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 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.

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.

scholarly journals Hepatic fibrinogen storage disease and hypofibrinogenemia caused by fibrinogen Aguadilla mutation: a case report

2020 ◽  
Vol 48 (1) ◽  
pp. 030006051989803 ◽  
Author(s):  
Leilei Gu ◽  
Bin Wang ◽  
Lu Liu ◽  
Qiaorong Gan ◽  
Xiaolong Liu ◽  
...  
Keyword(s):  
Liver Disease ◽  
Inclusion Bodies ◽  
Storage Disease ◽  
Genetic Disorder ◽  
Periodic Acid ◽  
Abnormal Liver Function Test ◽  
Test Results ◽  
Periodic Acid Schiff ◽  
Single Nucleotide ◽  
Alpha 1 Antitrypsin

Hepatic fibrinogen storage disease is a rare autosomal dominant genetic disorder characterized by hypofibrinogenemia, as well as the retention of variant fibrinogen within the hepatocellular endoplasmic reticulum. Here, we describe an asymptomatic 4-year-old boy with abnormal liver function test results and unexpected hypofibrinogenemia. Liver biopsy showed circular eosinophil inclusion bodies in the hepato-cytoplasm. Immunostaining results of eosinophil inclusion bodies were positive for fibrinogen. Following pretreatment with diastase, the inclusion bodies failed to stain with the periodic acid–Schiff technique; moreover, immunostaining results were positive for fibrinogen, but negative for alpha-1-antitrypsin. Genetic analysis identified a heterozygous missense mutation c.1201C > T (p. Arg401Trp) within the fibrinogen γ-chain ( FGG) gene and an additional single nucleotide polymorphism c.-58 A > G within the 5′-untranslated region of the fibrinogen Aα-chain ( FGA) gene. Thus, the patient was diagnosed with hepatic fibrinogen storage disease. Our results indicate that, for patients who exhibit chronic liver disease with unexpected hypofibrinogenemia, hepatic fibrinogen storage disease should be considered in the differential diagnosis. Moreover, our findings emphasize the importance of molecular diagnosis in patients with cryptogenic liver disease.

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.

A Nylon Filter A-ELISA for Detecting Viruses in Water

1993 ◽  
Vol 27 (7-8) ◽  
pp. 135-141 ◽  
Author(s):  
Abidelfatah M. Nasser ◽  
Yehudit Elkana ◽  
Leon Goldstein
Keyword(s):  
Positive Result ◽  
Solid Phase ◽  
Enteric Viruses ◽  
High Specificity ◽  
Negative Results ◽  
Order Of Magnitude ◽  
Lower Background

This study was designed to develop a modification of A-ELISA performed in microtitre plates. Nylon filters have been utilized successfully as a solid phase for the performance of A-ELISA. The use of nylon filters resulted in lower background than nitro-cellulose and paper filters, indicating their suitability as a solid phase for developing A-ELISA. With enteric viruses, human rotaviruses and MS-2 coliphage, negative results were obtained, suggesting high specificity of the developed technique for poliovirus 1. The sensitivity of the developed A-ELISA has been shown to be at least one order of magnitude greater than ordinary ELISA. A positive result with the nylon A-ELISA can be obtained with samples containing 100-1000 pfu/ml of poliovirus. Up to date methods used for detecting viruses in water are elaborate, time consuming and costly. Applying the nylon A-ELISA may overcome some of these disadvantages.

scholarly journals Disposition of MDMA and Metabolites in Human Sweat Following Controlled MDMA Administration

2009 ◽  
Vol 55 (3) ◽  
pp. 454-462 ◽  
Author(s):  
Allan J Barnes ◽  
Bruno S De Martinis ◽  
David A Gorelick ◽  
Robert S Goodwin ◽  
Erin A Kolbrich ◽  
...  
Keyword(s):  
Solid Phase ◽  
Random Order ◽  
Research Unit ◽  
Sweat Test ◽  
Test Results ◽  
Double Blind ◽  
Scientific Database ◽  
Sweat Testing ◽  
Clinical Research Unit ◽  
Sweat Tests

Abstract Background: Understanding the excretion of 3,4-methylenedioxymethamphetamine (MDMA) and metabolites in sweat is vital for interpretation of sweat tests in drug treatment, criminal justice, and workplace programs. Methods: Placebo, low (1.0 mg/kg), and high (1.6 mg/kg) doses of oral MDMA were given double-blind in random order to healthy volunteers (n = 15) with histories of MDMA use. Participants resided on the closed clinical research unit for up to 7 days after each dose. Volunteers wore PharmChek® sweat patches (n = 640) before, during, and after controlled dosing. Patches were analyzed by solid phase extraction and GC-MS for MDMA, methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA). Limits of quantification (LOQ) were 2.5 ng/patch for MDMA and 5 ng/patch for HMA, HMMA, and MDA. Results: MDMA was the primary analyte detected in 382 patches (59.7%), with concentrations up to 3007 ng/patch. MDA was detected in 188 patches (29.4%) at <172 ng/patch, whereas no HMMA or HMA was detected; 224 patches (35.0%) and 60 patches (9.4%) were positive for MDMA and MDA, respectively, at the 25-ng/patch threshold proposed by the Substance Abuse and Mental Health Services Administration. Conclusions: Sweat testing was shown to be an effective and reliable method for monitoring MDMA use in this controlled MDMA administration study. However, variability in sweat excretion suggests that results should be interpreted qualitatively rather than quantitatively. These data provide a scientific database for interpretation of MDMA sweat test results.

