Screening for Defined Cystic Fibrosis Mutations by Solid-Phase Minisequencing

1992 ◽  
Vol 38 (1) ◽  
pp. 39-43 ◽  
Author(s):  
Anu Jalanko ◽  
Juha Kere ◽  
Erkki Savilahti ◽  
Marianne Schwartz ◽  
Ann-Christine Syvänen ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Rapid Method ◽  
Solid Phase ◽  
Point Mutations ◽  
Accurate Method ◽  
Microtiter Plate ◽  
Quantitative Detection ◽  
Major Mutation ◽  
Allele Combination

Abstract We have developed a rapid method for the quantitative detection of point mutations and deletions. In this minisequencing method, enzymatically amplified DNA, 5'-biotinylated in one strand, is bound to a solid phase and denatured. A detection primer, constructed to end immediately before the mutation, is annealed to the immobilized single-stranded template and elongated with a single, labeled deoxynucleoside residue. We have applied the solid-phase minisequencing method to the detection of the major mutation, delta F508, causing cystic fibrosis (CF). In the presence of the allele with the delta F508 mutation, [3H]dTTP is incorporated; with the nonmutated allele, [3H]dCTP is incorporated. Thus, samples from heterozygous individuals allow the incorporation of both labels. The method was evaluated by analyzing 59 coded DNA specimens collected from 20 Finnish CF patients and their parents. The ratio of [3H]C to [3H]T gave unambiguously the allele combination. The solid-phase minisequencing method was also applicable to the analysis of three CF mutations simultaneously, i.e., delta F508, G542X, and G551D. We conclude that the microtiter-plate-based minisequencing test is an accurate method for the screening of defined sequence alterations in the CF gene.

Colorimetric solid-phase minisequencing assay illustrated by detection of alpha 1-antitrypsin Z mutation

1993 ◽  
Vol 39 (11) ◽  
pp. 2282-2287 ◽  
Author(s):  
L Harju ◽  
T Weber ◽  
L Alexandrova ◽  
M Lukin ◽  
M Ranki ◽  
...  
Keyword(s):  
Solid Phase ◽  
Colorimetric Assay ◽  
Point Mutations ◽  
High Specificity ◽  
Microtiter Plate ◽  
Test Results ◽  
Single Nucleotide ◽  
Nitrophenyl Phosphate ◽  
Simple Alternative ◽  
Alpha 1 Antitrypsin

Abstract In solid-phase minisequencing, a defined point mutation is detected in microtiter plate-immobilized DNA by a single nucleotide primer extension reaction. We have here developed the method into a colorimetric assay and applied it to the detection of the Z mutation of the alpha 1-antitrypsin gene. We used novel nucleoside triphosphates modified with dinitrophenyl (DNP) hapten, permitting detection by anti-DNP-alkaline phosphatase conjugate, with p-nitrophenyl phosphate as substrate. The Z mutation is detected in two reactions: DNP-labeled dCTP is incorporated when the template is normal, DNP-dUTP when the Z mutation is present. Both modified nucleotides were incorporated with high specificity and with an efficiency similar to that of unmodified nucleotides. The test results are measured by spectrophotometry, yielding quantitative absorbance values. Calculation of the ratio of C to U signal permitted unambiguous distinction of normal homozygous, ZZ homozygous, and ZM heterozygous genotypes. The colorimetric minisequencing assay is rapid, standardized, and automatable, and thus provides an accurate and simple alternative for the analysis of known point mutations.

scholarly journals Quantitative Determination of Cd in Soil Using Laser-Induced Breakdown Spectroscopy in Air and Ar Conditions

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2492 ◽  
Author(s):  
Xiaodan Liu ◽  
Fei Liu ◽  
Weihao Huang ◽  
Jiyu Peng ◽  
Tingting Shen ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Least Squares ◽  
Three Dimensional ◽  
Accurate Method ◽  
Laser Induced Breakdown Spectroscopy ◽  
Quantitative Detection ◽  
Support Vector ◽  
Least Squares Regression ◽  
Breakdown Spectroscopy ◽  
Laser Induced Breakdown

Rapid detection of Cd content in soil is beneficial to the prevention of soil heavy metal pollution. In this study, we aimed at exploring the rapid quantitative detection ability of laser- induced breakdown spectroscopy (LIBS) under the conditions of air and Ar for Cd in soil, and finding a fast and accurate method for quantitative detection of heavy metal elements in soil. Spectral intensity of Cd and system performance under air and Ar conditions were analyzed and compared. The univariate model and multivariate models of partial least-squares regression (PLSR) and least-squares support vector machine (LS-SVM) of Cd under the air and Ar conditions were built, and the LS-SVM model under the Ar condition obtained the best performance. In addition, the principle of influence of Ar on LIBS detection was investigated by analyzing the three-dimensional profile of the ablation crater. The overall results indicated that LIBS combined with LS-SVM under the Ar condition could be a useful tool for the accurate quantitative detection of Cd in soil and could provide reference for environmental monitoring.

scholarly journals Solid-Phase Extraction and Gas Chromatography with Mass Spectrometric Determination of Capsaicin and Some of Its Analogues from Chili Peppers (Capsicum spp.)

