scholarly journals Methacarn Fixation for Genomic DNA Analysis in Microdissected, Paraffin-embedded Tissue Specimens

2002 ◽  
Vol 50 (9) ◽  
pp. 1237-1245 ◽  
Author(s):  
Chikako Uneyama ◽  
Makoto Shibutani ◽  
Naoya Masutomi ◽  
Hironori Takagi ◽  
Masao Hirose
Keyword(s):  
Nested Pcr ◽  
Genomic Dna ◽  
Hippocampal Neurons ◽  
Dna Analysis ◽  
Extraction Procedure ◽  
Pcr Amplification ◽  
Single Step ◽  
Superior Performance ◽  
Cresyl Violet ◽  
Rat Tissues

We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 × 1-mm area of cerebral cortex of a 10-μm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.

scholarly journals Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

2001 ◽  
Vol 67 (6) ◽  
pp. 2453-2459 ◽  
Author(s):  
Roger H. Williams ◽  
Elaine Ward ◽  
H. Alastair McCartney
Keyword(s):  
Nested Pcr ◽  
Southern Blotting ◽  
Dna Analysis ◽  
Fungal Spores ◽  
Air Sampling ◽  
Single Step ◽  
Detection Sensitivity ◽  
Dna Purification ◽  
Single Spore ◽  
Airborne Fungal Spores

ABSTRACT Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.

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

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 961-972 ◽  
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.

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

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.

Microbial characteristics of a methanogenic phenol-degrading sludge

2005 ◽  
Vol 52 (1-2) ◽  
pp. 73-78 ◽  
Author(s):  
T. Zhang ◽  
S. Z. Ke ◽  
Y. Liu ◽  
H.P. Fang
Keyword(s):  
Dna Analysis ◽  
Pcr Amplification ◽  
Upflow Anaerobic Sludge Blanket ◽  
Anaerobic Sludge ◽  
Chain Reaction ◽  
Microbial Characteristics ◽  
Polymerase Chain ◽  
Anaerobic Sludge Blanket

Microbial properties of a methanogenic granular phenol-degrading sludge were characterized using the 16S rRNA/DNA-based techniques, including polymerase chain reaction (PCR) amplification, cloning, DNA sequencing, and fluorescence in situ hybridization (FISH). The sludge was sampled from an upflow anaerobic sludge blanket reactor, which removed 98% of phenol (up to 1260 mg/l) in wastewater at 26°C with 12 hours of hydraulic retention. Based on DNA analysis, the Eubacteria in the sludge was composed of 13 operational taxonomy units (OTUs). Two OTUs, one resembling Clostridium and the other remotely resembling Desulfotomaculum, were likely responsible for the conversion of phenol to benzoate, which was further degraded by five Syntrophus-resembling OTUs to acetate and H2/CO2; methanogens lastly converted acetate and H2/CO2 into methane. The role of six remaining OTUs remains unclear. Overall, the sludge was composed of 26±6% Eubacteria and 74±9% methanogens, of which 54±6% were acetotrophic Methanosaetaceae, 14±3% and 3±2% were hydrogenotrophic Methanomicrobiales and Methanobacteriaceae, respectively.

scholarly journals Prenatal diagnosis of pyruvate kinase deficiency

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2354-2356 ◽  
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.

scholarly journals Variability of D2/D3 segment sequences of several populations and pathotypes of potato cyst nematodes (Globodera rostochiensis, Globodera pallida)

2010 ◽  
Vol 46 (No. 4) ◽  
pp. 171-180 ◽  
Author(s):  
O. Douda ◽  
M. Zouhar ◽  
E. Nováková ◽  
J. Mazáková ◽  
P. Ryšánek
Keyword(s):  
Dna Analysis ◽  
Pcr Amplification ◽  
Globodera Rostochiensis ◽  
Globodera Pallida ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes ◽  
Routine Diagnostics ◽  
Pair Comparisons ◽  
Single Fragment ◽  
Direct Management

Potato cyst nematodes (Globodera rostochiensis, Globodera pallida) remain a key pest in the main potato growing regions of the Czech Republic. Due to difficult direct management and presence of diverse pathotypes attacking different potato cultivars the rapid and reliable diagnostics is of crucial importance. Currently, efforts are aimed at a description of different pathotypes based on DNA analysis. The main objective of this study was to evaluate the homogeneity of sequences of D2/D3 segments of the 28S rDNA gene obtained from 3 populations of G. rostochiensis and 5 populations of G. pallida and estimate their value for diagnostic purposes. PCR amplification yielded a single fragment of the length of 700 bp approximately in all populations. The alignment score of the vast majority of all pair comparisons of G. rostochiensis and G. pallida populations varied from 98 to 99. In total 14 point deletions and 3 substitutions were observed. The variability of D2/D3 segments of potato cyst nematodes is rather low and this DNA region can be used for diagnostics on a species level because more differences were found after comparing with G. tabacum and G. millefolii sequences obtained from Gene Bank; however the applicability of D2/D3 sequences to routine diagnostics of potato cyst nematodes could be complicated by its similarity to corresponding sequences of the nematode G. artemisiae.

XL PCR Amplification of Long Targets From Genomic DNA

2003 ◽  
pp. 17-30
Author(s):  
Suzanne Cheng ◽  
Lori A. Kolmodin
Keyword(s):  
Genomic Dna ◽  
Pcr Amplification

Genetic variation in populations of two Mediterranean annual pasture legumes (Biserrula pelecinus L. and Ornithopus compressus L.) and associated rhizobia

1999 ◽  
Vol 50 (3) ◽  
pp. 303 ◽  
Author(s):  
A. Loi ◽  
J. G. Howieson ◽  
P. S. Cocks ◽  
S. J. Carr
Keyword(s):  
Genetic Variation ◽  
Flowering Time ◽  
Morphological Traits ◽  
Root Nodule ◽  
Dna Analysis ◽  
Phenotypic Variability ◽  
Pcr Amplification ◽  
Nodule Bacteria ◽  
Pasture Legumes ◽  
Ornithopus Compressus

Genetic variation between and within populations of Biserrula pelecinus L. (biserrula) and Ornithopus compressus L. (yellow serradella) and associated rhizobia was studied using germplasm collected from sites in central-eastern and south-eastern Sardinia (Italy). Pods and root-nodule bacteria were collected on diagonal transects at each site. Plants were characterised in nursery rows and the rhizobia were isolated and tested for their effectiveness. Thirteen morphological traits were recorded and the results were analysed using cluster analysis. Genetic and phenotypic variation of rhizobia were assessed using DNA analysis (PCR, RAPDs) and effectiveness indices, respectively. Genetic variation based on morphological traits was found between and within sites for both species. Pod characteristics and flowering time were the most important traits assisting in discriminating between accessions. Flowering time varied more in serradella than in biserrula, particularly at Cantoniera Cannas. Although all rhizobial strains nodulated all accessions of biserrula, great variability in capacity to fix nitrogen was evident between and within sites. Distinct PCR amplification profiles were generated for individual rhizobial strains, which confirmed the phenotypic variability (effectiveness indices) of the strains. No relationship was found between host and rhizobia variation. The results are discussed in terms of (a) genetic differences for each species within and between sites; (b) differences in behaviour in respect to genetic variation between biserrula, serradella, and other Mediterranean annual legumes; and (c) spatial variability and symbiotic effectiveness of rhizobia.

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