scholarly journals Sensitive quantitative assay for point mutations in the rat H-ras gene based on single nucleotide primer extension

2010 ◽  
Vol 1 (4) ◽  
pp. 657-661 ◽  
Author(s):  
YOSHIHISA YANO ◽  
TOMOHIRO YANO ◽  
ANNA KINOSHITA ◽  
AI MATOBA ◽  
TADAYOSHI HASUMA ◽  
...  
Keyword(s):  
Point Mutations ◽  
Primer Extension ◽  
Quantitative Assay ◽  
Ras Gene ◽  
Single Nucleotide ◽  
Single Nucleotide Primer Extension

scholarly journals Competitive SNP-LAMP probes for rapid and robust single-nucleotide polymorphism detection

2021 ◽  
Author(s):  
Leland B Hyman ◽  
Clare R Christopher ◽  
Philip A Romero
Keyword(s):  
Point Of Care ◽  
High Specificity ◽  
Common Source ◽  
Lamp Method ◽  
Universal Method ◽  
Nucleotide Polymorphisms ◽  
One Pot ◽  
Single Nucleotide ◽  
Specificity And Sensitivity ◽  
And Shifts

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation between individuals and have implications in human disease, pathogen drug resistance, and agriculture. SNPs are typically detected using DNA sequencing, which requires advanced sample preparation and instrumentation, and thus cannot be deployed for on-site testing or in low-resource settings. In this work we have developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines LAMP-based target amplification with fluorescent probes to detect SNPs with high specificity in a one-pot reaction format. A competitive "sink" strand preferentially binds to off-target products and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible SARS-CoV-2 variants. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity.

scholarly journals Museum Genomics Illuminate the High Specificity of a Bioluminescent Symbiosis for a Genus of Reef Fish

2021 ◽  
Vol 9 ◽  
Author(s):  
Alison L. Gould ◽  
Allison Fritts-Penniman ◽  
Ana Gaisiner
Keyword(s):  
High Specificity ◽  
Bacterial Symbiont ◽  
Light Organ ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
New Approach ◽  
Host Genus ◽  
Symbiotic Relationships ◽  
Luminous Bacteria ◽  
Formalin Fixed

Symbiotic relationships between bioluminescent bacteria and fishes have evolved multiple times across hundreds of fish taxa, but relatively little is known about the specificity of these associations and how stable they are over host generations. This study describes the degree of specificity of a bioluminescent symbiosis between cardinalfishes in the genus Siphamia and luminous bacteria in the Vibrio family. Primarily using museum specimens, we investigated the codivergence of host and symbiont and test for patterns of divergence that correlate with both biogeography and time. Contrary to expectations, we determined that the light organ symbionts of all 14 Siphamia species examined belong to one genetic clade of Photobacterium mandapamensis (Clade II), indicating that the association is highly specific and conserved throughout the host genus. Thus, we did not find evidence of codivergence among hosts and symbionts. We did observe that symbionts hosted by individuals sampled from colder water regions were more divergent, containing more than three times as many single nucleotide polymorphisms than the rest of the symbionts examined. Overall, our findings indicate that the symbiosis between Siphamia fishes and P. mandapamensis Clade II has been highly conserved across host taxa and over a broad geographic range despite the facultative nature of the bacterial symbiont. We also present a new approach to simultaneously recover genetic information from a bacterial symbiont and its vertebrate host from formalin-fixed specimens, enhancing the utility of museum collections.

scholarly journals PEMBUATAN SUMBER RADIASI GAMMA 137Cs DENGAN AKTIVITAS 20 mCi DARI PEB U3Si2-Al PASCA IRADIASI DALAM CONTAINER STAINLESS STEEL

2016 ◽  
Vol 21 (3) ◽  
Author(s):  
Aslina Br. Ginting ◽  
Yanlinastuti Yanlinastuti ◽  
Noviarty Noviarty ◽  
Boybul Boybul ◽  
Arif Nogroho ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Gamma Radiation ◽  
Cation Exchange ◽  
High Activity ◽  
Radiation Source ◽  
Solid Phase ◽  
Test Results ◽  
Spent Fuel ◽  
Post Irradiation ◽  
Outer Capsule

