Genomic DNA PCR analysis to assess xenograft development in mouse mammary gland

The mouse transplantation model remains the most relevant methodology to assess the functional capacities of mammary cells and is particularly appropriate for investigations regarding mammary stem cells, whatever the species studied. Following xenotransplantation in mice mammary fat pad, the development of the xenograft is commonly evaluated by immunohistology. Here, we present a simple and rapid method to control the species specificity of a xenograft based on genomic DNA PCR amplification. DNA is extracted from the fixed samples intended for histology, thus allowing the reuse of precious samples. Standard and digital droplet PCR (requiring low DNA quantities) methods have been used to make the present method suitable for the analysis of xenotransplanted samples.

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Comparison Between Currently Used Blood Samples And New Saliva Dna Collection Method For Quality Of Genomic Dna And Genotyping

ARS Medica Tomitana ◽

10.2478/v10307-012-0003-0 ◽

2012 ◽

Vol 18 (1) ◽

pp. 19-23

Author(s):

Loredana Mariana Aftenie ◽

Irina Franciuc ◽

Alina Martinescu ◽

Adina Honcea

Keyword(s):

Genomic Dna ◽

Response Rate ◽

Saliva Sample ◽

Pcr Amplification ◽

Good Alternative ◽

Pcr Analysis ◽

Blood Samples ◽

Biospecimen Collection ◽

Whole Saliva

AbstractObtaining blood biospecimens presents logistical and financial challenges. As a result, saliva biospecimen collection is becoming more frequent because of the ease of collection and lower cost. This article describes an assessment of two different methods for collecting samples: whole blood and whole saliva samples used further for DNA extraction and HLA genotyping in immunogenic disease on a group of patients registered at our Molecular Genetics Laboratory Faculty of Medicine “Ovidius” University Constanţa. Our data show that only 81% of the requested participants delivered a blood sample, whereas 19% delivered a saliva sample because they refuse the first sampling method. Analysis of purified genomic DNA by Nano Photometer and agarose gel electrophoresis revealed that blood and saliva samples resulted in DNA with the best quality. PCR analysis showed that DNA from 100% of the blood samples and 93% of the saliva samples could be subsequently amplified. Our study shows that the response rate of self-collection saliva samples had to be considering for the patients that have a low response rate of blood sampling. The quality of genomic DNA from saliva samples was comparable with blood samples as assessed by purity, concentration, yield and PCR amplification. We conclude that the use of saliva samples is a good alternative to blood samples to obtain genomic DNA of high quality and it will considerably increase the participant’s response rate for genetic studies.

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Selection of the most suitable method for the extraction of DNA from foods and feeds for species identification

Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis ◽

10.11118/actaun201058020169 ◽

2010 ◽

Vol 58 (2) ◽

pp. 169-174

Author(s):

Michaela Nesvadbová ◽

Aleš Knoll ◽

Anna Vašátková

Keyword(s):

Species Identification ◽

Genomic Dna ◽

Pcr Amplification ◽

Degraded Dna ◽

Dna Purification ◽

Pcr Analysis ◽

Isolation System ◽

Dna Yield ◽

Pcr Products ◽

Food And Feed

High quality and purity of DNA isolated from food and feed is essential for species identification and has unpredictable influences an effect of analysis. In this study, the efficiency of eight different methods for DNA isolation was investigated. For DNA extraction, the raw chicken meet, ham, sausages, tinned lunch meat, pate, tinned feeds for dogs, complete granulated feeds for dogs and chicken flour were used. Kits of several different producers, i.e.: NucleoSpin Food (Marchery-Nagel), Wizard Genomic DNA Purification Kit (Promega), Invisorb Spin Food Kit I (Invitek), Wizard SV Genomic DNA Purification System (Promega), JetQuick Tissue DNA Spin Kit (Genomed), RNA Blue (Top-Bio), JetQuick Blood & Cell Culture Kit (Genomed), QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit (Qiagen) were employed in the study. Gel agarose electrophoresis for primary verification of DNA quality was performed. The isolates were subsequently assessed for quantity and quality using by spectrophotometer Nanodrop 2000 (Thermo Scientific). To verify of template usability and quality of isolated DNA, the polymerase chain reaction (PCR) was used.Differences between isolated DNA from tinned products and meat, ham, sausage, granulated dog feed and chicken flour were found. In tinned food and feed, the DNA was more degraded, DNA content and DNA purity was lower and also PCR amplification was the most difficult. Overall DNA yield and quality have important influence on PCR products amplification. The best results were obtained with NucleoSpin Food and JetQuick Tissue DNA Spin Kit. DNA extracted by these methods proved highest yields, purity and template quality in all foods and feeds and the results of PCR analysis are excellent reproducible. Analyses showed that results depended on different food or feed using and different isolation system.The results of this work will be utilized to choose the suitable isolating kit for educational course, which is designed for students and also for following research and analyses.

