scholarly journals Rapid Genotyping of the Human Renin (REN) Gene by the LightCycler® Instrument: Identification of Unexpected Nucleotide Substitutions within the Selected Hybridization Probe Area

2010 ◽  
Vol 29 (5) ◽  
pp. 243-249 ◽  
Author(s):  
Line Wee ◽  
Hege Vefring ◽  
Grete Jonsson ◽  
Astanand Jugessur ◽  
Rolv Terje Lie
Keyword(s):  
Melting Curve ◽  
Renin Angiotensin System ◽  
Western World ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Substitutions ◽  
Hybridization Probe ◽  
Hybridization Probes ◽  
Specific Hybridization ◽  
Allele Specific

Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS) may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN) that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm). Ninety-two mother-father-child triads (n=276) from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs) inREN. All three htSNPs (rs5705, rs1464816 and rs3795575) were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041) were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

scholarly journals Direct Molecular Haplotyping of the IVS-8 Poly(TG) and PolyT Repeat Tracts in the Cystic Fibrosis Gene by Melting Curve Analysis of Hybridization Probes

2005 ◽  
Vol 51 (9) ◽  
pp. 1619-1623 ◽  
Author(s):  
Alison Millson ◽  
Genevieve Pont-Kingdon ◽  
Sam Page ◽  
Elaine Lyon
Keyword(s):  
Perfect Match ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Clinical Diagnostics ◽  
Cystic Fibrosis Gene ◽  
Cftr Gene ◽  
Melting Curve Analysis ◽  
Hybridization Probe ◽  
Hybridization Probes

Abstract Background: Molecular haplotyping is a developing technology with great potential for use in clinical diagnostics. We describe a haplotyping method that uses PCR combined with hybridization probes. Methods: We designed a LightCycler assay that uses fluorescence resonance energy transfer hybridization probes to haplotype the poly(TG) and polyT (TG-T) tract in the IVS-8 region of the CFTR gene. The reporter probe was designed as a perfect match to the TG12-5T allele. Results: Analysis of 132 samples revealed 9 unique derivative melting temperatures (Tms); the lowest was 42.4 °C and the highest was 63.6 °C. The lowest Tms were in the TGn-9T group, the intermediate Tms in the TGn-7T group, and the highest Tms in the TGn-5T group. Haplotype frequencies were highest (39%) for TG11-7T and lowest (0.4%) for TG13-5T. Conclusions: Different combinations of polymorphisms under the reporter hybridization probe had unique and characteristic Tms. This property enables genotyping as well as determination of the phase of multiple variants under the probe, a principle we demonstrated by haplotyping the TG-T repeat tract in the IVS-8 region of the CFTR gene.

scholarly journals A Novel Test System for Genotyping rs43703016 Single-nucleotide Substitutions in the Bovine CSN3 Gene

2019 ◽  
pp. 1-5
Author(s):  
Svetlana Kovalchuk ◽  
Arina Tagmazyan ◽  
Eugene Klimov
Keyword(s):  
Nucleotide Substitution ◽  
Milk Proteins ◽  
Test System ◽  
Nucleotide Polymorphisms ◽  
Dna Testing ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Allele Specific ◽  
Selection Of

Aims: Caseins are among the main milk proteins that determine many of its properties. Bovine kappa-casein (CSN3) is associated with the qualitative composition of milk, as well as with the quality of cheese obtained from this milk. The rs43703016 single-nucleotide substitution (g.88532332A>C; Asp148Ala) in exon 4 of the bovine CSN3 gene plays an important role in the production of quality hard cheeses. Various methods for the DNA testing of this substitution have been developed in the last three decades. Emergent DNA technologies provide an opportunity to modernize methods of genotyping single-nucleotide polymorphisms. Results: We have developed and verified a method to differentiate A/C alleles of the rs43703016 substitution in the bovine CSN3 gene by real-time PCR using allele-specific fluorescent probes. Conclusion: Our new method allows fast genotyping of animals, and may be used for selection of cows carrying the CC genotype, which determines good cheese-making properties of milk.

