scholarly journals Development and evaluation of an unlabeled probe high-resolution melting assay for detection of ATP7B mutations in Wilson's disease

2016 ◽  
Vol 31 (4) ◽  
pp. e22064 ◽  
Author(s):  
Anjian Xu ◽  
Tingxia Lv ◽  
Bei Zhang ◽  
Wei Zhang ◽  
Xiaojuan Ou ◽  
...  
Keyword(s):  
High Resolution ◽  
Wilson’S Disease ◽  
High Resolution Melting ◽  
Wilson's Disease ◽  
Unlabeled Probe

A High-Resolution Melting Analysis with an Unlabeled Probe for CRISPR/Cas9-Induced ZBED6 Knockout Pigs Detection

2020 ◽  
Author(s):  
Xiaofei Liu ◽  
Songyin Qiu ◽  
Lin Mei ◽  
Hongli Jing ◽  
Xiangmei Lin ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Limit Of Detection ◽  
Economic Traits ◽  
Established Method ◽  
Hrm Analysis ◽  
Unlabeled Probe ◽  
Single Base Deletion ◽  
Melting Analysis ◽  
Tissue Specimens

Abstract Background The zinc finger BED-type containing 6 knockout (ZBED6-KO) pigs were created to improve economic traits by increasing the expression of insulin-like growth factor 2. They were generated by CRISPR/CRISPR-associated protein 9 (Cas9) technology and a single-base deletion of ZBED6 was found. An efficient and rapid method was needed to detect this type of pig. Objective This study aimed to develop a high-resolution melting (HRM) method to detect ZBED6-KO pigs. Methods An unlabeled probe and two primers were designed to develop HRM method. The limit of detection, specificity and accuracy of established method were tested by the constructed plasmid and DNA extracts of tissue specimens. Results The limit of detection by established method was 102 copies/µL. The HRM method with an unlabeled probe showed good specificity and high accuracy. Conclusions The established HRM analysis with an unlabeled probe showed it to be a highly effective, rapid and reliable to distinguish ZBED6-KO pigs with wild-type pigs. Highlights It is the first time that HRM analysis with an unlabeled probe has been used in the detection of genome editing pigs by the CRISPR/Cas9 technology.

scholarly journals Identification of TNIP1 Polymorphisms by High Resolution Melting Analysis with Unlabelled Probe: Association with Systemic Lupus Erythematosus

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Jie Zhang ◽  
Yuewen Chen ◽  
Yong Shao ◽  
Qi Wu ◽  
Ming Guan ◽  
...  
Keyword(s):  
High Resolution ◽  
Lupus Erythematosus ◽  
Chinese Population ◽  
High Resolution Melting ◽  
Disease Risk ◽  
High Resolution Melting Analysis ◽  
Negative Regulator ◽  
Hrm Analysis ◽  
Unlabeled Probe ◽  
Melting Analysis

Background. TNFα-induced protein 3 (TNFAIP3) interacting with protein 1 (TNIP1) acts as a negative regulator of NF-κB and plays an important role in maintaining the homeostasis of immune system. A recent genome-wide association study (GWAS) showed that the polymorphism of TNIP1 was associated with the disease risk of SLE in Caucasian. In this study, we investigated whether the association of TNIP1 with SLE was replicated in Chinese population.Methods. The association of TNIP1 SNP rs7708392 (G/C) was determined by high resolution melting (HRM) analysis with unlabeled probe in 285 SLE patients and 336 healthy controls.Results. A new SNP rs79937737 located on 5 bp upstream of rs7708392 was discovered during the HRM analysis. No association of rs7708392 or rs79937737 with the disease risk of SLE was found. Furthermore, rs7708392 and rs79937737 were in weak linkage disequilibrium (LD). Hypotypes analysis of the two SNPs also showed no association with SLE in Chinese population.Conclusions. High resolution melting analysis with unlabeled probes proves to be a powerful and efficient genotyping method for identifying and screening SNPs. No association of rs7708392 or rs79937737 with the disease risk of SLE was observed in Chinese population.

Genotyping cytomegalovirus UL97 mutations by high-resolution melting analysis with unlabeled probe

2011 ◽  
Vol 157 (3) ◽  
pp. 475-481 ◽  
Author(s):  
Xiao-Tao Zhao ◽  
Dan-Qiu Zhou ◽  
Shuai Wu ◽  
Yue-Wen Chen ◽  
Yong Shao ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Unlabeled Probe ◽  
Melting Analysis

Differential alteration of the nigrostriatal dopaminergic system in Wilson's disease investigated with [123I]ß-CIT and high-resolution SPET

2001 ◽  
Vol 28 (11) ◽  
pp. 1656-1663 ◽  
Author(s):  
Henryk Barthel ◽  
Dietlind Sorger ◽  
Hans-Jürgen Kühn ◽  
Armin Wagner ◽  
Regine Kluge ◽  
...  
Keyword(s):  
High Resolution ◽  
Wilson’S Disease ◽  
Dopaminergic System ◽  
Wilson's Disease

scholarly journals The Association of IL-12b Polymorphisms with Systemic Lupus Erythematosus in Chinese Han Population

