Resolution ◽  
Mutation Screening ◽  
Melting Curve ◽  
Single Nucleotide ◽  
Mutation Scanning ◽  
Double Stranded Dna ◽  
Closed Tube ◽  
Pcr Products ◽  
Melting Analysis ◽  
Detection Of Mutations

Abstract Background: High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396–406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides. Methods: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), β-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR. Results: The six common β-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1–2 min, amplification and analysis were achieved in 10–20 min with rapid cycling conditions. Conclusions: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.

scholarly journals Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye

2004 ◽  
Vol 50 (8) ◽  
pp. 1328-1335 ◽  
Author(s):  
Luming Zhou ◽  
Alexander N Myers ◽  
Joshua G Vandersteen ◽  
Lesi Wang ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Oligonucleotide Probes ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Melting Curves ◽  
Unlabeled Probe ◽  
Closed Tube ◽  
Melting Analysis

Abstract Background: Homogeneous PCR methods for genotyping usually require fluorescently labeled oligonucleotide probes. Amplicon melting with the DNA dye LCGreen™ I was recently introduced as a closed-tube method of genotyping that does not require probes or real-time PCR. However, some single-nucleotide polymorphisms (SNPs) could not be completely genotyped without addition of a known genotype, and high-resolution melting techniques were necessary. Methods: A 3′-blocked, unlabeled oligonucleotide probe and the saturating dye, LCGreen I, were added to standard PCR reagents before amplification. After PCR, the samples were melted at 0.1–0.3 °C/s in high-resolution (HR-1™), high-throughput (LightTyper™), and rapid-cycle, real-time (LightCycler®) instruments, and fluorescence melting curves were recorded. Results: Derivative melting curves of the probe–target duplexes were characteristic of the genotype under the probe. With synthetic plasmid templates, all SNP base combinations could be genotyped. For human genomic DNA, the technique was demonstrated with mutations associated with cystic fibrosis, including SNPs (G542X, I506V, and F508C) and 3-bp deletions (F508del and I507del). Conclusions: Genotyping of SNPs and small deletions by melting analysis of an unlabeled probe in the presence of LCGreen I is simple and rapid. Only three unlabeled oligonucleotides (two primers and one probe), a saturating DNA dye, PCR, and a melting instrument are required. The method is closed-tube, does not require fluorescently labeled probes or real-time PCR, and can be completed in <10 min on any instrument capable of monitoring melting curves by fluorescence.

scholarly journals Mutation Scanning of the RET Protooncogene Using High-Resolution Melting Analysis

2006 ◽  
Vol 52 (1) ◽  
pp. 138-141 ◽  
Author(s):  
Rebecca L Margraf ◽  
Rong Mao ◽  
W Edward Highsmith ◽  
Leonard M Holtegaard ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
Mutation Detection ◽  
Melting Curve ◽  
Curve Shape ◽  
Wild Type ◽  
Ret Protooncogene ◽  
Mutation Scanning ◽  
Ret Mutation ◽  
Sequence Variations ◽  
Melting Analysis

Abstract Background: Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. The current widely used approach for RET mutation detection is sequencing of the exons. Methods: Because RET mutations are rare and the majority are heterozygous mutations, we investigated RET mutation detection by high-resolution amplicon melting analysis. This mutation scanning technique uses a saturating double-stranded nucleic acid binding dye, LCGreen®, and the high-resolution melter, HR-1™, to detect heterozygous and homozygous sequence variations. Mutant genotypes are distinguished from the wild-type genotype by an altered amplicon melting curve shape or position. Results: Samples of 26 unique RET mutations, 4 nonpathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mutation-scanning assay differentiated RET sequence variations from the wild-type genotype by altered derivative melting curve shape or position. A blinded study of 80 samples (derived from the 35 mutant, polymorphism, or wild-type samples) demonstrated that 100% of RET sequence variations were differentiated from wild-type samples. For exons 11 and 13, the nonpathogenic polymorphisms could be distinguished from the pathogenic RET mutations. Some RET mutations could be directly genotyped by the mutation scanning assay because of unique derivative melting curve shapes. Conclusion: RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.

scholarly journals Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High-Resolution Melting Analysis

2004 ◽  
Vol 50 (10) ◽  
pp. 1748-1754 ◽  
Author(s):  
Gudrun H Reed ◽  
Carl T Wittwer
Keyword(s):  
Single Nucleotide Polymorphism ◽  
High Resolution ◽  
Sensitivity And Specificity ◽  
High Resolution Melting ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Product Size ◽  
Pcr Products ◽  
Melting Analysis