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Bulk Genomic DNA PCR Analysis—A Rapid Method to Estimate Genetic Relatedness among Heterogeneous Lucerne (Medicago sativa L.) Cultivars

CYTOLOGIA ◽

10.1508/cytologia.72.363 ◽

2007 ◽

Vol 72 (3) ◽

pp. 363-368 ◽

Cited By ~ 3

Author(s):

Amaresh Chandra

Keyword(s):

Medicago Sativa ◽

Rapid Method ◽

Genomic Dna ◽

Genetic Relatedness ◽

Pcr Analysis ◽

Medicago Sativa L ◽

Dna Pcr

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Single and Double Infections with Wolbachia in the Parasitic Wasp Nasonia vitripennis Effects on Compatibility

Genetics ◽

10.1093/genetics/143.2.961 ◽

1996 ◽

Vol 143 (2) ◽

pp. 961-972 ◽

Cited By ~ 9

Author(s):

Marie-Jeanne Perrot-Minnot ◽

Li Rong Guo ◽

John H Werren

Keyword(s):

Genomic Dna ◽

Rapid Development ◽

Parasitic Wasp ◽

Pcr Amplification ◽

Bacterial Strains ◽

Nasonia Vitripennis ◽

Larval Diapause ◽

Wide Range ◽

Reproductive Incompatibility ◽

Infected Line

Abstract Wolbachia are cytoplasmically inherited bacteria responsible for reproductive incompatibility in a wide range of insects. There has been little exploration, however, of within species Wolbachia polymorphisms and their effects on compatibility. Here we show that some strains of the parasitic wasp Nasonia vitripennis are infected with two distinct bacterial strains (A and B) whereas others are singly infected (A or B). Double and single infections are confirmed by both PCR amplification and Southern analysis of genomic DNA. Furthermore, it is shown that prolonged larval diapause (the overwintering stage of the wasp) of a double-infected strain can lead to stochastic loss of one or both bacterial strains. After diapause of a double-infected line, sublines were produced with AB, A only, B only or no Wolbachia. A and B sublines are bidirectionally incompatible, whereas males from AB lines are unidirectionally incompatible with females of A and B sublines. Results therefore show rapid development of bidirectional incompatibility within a species due to segregation of associated symbiotic bacteria.

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Suitability of different tissue fixatives for subsequent PCR analysis of Cysticercus ovis

Helminthologia ◽

10.2478/s11687-012-0014-1 ◽

2012 ◽

Vol 49 (2) ◽

pp. 67-70 ◽

Cited By ~ 1

Author(s):

M. Kolesárová ◽

R. Herich ◽

M. Levkut ◽

J. Čurlík ◽

M. Levkut

Keyword(s):

Retrospective Studies ◽

Pcr Amplification ◽

Pcr Analysis ◽

Chain Reaction ◽

Fresh Tissue ◽

Carnoy’S Solution ◽

Negative Results ◽

Pcr Product ◽

Polymerase Chain ◽

Cysticercus Ovis

AbstractPCR amplification of specific DNA regions is a powerful tool for retrospective studies, but not all preservation or fixation methods render DNA that is suitable for subsequent amplification. Several factors affect sensitivity of polymerase chain reaction (PCR) amplification. There were reported the effects of commonly used fixation solutions — 10 % neutral buffered formalin, 20 % neutral buffered formalin and Carnoy’s solution and the efficiency of PCR amplification in fresh tissue and paraffin (or wax) embedded samples of Cysticercus ovis. DNA from samples was isolated and PCR product of 1300 bp was amplified. Results indicated that the samples fixed in Carnoy’s solution produced reliable amplification of desired fragments. The samples that were fixed in 10 % and 20 % neutral buffered formalin brought negative results.