1999 ◽  
Vol 82 (6) ◽  
pp. 1399-1405 ◽  
Author(s):  
Philemon Manirakiza ◽  
Adrian Covaci ◽  
Paul Schepens
Keyword(s):  
Gas Chromatography ◽  
Quantitative Determination ◽  
Solid Phase ◽  
Accurate Method ◽  
Mass Spectrometric ◽  
Phase Extraction ◽  
Capsicum Spp ◽  
The Mean ◽  
Chili Peppers

Abstract A rapid and accurate method has been developed for the quantitative determination of capsaicin and its most important analogues, dihydrocapsaicin and nordihydrocapsaicin in chili peppers. These components were extracted with methylene chlo ride and separated from interfering substances with activated charcoal. Further cleanup on Florisil cartridges and elution with ethyl acetate were performed before gas chromatographic with mass spectrometric quantitation. The concentrations found were 440 ± 64 μg/g capsaicin, 81 ± 10 μg/g dihydrocapsaicin, and 11 ± 2 μg/g nordihydrocapsaicin. The mean recovery values for triplicate analysis were between 85-94%.

scholarly journals Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation

1996 ◽  
Vol 42 (9) ◽  
pp. 1391-1397 ◽  
Author(s):  
T Pastinen ◽  
J Partanen ◽  
A C Syvänen
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Solid Phase ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Dna Templates ◽  
Single Nucleotide ◽  
Large Numbers ◽  
Dna Sequence Variation ◽  
Hla Dqa1 ◽  
Efficient Screening

Abstract We developed a multiplex, solid-phase minisequencing method to detect multiple single-nucleotide polymorphisms in an undivided sample. The amplified DNA templates are first captured on a manifold. Then, with multiple minisequencing primers of various sizes, single-nucleotide extension reactions are carried out simultaneously with fluorescently labeled dideoxynucleotides. The size of the extended product, determined by using a DNA sequencing instrument, defines the site of the polymorphisms, and the incorporated nucleotide gives the identity of the nucleotide at each site. HLA-DQA1 typing was used as a model system to evaluate the method. The DR2 subgroup of the HLA-DRB1 gene was typed along with the DQA1 gene to demonstrate the feasibility of the method in analyzing multiple genes at multiple sites simultaneously. The method is generally applicable for screening any single-nucleotide polymorphisms or point mutations, and its manifold format allows practical handling of large numbers of samples.

Competitive enzyme-linked immunoassay for Factor VIII antigen.

1979 ◽  
Vol 25 (11) ◽  
pp. 1924-1927 ◽  
Author(s):  
L D Yorde ◽  
C V Hussey ◽  
D E Yorde ◽  
E A Sasse
Keyword(s):  
Horseradish Peroxidase ◽  
Factor Viii ◽  
Peroxidase Activity ◽  
Solid Phase ◽  
Microtiter Plate ◽  
Antigen Binding ◽  
Normal Range ◽  
Antigen Concentration ◽  
Soluble Complex ◽  
Factor Viii Antigen

Abstract We describe a competitive enzyme-linked immunoassay for Factor VIII antigen. Binding of anti-factor VIII to solid-phase Factor VIII antigen is competitively inhibited by the free factor VIII antigen that is to be measured. The amount of anti-Factor VIII bound to solid-phase VIII is measured by applying in sequence a heterologous bridging antibody and a soluble antibody/enzyme immune complex. The soluble complex used was rabbit antiperoxidase/horseradish peroxidase. Peroxidase activity is inversely proportional to the Factor VIII antigen concentration in the original test plasma and is measured spectrophotometrically. The assay can be performed in as little as 4 h with only a microtiter plate, antisera, antigen, and a spectrophotometer. It is sensitive to 0.05 units of Factor VIII antigen per milliliter, and reproducibility, linearity, and normal range are similar to those reported for other techniques.

scholarly journals Solid-Phase Amplification for Detection of C282Y and H63D Hemochromatosis (HFE) Gene Mutations

2001 ◽  
Vol 47 (8) ◽  
pp. 1384-1389 ◽  
Author(s):  
Mark S Turner ◽  
Sarah Penning ◽  
Angela Sharp ◽  
Valentine J Hyland ◽  
Ray Harris ◽  
...  
Keyword(s):  
Solid Phase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Pcr Primers ◽  
Clinical Samples ◽  
Hfe Gene ◽  
Target Sequence ◽  
Nucleotide Polymorphisms ◽  
Nitrophenyl Phosphate ◽  
Hemochromatosis Gene

Abstract Background: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples. Methods: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate. Results: Two different amplicon-labeling methods were evaluated. Analysis of 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment length polymorphism assay results. Conclusions: The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.

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