Abstract Background: Screening for heterozygous sequence changes in PCR products, also known as “mutation scanning”, is an important tool for genetic research and clinical applications. Conventional methods require a separation step. Methods: We evaluated the sensitivity and specificity of homogeneous scanning, using a saturating DNA dye and high-resolution melting. Heterozygous single-nucleotide polymorphism (SNP) detection was studied in three different sequence backgrounds of 40%, 50%, and 60% GC content. PCR products of 50–1000 bp were generated in the presence of LCGreen™ I. After fluorescence normalization and temperature overlay, melting curve shape was used to judge the presence or absence of heterozygotes among 1632 cases. Results: For PCR products of 300 bp or less, all 280 heterozygous and 296 wild-type cases were correctly called without error. In 672 cases between 400 and 1000 bp with the mutation centered, the sensitivity and specificity were 96.1% and 99.4%, respectively. When the sequence background and product size with the greatest error rate were used, the sensitivity of off-center SNPs (384 cases) was 95.6% with a specificity of 99.4%. Most false negatives occurred with SNPs that were compared with an A or T wild type sequence. Conclusions: High-resolution melting analysis with the dye LCGreen I identifies heterozygous single-base changes in PCR products with a sensitivity and specificity comparable or superior to nonhomogeneous techniques. The error rate of scanning depends on the PCR product size and the type of base change, but not on the position of the SNP. The technique requires only PCR reagents, the dye LCGreen I, and 1–2 min of closed-tube, post-PCR analysis.

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

2007 ◽  
Vol 53 (2) ◽  
pp. 349-352 ◽  
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.

scholarly journals Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dyes

2006 ◽  
Vol 52 (3) ◽  
pp. 494-503 ◽  
Author(s):  
Mark G Herrmann ◽  
Jacob D Durtschi ◽  
L Kathryn Bromley ◽  
Carl T Wittwer ◽  
Karl V Voelkerding
Keyword(s):  
High Resolution ◽  
Globin Gene ◽  
Melting Curve ◽  
Sybr Green ◽  
Sybr Green I ◽  
Dna Melting ◽  
Curve Shape ◽  
Comparative Performance ◽  
Mutation Scanning ◽  
Melting Analysis

Abstract Background: DNA melting analysis for genotyping and mutation scanning of PCR products by use of high-resolution instruments with special “saturation” dyes has recently been reported. The comparative performance of other instruments and dyes has not been evaluated. Methods: A 110-bp fragment of the β-globin gene including the sickle cell anemia locus (A17T) was amplified by PCR in the presence of either the saturating DNA dye, LCGreen Plus, or SYBR Green I. Amplicons of 3 different genotypes (wild-type, heterozygous, and homozygous mutants) were melted on 9 different instruments (ABI 7000 and 7900HT, Bio-Rad iCycler, Cepheid SmartCycler, Corbett Rotor-Gene 3000, Idaho Technology HR-1 and LightScanner, and the Roche LightCycler 1.2 and LightCycler 2.0) at a rate of 0.1 °C/s or as recommended by the manufacturer. The ability of each instrument/dye combination to genotype by melting temperature (Tm) and to scan for heterozygotes by curve shape was evaluated. Results: Resolution varied greatly among instruments with a 15-fold difference in Tm SD (0.018 to 0.274 °C) and a 19-fold (LCGreen Plus) or 33-fold (SYBR Green I) difference in the signal-to-noise ratio. These factors limit the ability of most instruments to accurately genotype single-nucleotide polymorphisms by amplicon melting. Plate instruments (96-well) showed the greatest variance with spatial differences across the plates. Either SYBR Green I or LCGreen Plus could be used for genotyping by Tm, but only LCGreen Plus was useful for heterozygote scanning. However, LCGreen Plus could not be used on instruments with an argon laser because of spectral mismatch. All instruments compatible with LCGreen Plus were able to detect heterozygotes by altered melting curve shape. However, instruments specifically designed for high-resolution melting displayed the least variation, suggesting better scanning sensitivity and specificity. Conclusion: Different instruments and dyes vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole-amplicon melting analysis.

scholarly journals PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method

2010 ◽  
Vol 12 (5) ◽  
pp. 697-704 ◽  
Author(s):  
Panagiotis A. Vorkas ◽  
Nikoleta Poumpouridou ◽  
Sophia Agelaki ◽  
Christos Kroupis ◽  
Vassilis Georgoulias ◽  
...  
Keyword(s):  
High Resolution ◽  
Analysis Method ◽  
Mutation Scanning ◽  
Highly Sensitive ◽  
Melting Analysis ◽  
Hotspot Mutation

Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis

2007 ◽  
Vol 2 (1) ◽  
pp. 59-66 ◽  
Author(s):  
Jesse Montgomery ◽  
Carl T Wittwer ◽  
Robert Palais ◽  
Luming Zhou
Keyword(s):  
High Resolution ◽  
Dna Melting ◽  
Mutation Scanning ◽  
Melting Analysis
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