Homogeneous Multiplex Genotyping of Hemochromatosis Mutations with Fluorescent Hybridization Probes

Bernard, Philip S.; Ajioka, Richard S.; Kushner, James P.; Wittwer, Carl T.

doi:10.1016/s0002-9440(10)65650-7

Cited by 146 publications

(55 citation statements)

References 39 publications

(30 reference statements)

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“…[17][18][19][20] Heterozygotes are easily identified by a change in shape of the melting curve. 3 Homozygous variants are more difficult to detect and may produce only small differences in T m that are best detected on high-resolution melting instruments.…”

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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“…An assay for hereditary hemochromatosis was described distinguishing the HFE C282Y, H63D and S65L mutant and normal alleles with unique Tms for all four possible alleles. 17 Later, multiplexed assays with different fluorophores used multiple channels of the LightCycler for MTHFR (C677T and A1298C) 18 and HFE (C282Y, H63D, and S65C). 19 With only minor changes, both assays are currently used in clinical laboratories.…”

Section: Genotypingmentioning

confidence: 99%

LightCycler Technology in Molecular Diagnostics

Lyon

Wittwer

2009

The Journal of Molecular Diagnostics

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From the first report of the LightCycler as a real-time polymerase chain reaction (PCR) instrument 1 clinical laboratories quickly realized its potential as a diagnostic tool. Quantitative applications important for infectious disease and oncology applications were followed by genotyping assays that took advantage of accurate temperature control to melt DNA amplicons or probe/amplicon duplexes. The first Food and Drug Administration-cleared molecular genetic assays were developed for this platform, specifically genotyping single base variants in F5 and F2. According to a College of American Pathologists survey, 2 ϳ30% of clinical laboratories report using the LightCycler for these two assays. This article will review the history of LightCycler technology, focusing on those applications widely incorporated into molecular diagnostics. In addition we will review recent developments that may impact future clinical testing.Real-time PCR instruments simultaneously amplify and detect, eliminating the need to open tubes containing PCR amplicon and therefore reducing the risk of future contamination. Fluorescent dyes or probes allow continuous monitoring as template DNA is amplified.3 By monitoring fluorescence every cycle, the amount of original target can be calculated. By monitoring fluorescence as the temperature changes, genotyping and heterozygote scanning can be performed by melting analysis, often removing the need for downstream analysis.The first two platforms developed for real-time PCR were the LightCycler (Roche, Indianapolis, IN) and the ABI 7700 (Applied Biosystems, Foster City, CA). The instruments were as different as the groups that created them. At the time, ABI was the undisputed corporate leader of PCR technology and the 7700 was a large 96-well laser-based instrument focused on throughput.

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“…Dyes that stain double-stranded DNA are commonly used to identify products by their melting temperature (T m ) 1 (2 ). Alternatively, hybridization probes allow genotyping by melting of product/probe duplexes (3)(4)(5)(6).…”

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confidence: 99%

High-Resolution DNA Melting Analysis for Simultaneous Mutation Scanning and Genotyping in Solution

et al. 2005

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Background: High-resolution DNA melting analysis with saturation dyes for either mutation scanning of PCR products or genotyping with unlabeled probes has been reported. However, simultaneous PCR product scanning and probe genotyping in the same reaction has not been described. Methods: Asymmetric PCR was performed in the presence of unlabeled oligonucleotide probes and a saturating fluorescent DNA dye. High-resolution melting curves for samples in either capillaries (0.3°C/s) or microtiter format (0.1°C/s) were generated in the same containers used for amplification. Melting curves of the factor V Leiden single-nucleotide polymorphism (SNP) and several mutations in exons 10 and 11 of the cystic fibrosis transconductance regulator gene were analyzed for both PCR product and probe melting transitions. Results: Independent verification of genotype for simple SNPs was achieved by either PCR product or probe melting transitions. Two unlabeled probes in one reaction could genotype many sequence variants with simultaneous scanning of the entire PCR product. For example, analysis of both product and probe melting transitions genotyped ⌬F508, ⌬I507, Q493X, I506V, and F508C variants in exon 10 and G551D, G542X, and R553X variants in exon 11. Unbiased hierarchal clustering of the melting transitions identified the specific sequence variants. Conclusions: When DNA melting is performed rapidly and observed at high resolution with saturating DNA dyes, it is possible to scan for mutations and genotype at the same time within a few minutes after amplification.

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Homogeneous Multiplex Genotyping of Hemochromatosis Mutations with Fluorescent Hybridization Probes

Cited by 146 publications

References 39 publications

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

LightCycler Technology in Molecular Diagnostics

High-Resolution DNA Melting Analysis for Simultaneous Mutation Scanning and Genotyping in Solution

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