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

Gundry, Cameron N.; Vandersteen, Joshua G.; Reed, Gudrun H.; Pryor, Robert J.; Chen, Jian; Wittwer, Carl T.

doi:10.1373/49.3.396

Cited by 315 publications

(251 citation statements)

References 31 publications

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“…However, they will not control for T m variation secondary to the concentration of amplified DNA. Luckily, this variation is minor, 10 and the PCR plateau tends to equalize any difference in starting DNA concentration.…”

Section: Discussionmentioning

confidence: 99%

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

Seipp

Durtschi

Liew

et al. 2007

The Journal of Molecular Diagnostics

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Amplicon melting is a closed-tube method for genotyping that does not require probes , real-time analysis , or allele-specific polymerase chain reaction. However , correct differentiation of homozygous mutant and wild-type samples by melting temperature (T m ) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood , amplicon T m differences of 0.03 to 0.39°C attributable to the extractions were observed. To correct for solution chemistry differences between samples , complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. Highand low-temperature controls bracketing the amplicon melting region decreased the T m SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included , whereas a 3% error rate was observed without temperature correction. In conclusion , internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments. Amplicon melting analysis is a simple closed-tube genotyping method that uses a saturating DNA binding dye instead of fluorescently labeled primers or probes.1 Highresolution melting analysis can detect single base changes and other variations in single or multiplex polymerase chain reaction (PCR).2 Wild-type and homozygous mutant samples typically have sharp, symmetric melting transitions, whereas heterozygous samples have more complex, gradual melting curves. Homozygous sequence changes result in characteristic shifts in melting temperature (T m ). [2][3][4][5] In contrast, heterozygous samples are identified by melting peak shape and width and not by T m . Correct identification of sample genotype by amplicon melting requires standardization of reaction conditions to achieve reproducible, characteristic melting profiles. Reaction conditions can vary between lots of PCR reagents, including different buffers introduced by the DNA isolation method. Ionic strength, in particular, significantly affects T m . -10The current study introduces the use of one or more internal controls for temperature calibration between reactions. Complimentary, unlabeled oligonucleotides that do not interfere with the PCR were designed so that they melt outside the temperature region of PCR product melting. Any buffer differences that affect duplex T m s affects both the amplicon and the internal temperature controls, allowing subsequent temperature correction of melting profiles. As a genotyping target, the 1298AϾC and 677CϾT variants of the methylenetetrahydrofolate reductase (MTHFR) gene were used. A single-color duplex amplicon melting assay (with a...

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Section: Discussionmentioning

confidence: 99%

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

Seipp

Durtschi

Liew

et al. 2007

The Journal of Molecular Diagnostics

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“…A first blood sample was drawn through an antecubital cannula immediately after patients sat down and a 2nd blood sample after 30 min of supine rest. Plasma samples were assayed for concentrations of metanephrines (6 ).…”

Section: Introductionmentioning

confidence: 99%

“…LC Green Plus (Idaho Technology) can be used at concentrations that do not inhibit PCR amplification but efficiently saturate PCR products. HRM analysis of PCR products amplified in the presence of LC Green Plus can detect heterozygous and most homozygous sequence variations by the difference in shape and position of the melting curve when compared with a wild-type melt profile (4,6 ). The method would therefore be amenable to screening an X-linked dominant disorder with heterozygous females and hemizygous male patients.…”

mentioning

confidence: 99%

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

Kennerson

Warburton²,

Nelis³

et al. 2007

Clinical Chemistry

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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 DNAbinding 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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“…These features provide real advantages for the clinical microbiology laboratory. A recent development in real-time PCR technology is high-resolution DNA melting (HRM) analysis (8,9 ). Although melt curves are predominantly used to determine the melting temperature (T m ) of amplified double-stranded DNA, it is recognized that the precise shape of a melting curve is a function of the DNA sequence, and this characteristic forms the basis of HRM analysis (10 ).…”

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confidence: 99%

High-Resolution Melting Analysis of the spa Repeat Region of Staphylococcus aureus

et al. 2008

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BACKGROUND:The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequencebased genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. We describe here a method for genotyping S. aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X.

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

Cited by 315 publications

References 31 publications

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

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

High-Resolution Melting Analysis of the spa Repeat Region of Staphylococcus aureus

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