Two-State Protein Melting Curve (N, P, T Ensemble)

CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA) 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC), two peripheral bloods (PB), 12 granulocyte products, natural killer (NK)-92, B-lymphocyte (CO88BV59-1), K-562 leukemia cell line, human embryonic stem cell (hESC), and human fibroblasts (HF). HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

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High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

Journal of Fish Diseases ◽

10.1111/jfd.13053 ◽

2019 ◽

Vol 42 (10) ◽

pp. 1359-1368 ◽

Cited By ~ 4

Author(s):

Yolanda Torres‐Corral ◽

Clara Fernández‐Álvarez ◽

Ysabel Santos

Keyword(s):

Real Time ◽

High Throughput ◽

Real Time Pcr ◽

Melting Curve ◽

Curve Analysis ◽

Sybr Green ◽

Sybr Green I ◽

Melting Curve Analysis ◽

Identification And Quantification

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Rapid genotypic detection ofBacillus anthracisand theBacillus cereusgroup by multiplex real-time PCR melting curve analysis

FEMS Immunology & Medical Microbiology ◽

10.1016/j.femsim.2004.10.005 ◽

2005 ◽

Vol 43 (2) ◽

pp. 301-310 ◽

Cited By ~ 36

Author(s):

Kijeong Kim ◽

Juwon Seo ◽

Katherine Wheeler ◽

Chulmin Park ◽

Daewhan Kim ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis

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Genotyping of a tri-allelic polymorphism by a novel melting curve assay in MTHFD1L: an association study of nonsyndromic Cleft in Ireland

BMC Medical Genetics ◽

10.1186/1471-2350-13-29 ◽

2012 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Stefano Minguzzi ◽

Anne M Molloy ◽

Kirke Peadar ◽

James Mills ◽

John M Scott ◽

...

Keyword(s):

Association Study ◽

Melting Curve ◽

Allelic Polymorphism

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Diagnosis of α-1-Antitrypsin Deficiency: An Algorithm of Quantification, Genotyping, and Phenotyping

Clinical Chemistry ◽

10.1373/clinchem.2006.072991 ◽

2006 ◽

Vol 52 (12) ◽

pp. 2236-2242 ◽

Cited By ~ 74

Author(s):

Melissa R Snyder ◽

Jerry A Katzmann ◽

Malinda L Butz ◽

Ping Yang ◽

D Brian Dawson ◽

...

Keyword(s):

Laboratory Testing ◽

Null Allele ◽

Melting Curve ◽

Curve Analysis ◽

Laboratory Evaluation ◽

Melting Curve Analysis ◽

Genotype Assay ◽

Antitrypsin Deficiency ◽

Phenotype Analysis ◽

A1at Deficiency

Abstract Background: Laboratory testing in suspected α-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency. Methods: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis. Results: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result. Conclusions: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.

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Intercistronic heterogeneity of the 16S–23S rRNA spacer region among Pseudomonas strains isolated from subterranean seeds of hog peanut (Amphicarpa bracteata)

Microbiology ◽

10.1099/mic.0.028274-0 ◽

2009 ◽

Vol 155 (8) ◽

pp. 2630-2640 ◽

Cited By ~ 7

Author(s):

J. T. Tambong ◽

R. Xu ◽

E. S. P. Bromfield

Keyword(s):

Root Zone ◽

Phylogenetic Analyses ◽

Melting Curve ◽

Pcr Amplification ◽

23S Rrna ◽

Melting Curve Analysis ◽

Its1 Region ◽

A Genome ◽

Pure Cultures ◽

Intercalating Dye

Intercistronic heterogeneity of the 16S–23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of Amphicarpa bracteata (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNAIle and tRNAAla in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the Pseudomonas community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus Pseudomonas, but the presence of non-identical rRNA operons within a genome may pose problems.

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