PEMBUATAN SUMBER RADIASI GAMMA ISOTOP 137Cs DENGAN AKTIVITAS 20 mCi DARI PEB U3Si2-Al PASCA IRADIASI DALAM CONTAINER STAINLESS STEEL. Kegiatan uji pasca iradiasi pelat elemen bakar (PEB) U3Si2-Al banyak menghasilkan larutan dengan keaktifan yang sangat tinggi. Larutan tersebut mengandung isotop 137Cs, uranium serta transuranium yang mempunyai waktu paroh panjang dan berbahaya bagi lingkungan. Namun larutan tersebut memiliki nilai ekonomis tinggi karena dapat dimanfaatkan sebagai bahan baku untuk pembuatan sumber radiasi sinar gamma isotop 137Cs. Hal ini dapat membantu bidang industri dalam memenuhi kebutuhan sumber radioaktif dalam negeri karena selama ini kebutuhan isotop 137Cs di Indonesia masih tergantung dari industri luar negeri. Selain itu, pengadaan dan transportasi isotop 137Cs dari luar negeri serta dalam penggunaannya memerlukan persyaratan yang cukup ketat dari Badan Pengawas Tenaga Nuklir Nasional (BAPETEN), sehingga menyebabkan harga isotop 137Cs menjadi mahal sampai di Indonesia. Dengan alasan tersebut, BATAN sebagai lembaga litbang nuklir di Indonesia perlu mempelajari pembuatan sumber radiasi gamma isotop 137Cs dari larutan hasil pengujian bahan bakar nuklir U3Si2-Al pasca iradiasi. Manfaat isotop 137Cs sangat luas antara lain digunakan dalam menganalisis sampel lingkungan, industri migas, konstruksi, radiografi, perikanan, rumah sakit dan pertambangan. Pembuatan sumber radiasi gamma isotop 137Cs dimulai dari pengumpulan larutan hasil pengujian PEB U3Si2-Al. Larutan larutan hasil pengujian mengandung isotop 137Cs dan isotop lainnya dikumpulkan menjadi satu dalam botol dengan volume 65 mL. Pemisahan isotop 137Cs dari hasil fisi lainnya dilakukan dengan metode penukar kation menggunakan zeolit Lampung dengan berat 45 gr. Hasil pemisahan diperoleh 137Cs-zeolit dalam fasa padat dan isotop lainnya berada dalam fasa cair. Padatan137Cs-zeolit kering kemudian kemudian ditimbang dan diukur aktivitasnya menggunakan spektrometer-g. Hasil analisis dengan spektrometer-g diperoleh aktivitas padatan 137Cs-zeolit sebesar 20 mCi. Untuk menjadi sumber radiasi gamma 137Cs, padatan 137Cs-zeolit dengan aktivitas 20 mCi dikemas dengan cara memasukkan ke dalam inner-outer capsule terbuat dari stainless steel yang telah dirancang sebelumnya. Container stainless steel diproses menjadi sumber radiasi gamma tertutup (shield source) untuk selanjutnya disertifikasi oleh PTKMR-BATAN sebagai lembaga kalibtrator bahan radioaktif di BATAN.Kata kunci: Larutan proses PEB U3Si2-Al, radioaktif gamma, isotop 137Cs, penukar kation, zeolit Lampung dan container. MANUCFACTURING OF 137Cs GAMMA RAY SOURCE WITH ACTIVITY 20 mCi FROM PEB U3Si2-Al POST IRRADIATION IN STAINLESS STEEL CONTAINER. In the post-irradiation examination of fuel element plate (PEB) U3Si2-Al), a solution of high activity as a result of testing nuclear fuel stored in hotcell with enough volume. The solution can not be discarded as waste because it still contains fission isotop such as137Cs, uranium and transuranium, which has a long half life and dangerous for the environment. This can help the industry in order to fulfill the needs of a radioactive source in Indonesia, because until now 137Cs isotope is derived from foreign industries. In addition, the procurement and transportation of isotopes 137Cs require stringent requirements, because they have toget permission from the National Nuclear Energy Agency (BAPETEN), thus causing the price of high activity 137Cs isotopes becomes expensive to Indonesia. For these reasons, BATAN as nuclear R&D institutions in Indonesia need to study make isotopes 137Cs gamma radiation source, which is contained in the waste from spent fuel test results U3Si2-Al. Isotope 137Cs can be used very widely, such as in the analysis of environmental samples, the oil and gas industry, construction, radiography, fisheries, hospitals, and mining. Making isotope 137Cs gamma radiation source starting from the collection of waste from the test results PEB U3Si2- Al. Waste solution was collected in a bottle with volume 65 mL. Collection of 137Cs isotopes of other fission carried out using the method of cation exchange with weight 45 gr of zeolite Lampung. The results of separation are 137Cs-zeolite in the solid phase and the other isotopes are in the liquid phase. 137Cs-zeolite solid is then dried and then weighed and measured its activity using a spectrometer-g. Result of analisys by spectrometer-g was obtained acitivity of 137Cs-zeolite solids was 20 mCi.137Cs-zeolite solids then packed in sealed containers (shield source) capsule-shaped stainless steel and than certificate by PTKMR-BATAN.Keywords: Process wastle of PEB U3Si2-Al, gamma radioactive, isotope 137Cs, cation exchange, zeolite Lampung and container.

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