Snapback Primer Genotyping with Saturating DNA Dye and Melting Analysis

Zhou, Luming; Errigo, Roscoe J.; Lu, Hongzhe; Poritz, Mark A.; Seipp, Michael T.; Wittwer, Carl T.

doi:10.1373/clinchem.2008.107615

Cited by 49 publications

(48 citation statements)

References 39 publications

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“…D: The snapback primer design only requires two primers, one with a 5Ј extension that is complementary to its own extension product. 39 Asymmetric PCR overproduces one strand, so that both full-length PCR product (amplicon) and an excess of one strand are formed. The excess single strand snaps back on itself (dotted line) so that its complementary regions (light gray lines) anneal to form a hairpin stem.…”

Section: Reducing Complexitymentioning

confidence: 99%

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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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Section: Reducing Complexitymentioning

confidence: 99%

“…39 Such snapback primers form intramolecular hairpins (similar to the more complicated Scorpion primers) and allow genotyping by melting. With snapback primers, only two unlabeled oligonucleotides are necessary and 3Ј-blocking is not required ( Figure 3D).…”

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confidence: 99%

LightCycler Technology in Molecular Diagnostics

Lyon

Wittwer

2009

The Journal of Molecular Diagnostics

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“…[36][37][38] However, several strategies can be used to achieve better resolution: genotyping using small amplicons, unlabelled probes, snapback primers, internal temperature calibrators and/or mixing patient samples with the reference control genotype. [39][40][41][42][43][44] We used internal calibrators and DNA mixing to improve the resolution of individual genotypes for four amplicons of the COX genes. However, the majority of examined amplicons did not require these adjustments.…”

Section: Discussionmentioning

confidence: 99%

High-resolution melting analysis of 15 genes in 60 patients with cytochrome-c oxidase deficiency

et al. 2012

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Cytochrome-c oxidase (COX) deficiency is one of the common childhood mitochondrial disorders. Mutations in genes for the assembly factors SURF1 and SCO2 are prevalent in children with COX deficiency in the Slavonic population. Molecular diagnosis is difficult because of the number of genes involved in COX biogenesis and assembly. The aim of this study was to screen for mutations in 15 nuclear genes that encode the 10 structural subunits, their isoforms and two assembly factors of COX in 60 unrelated Czech children with COX deficiency. Nine novel variants were identified in exons and adjacent intronic regions of COX4I2, COX6A1, COX6A2, COX7A1, COX7A2 and COX10 using high-resolution melting (HRM) analysis. Online bioinformatics servers were used to predict the importance of the newly identified amino-acid substitutions. The newly characterized variants updated the contemporary spectrum of known genetic sequence variations that are present in the Czech population, which will be important for further targeted mutation screening in Czech COX-deficient children. HRM and predictive bioinformatics methodologies are advantageous because they are low-cost screening tools that complement largescale genomic studies and reduce the required time and effort.

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“…HRMA can effectively distinguish between different alleles and genotypes at single nucleotide polymorphism (SNP) loci (13,15,16). To date, three novel methods have been developed for HRMA: The small-fragment amplification method (17), the non-labeled probe method (18) and the snapback probe primer method (19). A recent study showed that the method of genomic DNA extraction influences the polymerase chain reaction (PCR)-HRMA product (20).…”

Section: Introductionmentioning

confidence: 99%

Correlation between thyroglobulin gene polymorphisms and autoimmune thyroid disease

Wang¹,

Wang²,

Qilin³

et al. 2015

Molecular Medicine Reports

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Abstract. The aim of the present study was to detect thyroglobulin (Tg) gene polymorphisms in a Han Chinese population from the Northern regions of Henan province, China, and to study the correlation between Tg gene polymorphisms and autoimmune thyroid disease (AITD). A total of 270 patients with AITD and 135 healthy controls were enrolled. Genomic DNA was extracted and fluorescence polymerase chain reaction analysis was performed; high-resolution melting curve analysis (HRMA) was used to detect single-nucleotide polymorphisms (SNPs) in exons 10, 12 and 33 of the Tg gene. SNPs were then correlated with AITD. Han people from the Northern regions of Henan displayed four Tg exon SNPs: E10SNP24 T/G, E10SNP158 T/C, E12SNP A/G and E33SNP C/T. Several allele and genotype frequencies differed between the AITD group and the healthy control group (Tg E10SNP: Allele T, P<0.01; allele G, P<0.01; and Tg genotype GG, P<0.01; genotype TG, P<0.01. Tg E12SNP: Allele A, P<0.01; allele G, P<0.01; Tg genotype GG, P<0.01; genotype AG, P<0.01). A statistically significant difference in the frequency of selected Tg SNPs haplotypes was also present between AITD patients and healthy controls (P<0.05). There was no significant difference in haplotypes between various types of AITD (hypothyroidism, hyperthyroidism and Hashimoto's disease). The Tg SNP frequency distribution was significantly different between Han populations of the Northern regions of Henan province and the Xi'an regions of Shaanxi province. The results of the present study suggested that specific Tg gene alleles or genotypes were correlated with AITD; specific Tg SNP haplotypes were associated with hypothyroidism, hyperthyroidism and Hashimoto's disease, and the Tg SNP frequency distribution differed depending on the geographical location of the Han Chinese populations.

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Snapback Primer Genotyping with Saturating DNA Dye and Melting Analysis

Cited by 49 publications

References 39 publications

LightCycler Technology in Molecular Diagnostics

LightCycler Technology in Molecular Diagnostics

High-resolution melting analysis of 15 genes in 60 patients with cytochrome-c oxidase deficiency

Correlation between thyroglobulin gene polymorphisms and autoimmune thyroid disease

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