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Molecular Identification of Aflatoxigenic Fungi in Some Foods from Selected Markets in Lagos

Asian Journal of Biochemistry, Genetics and Molecular Biology ◽

10.9734/ajbgmb/2020/v5i430139 ◽

2020 ◽

pp. 42-50

Author(s):

Oluwatosin Bidemi Ajiboye ◽

Wahab Oluwanisola Okunowo ◽

Emmanuel Gboyega Ajiboye ◽

Abiola Olajumoke Oyedeji

Keyword(s):

Molecular Identification ◽

Genomic Dna ◽

Regulatory Gene ◽

Pcr Amplification ◽

High Specificity ◽

Food Samples ◽

Local Market ◽

Nested Polymerase Chain Reaction ◽

Aflatoxigenic Fungi ◽

Food Commodities

Aflatoxigenic fungi are species of fungi that produce aflatoxins in food commodities. This study was aimed at screening different food samples in our local market for aflatoxigenic fungi using the aflatoxin regulatory gene (aflR gene). Six food samples (wheat, cowpea, rice, maize, melon and groundnut), were sourced from three different markets in Lagos metropolis (Mushin, Oyingbo and Mile 12). Fungi were isolated from these food samples and identified morphologically and microscopically. The genomic DNA was obtained using DNA isolation kits. The aflR gene was amplified from genomic DNA, nested, subjected to agarose gel electrophoresis and gel imaging. The Internal Transcribed Spacer (ITS) was also amplified from the genomic DNA for molecular identification of the organisms. The results showed that Aspergillus flavus were isolated from all the food samples from the three markets, while Aspergillus niger was present in rice, melon and wheat from Mile 12 market, maize and groundnut from Mushin market, rice and cowpea from Oyingbo market. A. flavus and A.niger were isolated from all the food samples when similar food samples from different market were mixed together. Only A. flavus amplicon from the nested polymerase chain reaction (PCR) showed approximately 400bp DNA fragment on the gel. This study has shown that PCR amplification of aflR gene has high specificity for detection of aflatoxigenic fungi in food samples thus, may be employed in screening food samples for contamination by aflatoxigenic fungi.

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XL PCR Amplification of Long Targets from Genomic DNA

PCR Cloning Protocols ◽

10.1385/1-59259-177-9:037 ◽

2003 ◽

pp. 037-051 ◽

Cited By ~ 1

Author(s):

Lori A. Kolmodin

Keyword(s):

Genomic Dna ◽

Pcr Amplification

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Prenatal diagnosis of pyruvate kinase deficiency

Blood ◽

10.1182/blood.v84.7.2354.2354 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2354-2356 ◽

Cited By ~ 14

Author(s):

L Baronciani ◽

E Beutler

Keyword(s):

Pyruvate Kinase ◽

Genomic Dna ◽

Direct Method ◽

Pcr Amplification ◽

Prenatal Testing ◽

Restriction Endonuclease Analysis ◽

Diagnostic Methods ◽

Pyruvate Kinase Deficiency ◽

A Direct Method ◽

Endonuclease Analysis

Abstract Prenatal testing for pyruvate kinase deficiency is often requested by parents who already have an affected child. However, before the development of molecular biologic techniques there were no suitable diagnostic methods. We present here two cases in which the diagnosis was established, one using amniotic fluid cells, the other cord blood. Two different approaches were used. The first, using a direct method of PCR amplification and restriction endonuclease analysis, detected mutations in fetus genomic DNA. The second method, using two polymorphic sites linked to the PKRL gene, enabled us to establish which chromosome had been inherited from each parent.

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