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

Farrar, Jared S.; Palais, Robert; Wittwer, Carl T.

doi:10.1373/clinchem.2011.166306

Cited by 16 publications

(19 citation statements)

References 37 publications

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“…It was successfully applied for the genotyping of Factor V Leiden [26], Gilbert Syndrome UGT1A1 (TA) n promoter polymorphism [24] and for the enrichment and detection of rare alleles [25]. As previously reported in other assays utilizing the snapback primer strategy [24]–[26], our method was also found to be optimal using asymmetric concentrations with an excess of snapback primer and limiting the reverse primer. Despite the fact that this assay is based on an intercalating dye (EvaGreen), it remains highly specific due to the snapback strategy which increased the specificity of the test to the similar level with the fluorescently labelled probe assays.…”

Section: Discussionsupporting

confidence: 56%

“…Indeed, the fixed data-acquisition capabilities (3 data points/°C) of the 7500 Fast Real-Time PCR System and the supplied version of the software (version 1.4) were not adequate for evaluating the snapback melting [24].…”

Section: Resultsmentioning

confidence: 99%

“…We investigated the possibility of using an unlabelled probe strategy based on a snapback primer and the intercalating dsDNA EvaGreen dye [24]–[26] for CaPV genotyping using the fluorescent melting curve analysis of the PCR products. Snapback primers are oligonucleotides that include as a probe element a 5′-tail that is complementary to the extension product of the primer and creating a hairpin loop in the single-stranded product [26].…”

Section: Introductionmentioning

confidence: 99%

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Development of a Cost-Effective Method for Capripoxvirus Genotyping Using Snapback Primer and dsDNA Intercalating Dye

et al. 2013

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Sheep pox virus (SPPV), goat pox virus (GTPV) and lumpy skin disease virus (LSDV) are very closely related viruses of the Capripoxvirus (CaPV) genus of the Poxviridae family. They are responsible for sheep pox, goat pox and lumpy skin disease which affect sheep, goat and cattle, respectively. The epidemiology of capripox diseases is complex, as some CaPVs are not strictly host-specific. Additionally, the three forms of the disease co-exist in many sub-Saharan countries which complicates the identification of the virus responsible for an outbreak. Genotyping of CaPVs using a low-cost, rapid, highly specific, and easy to perform method allows a swift and accurate identification of the causative agent and significantly assists in selecting appropriate control and eradication measures, such as the most suitable vaccine against the virus during the outbreaks. The objective of this paper is to describe the design and analytical performances of a new molecular assay for CaPV genotyping using unlabelled snapback primers in the presence of dsDNA intercalating EvaGreen dye. This assay was able to simultaneously detect and genotype CaPVs in 63 samples with a sensitivity and specificity of 100%. The genotyping was achieved by observing the melting temperature of snapback stems of the hairpins and those of the full-length amplicons, respectively. Fourteen CaPVs were genotyped as SPPVs, 25 as GTPVs and 24 as LSDVs. The method is highly pathogen specific and cross platform compatible. It is also cost effective as it does not use fluorescently labelled probes, nor require high-resolution melting curve analysis software. Thus it can be easily performed in diagnostic and research laboratories with limited resources. This genotyping method will contribute significantly to the early detection and genotyping of CaPV infection and to epidemiological studies.

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Section: Discussionsupporting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a Cost-Effective Method for Capripoxvirus Genotyping Using Snapback Primer and dsDNA Intercalating Dye

et al. 2013

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“…Alternate methods can been used to increase the detection sensitivity of melting assays, such as decreasing the PCR product length (10 ), calibration with an internal oligonucleotide duplex (13 ), quantitative heteroduplex analysis after mixing with a known genotype (25 ), or the use of unlabeled probes (14,15 ) or snapback primers (26,27 ). Nevertheless, melting of PCR products has an inherent simplicity that is very attractive.…”

Section: Discussionmentioning

confidence: 99%

Genotyping Accuracy of High-Resolution DNA Melting Instruments

Zhou

Palais

et al. 2014

Clinical Chemistry

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“…The stem-loop hairpin structure of the single-strand DNA amplicon facilitates the selective amplification of the mutant allele, providing the snapback primer system with the favorable sensitivity to identify the trace amount of mutations with as low as 0.01–1% mutation load. Moreover, since the test result is evaluated with melting curve analysis, the snapback primer system can precisely segregate almost all the snapback probe-flanked small sequence alternations, such as SNP, insert, deletion, and even tandem repeats [31], thus minimizing the risk of false negative results induced by the rare mutation. Moreover, the unique melting temperature of each PCR product in melting curve analysis also improves the detection specificity and eliminates underlying false positive results.…”

Section: Discussionmentioning

confidence: 99%

A Multiplex Snapback Primer System for the Enrichment and Detection ofJAK2V617F andMPLW515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

Zhang

et al. 2014

BioMed Research International

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A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.

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

Cited by 16 publications

References 37 publications

Development of a Cost-Effective Method for Capripoxvirus Genotyping Using Snapback Primer and dsDNA Intercalating Dye

Development of a Cost-Effective Method for Capripoxvirus Genotyping Using Snapback Primer and dsDNA Intercalating Dye

Genotyping Accuracy of High-Resolution DNA Melting Instruments

A Multiplex Snapback Primer System for the Enrichment and Detection ofJAK2V617F andMPLW515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

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