Closed-Tube SNP Genotyping Without Labeled Probes/A Comparison Between Unlabeled Probe and Amplicon Melting

Liew, Michael; Seipp, Michael T.; Durtschi, Jacob D.; Margraf, Rebecca L.; Dames, Shale; Erali, Maria; Voelkerding, Karl V.; Wittwer, Carl T.

doi:10.1309/p155-6428-5484-u0q1

Cited by 20 publications

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“…The method has been applied to many common clinical single-base variants 43 and compared against dual-hybridization probes 44 and unlabeled probes. 45 However, many small insertions/deletions and some single base changes can be difficult to distinguish from wild type when homozygous. For example, bp neutral single base changes with nearest neighbor symmetry are predicted to have identical Tms.…”

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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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confidence: 99%

LightCycler Technology in Molecular Diagnostics

Lyon

Wittwer

2009

The Journal of Molecular Diagnostics

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“…Additional closed tube methods that do not require labeled probes include snapback primers 8 and unlabeled probes. 3 SNE by high resolution melting has the advantage over these aforementioned methods in that it directly identifies the base substitution at a particular position, which would be most useful for nucleotide positions that are particularly variable.…”

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confidence: 99%

“…Genotyping for each assay was based on the melting pattern of the extension products, taken from the derivative melting temperature plots of the raw LS32 data. Melting temperatures and curves were calculated using both the analysis software from the LS32 as well as custom software previously used 3 in high resolution melting analysis.…”

Section: Genotypingmentioning

confidence: 99%

“…The original DNA binding dyes (e. g., SYBR green I) used for genotyping are accurate only when large differences in melting temperature are generated with allele specific PCR and GCrich tails. 1 However, better dyes and instruments are available today that recognize small differences in amplicon Tm and melting curve shape by increasing assay resolution, [2][3][4] rather than introducing artificial Tm differences by allele-specific PCR. Small amplicon melting is a minimalistic way to genotype most single nucleotide polymorphisms (SNPs).…”

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confidence: 99%

“…6 This is the case for the c.187CϾG (H63D) SNP identified in the hemochromatosis gene (HFE). 3 The sequence surrounding the SNP is ATCAT with the underlined base indicating the position of the SNP. The sequence from 5Ј to 3Ј in both sense and antisense directions is the same for both homozygotes.…”

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Nucleotide Extension Genotyping by High-Resolution Melting

Liew

Wittwer

Voelkerding

2010

The Journal of Molecular Diagnostics

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One limitation of small amplicon melting is the inability to genotype certain nearest-neighbor symmetric variations without manipulating the sample. We have developed a method for these exceptions: a high-resolution melting single nucleotide extension assay. Single nucleotide extension was performed in a new instrument , the LightScanner 32 (LS32) , which uses capillary reaction tubes and is capable of realtime PCR and sequential high-resolution melting of 32 samples. Asymmetric PCR used Platinum Taq and LC Green Plus in the master mix for target amplification. Dideoxynucleotides and extension oligonucleotides were sequestered in the tube cap and added post-PCR, maintaining a closed system. One dideoxynucleotides was used per capillary tube. Samples were cycled five times to incorporate dideoxynucleotides into the extension products using ThermoSequenase , followed by high-resolution melting. Single nucleotide polymorphisms from the RET proto-oncogene (n ‫؍‬ 7), hemochromatosis (HFE , n ‫؍‬ 30) , coagulation factor 2 (F2 , n ‫؍‬ 29) , coagulation factor 5 (F5 , n ‫؍‬ 30) , and methylenetetrahydrofolate reductase (MTHFR , n ‫؍‬ 60) genes were genotyped. The DNA melting profiles identified the target single nucleotide polymorphisms by the lowest melting temperature transition. All genotypes had a distinctive melting pattern. The method was 100% concordant with samples previously genotyped at HFE, MTHFR, and F2 and 90% concordant with F5. F5 discordants were genotyped correctly by redesigning the assay. Our results demonstrate that although single nucleotide polymorphisms can be successfully differentiated using this methodology , the method requires careful optimization.

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Streamlined assessment of gene variants by high resolution melt profiling utilizing the ornithine transcarbamylase gene as a model system

Dobrowolski¹,

Ellingson²,

Caldovic

et al. 2007

Hum. Mutat.

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Ornithine transcarbamylase (OTC) deficiency is an X-linked, semidominant genetic disorder and the most prevalent inherited defect of the urea cycle. Molecular genetic testing of the OTC gene is critically important for clinical diagnosis, carrier testing, and prenatal diagnosis. Private mutations are observed throughout the OTC gene with more than 340 reported disease-causing mutations. High-resolution melt profiling was adapted to perform homogeneous analysis of the 10 coding regions and their intronic flanks in a 96-well plate format. The 10 DNA fragments ranging from 146 bp to 266 bp are amplified in a PCR run. A common analysis condition simultaneously generates melting profiles from all 10 fragments. To streamline analysis, deviant profiles resulting from common polymorphic variants are triaged using redundant assessment with melt profile controls in selected whole-exon assays and a separate multiplex genotyping assay. The test is further streamlined by recovering dye-stained amplification product from the melt profiling plate to serve as DNA sequencing template. Described herein is the comprehensive analysis of the OTC gene in 23 OTC-deficient patients. This system provides a rapid means to localize sequence variants, markedly reducing the need for DNA sequencing, and is applicable to other genes and disorders.

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Closed-Tube SNP Genotyping Without Labeled Probes/A Comparison Between Unlabeled Probe and Amplicon Melting

Cited by 20 publications

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LightCycler Technology in Molecular Diagnostics

LightCycler Technology in Molecular Diagnostics

Nucleotide Extension Genotyping by High-Resolution Melting

Streamlined assessment of gene variants by high resolution melt profiling utilizing the ornithine transcarbamylase gene as a model system

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