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Yong Shao ◽  
Jie Zhang ◽  
Yuewen Chen ◽  
Qi Wu ◽  
Ming Guan ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
High Resolution ◽  
Lupus Erythematosus ◽  
Antinuclear Antibody ◽  
High Resolution Melting ◽  
Systemic Lupus ◽  
Chinese Han ◽  
Han Population ◽  
Unlabeled Probe ◽  
Melting Analysis

Background. Systemic lupus erythematosus (SLE) is a complex immune disease. The genetic variation in the IL-12b gene was found to associate with SLE in Caucasian population. In this study, we examined this association in Chinese Han population by a recently developed method, unlabeled probe-based high resolution melting analysis.Methods. A total of 297 SLE patients and 351 controls were recruited. Unlabeled probe-based high resolution melting analysis (HRMA) was used in genotyping.Results. Statistically significant differences were observed in both genotype and allele frequencies for rs6887695 in the SLE patients as compared with the controls. Minor allele (C) of rs6887695 (P=0.031, OR 0.78, [95% CI 0.63-0.98]) was found to be protective against SLE. The association of SNP rs6887695 with the diagnostic criteria of SLE was also examined. Minor allele (C) exerts protective effect on the incidence of arthritis (P=0.013, OR = 0.65, 95% CI = 0.47-0.92) and abnormalities of antinuclear antibody (P=0.022, OR = 0.68, 95% CI = 0.49–0.95). IL-12b SNPs were irrelevant to other diagnostic criteria of SLE.Summary. Polymorphisms of rs6887695 in IL-12b gene were associated with disease risk, as well as arthritis and antinuclear antibody synthesis, of systemic lupus erythematosus in Chinese population.

scholarly journals aac(6′)-Ib-cr Genotyping by Simultaneous High-Resolution Melting Analyses of an Unlabeled Probe and Full-Length Amplicon

2009 ◽  
Vol 54 (3) ◽  
pp. 1378-1380 ◽  
Author(s):  
Jan M. Bell ◽  
John D. Turnidge ◽  
Patiyan Andersson
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Full Length ◽  
Genotyping Assay ◽  
Unlabeled Probe ◽  
Closed Tube ◽  
One Step ◽  
Cost Efficient

ABSTRACT We have developed a time- and cost-efficient one-step closed-tube assay for genotyping of aac(6′)-Ib-cr that is capable of distinguishing between the two genetic aac(6′)-Ib-cr variants. Our genotyping assay uses the combined information of simultaneously acquired high-resolution melting data from an unlabeled probe and the full-length amplicon.

scholarly journals High-Resolution Melting Curve Analysis for High-Throughput Genotyping of NOD2/CARD15 Mutations and Distribution of These Mutations in Slovenian Inflammatory Bowel Diseases Patients

2011 ◽  
Vol 30 (5) ◽  
pp. 265-274 ◽  
Author(s):  
Mitja Mitrovič ◽  
Uroš Potočnik
Keyword(s):  
High Resolution ◽  
Inflammatory Bowel Diseases ◽  
High Resolution Melting ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Probe Design ◽  
Healthy Controls ◽  
Unlabeled Probe ◽  
Bowel Diseases ◽  
Inflammatory Bowel

Inflammatory bowel diseases (IBD) are usually classified into Crohn's disease (CD) and ulcerative colitis (UC). NOD2/CARD15 was the first identified CD-susceptibility gene and was confirmed as the most potent disease gene in CD pathogenesis. ThreeNOD2/CARD15variants, namely two missense polymorphisms R702W (rs2066844) and G908R (rs2066845), and a frame shift polymorphism L1007fs (rs2066847), were associated with CD in Caucasian populations. High resolution melting analysis (HRMA) with saturation LCGreen dyes was previously reported as a simple, inexpensive, accurate and sensitive method for genotyping and/or scanning of rare variants. For this reasons we used qPCR-HRMA for genotypingNOD2/CARD15variants in 588 Slovenian IBD patients and 256 healthy controls. PCR-RFLP was used as a reference method for genotyping of clinical samples. The optimization of an HRM experiment required careful design and adjustment of main parameters, such as primer concentration, MgCl2concentration, probe design and template DNA concentration. Different HRMA approaches were tested and used to develop a reliable and low-cost SNP genotyping assays for polymorphisms inNOD2/CARD15gene. Direct HRMA was the fastest and cheapest HRMA approach for L1007fs and R702W polymorphisms, yet for G908R polymorphism sufficient reliability was achieved after introduction of unlabeled probe. In association analysis, we found statistically significant association of L1007fs (p = 0.001, OR = 3.011, CI95%=1.494–6.071) and G908R (p = 2.62 × 10-4, OR = 14.117, CI95% = 1.884–105.799) polymorphisms with CD patients. At least one ofNOD2/CARD15polymorphisms was found in 78/354 (22.03%) in CD patients, 25/197 (12.69%) in UC patients and in 26/256 (10.15%) in healthy controls. We have successfully implementedNOD2/CARD15HRMA assays, which may contribute to the development of genetic profiles for risk prediction of developing CD and for differential diagnosis of CD vs. UC.

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