scholarly journals Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252887
Author(s):  
Renate Schneider ◽  
Aline Lamien-Meda ◽  
Herbert Auer ◽  
Ursula Wiedermann-Schmidt ◽  
Peter L. Chiodini ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Melting Curve ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Significant Rise ◽  
Quantitative Detection ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphisms

Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites/mL blood for P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.

scholarly journals High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

2021 ◽  
pp. 1-8
Author(s):  
Oytip Nathalang ◽  
Kamphon Intharanut ◽  
Sarisa Chidtrakoon
Keyword(s):  
High Resolution ◽  
Blood Group ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Blood Group Antigens ◽  
Red Cell ◽  
Nucleotide Polymorphisms ◽  
Hrm Analysis ◽  
Antibody Specificities

<b><i>Background:</i></b> High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. <b><i>Methods:</i></b> Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of <i>KEL</i>*<i>01</i> and <i>KEL</i>*<i>02</i>, <i>JK</i>*<i>01</i> and <i>JK</i>*<i>02</i>, <i>FY</i>*<i>01</i>, <i>FY</i>*<i>02</i>, and <i>FY</i>*<i>02 N.01</i>, <i>DI</i>*<i>01</i> and <i>DI</i>*<i>02</i>, <i>GYPB</i>*<i>03</i> and <i>GYPB</i>*<i>04</i>, <i>RHCE</i>*<i>E</i> and <i>RHCE</i>*<i>e,</i> and <i>DO</i>*<i>01</i> and <i>DO</i>*<i>02.</i> Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. <b><i>Results:</i></b> The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. <b><i>Conclusions:</i></b> We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

scholarly journals Rapid screening for targeted genetic variants via high-resolution melting curve analysis

2017 ◽  
Vol 55 (4) ◽  
Author(s):  
Allison B. Chambliss ◽  
Molly Resnick ◽  
Athena K. Petrides ◽  
William A. Clarke ◽  
Mark A. Marzinke
Keyword(s):  
Cytochrome P450 ◽  
High Resolution ◽  
Genetic Variants ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Rapid Screening ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphisms ◽  
Cytochrome P450 Enzymes ◽  
Confirmatory Testing

Abstract Background: Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5. Methods: Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (T Results: Intra-assay (n=5) precision of T Conclusions: The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of ‘reference’ or ‘variant’ available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.

scholarly journals High-Resolution Melting Curve Analysis of Genomic and Whole-Genome Amplified DNA

2008 ◽  
Vol 54 (12) ◽  
pp. 2055-2058 ◽  
Author(s):  
Michael H Cho ◽  
Dawn Ciulla ◽  
Barbara J Klanderman ◽  
Benjamin A Raby ◽  
Edwin K Silverman
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Dna Melting ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
High Resolution Melting Curve

Abstract Background: High-resolution melting curve analysis is an accurate method for mutation detection in genomic DNA. Few studies have compared the performance of high-resolution DNA melting curve analysis (HRM) in genomic and whole-genome amplified (WGA) DNA. Methods: In 39 paired genomic and WGA samples, 23 amplicons from 9 genes were PCR amplified and analyzed by high-resolution melting curve analysis using the 96-well LightScanner (Idaho Technology). We used genotyping and bidirectional resequencing to verify melting curve results. Results: Melting patterns were concordant between the genomic and WGA samples in 823 of 863 (95%) analyzed sample pairs. Of the discordant patterns, there was an overrepresentation of alternate melting curve patterns in the WGA samples, suggesting the presence of a mutation (false positives). Targeted resequencing in 135 genomic and 136 WGA samples revealed 43 single nucleotide polymorphisms (SNPs). All SNPs detected in genomic samples were also detected in WGA. Additional genotyping and sequencing allowed the classification of 628 genomic and 614 WGA amplicon samples. Heterozygous variants were identified by non–wild-type melting pattern in 98% of genomic and 97% of WGA samples (P = 0.11). Wild types were correctly classified in 99% of genomic and 91% of WGA samples (P &lt; 0.001). Conclusions: In WGA DNA, high-resolution DNA melting curve analysis is a sensitive tool for SNP discovery through detection of heterozygote variants; however, it may misclassify a greater number of wild-type samples.

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