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1

Lack of carriers of citrullinaemia and DUMPS in Indian Holstein cattle

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Patel R. K., Singh K. M., Soni K. J., Chauhan J. B., Sambasiva R. K. R. S.
Journal of Applied Genetics
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2006
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vol. 47
|
issue 3
239-242
EN
The present study investigated the occurrence of 2 autosomal recessive genetic diseases, bovine citrullinaemia and deficiency of uridine monophosphate synthase (DUMPS), in Indian Holstein cattle. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed on a group of 642 animals, mainly HF and HF crossbred cattle, to identify carriers of these diseases. None of the animals were carriers of citrullinaemia or DUMPS. It is possible that with the mounting selection pressure, the international gene pool may diminish, and consequently the risk of dissemination of inherited defects will increase. It is therefore recommended to screen breeding bulls for their breed-specific genetic diseases before they are inducted in artificial insemination programmes, to minimize the risk.
2
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Genetic similarity of Pseudomonas syringae pv. tomato strains showing various virulence

100%
Kozik E. U., Puławska J., Sobiczewski P.
Journal of Plant Protection Research
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2006
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vol. 46
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issue 4
3

The retinal fascin gene 2 (FSCN2) ? partial structural analysis and polymorphism detection in dogs with progressive retinal atrophy (PRA)

100%
Horak P., Knoll A., Dvorak J.
Journal of Applied Genetics
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2006
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vol. 47
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issue 4
361-364
EN
The retinal fascin 2 gene (FSCN2) underwent a molecular analysis, a search for polymorphisms and an evaluation as a candidate gene for retinopathies in dogs. Specific fragments of the gene encompassing partial exon 1 and intron 1, and exons 2?5 with respective introns were sequenced and these data were deposited in the GenBank database. Three distinct polymorphic sites detectable with PCR-RFLP were found ? AM050719: g.237G > A, AM050719: g.525A > G, and AM050720: g.1071A > G. No positive associations between these polymorphisms and the PRA-clinical status were observed in the investigated population consisting of Poodles, American Cocker Spaniels, and English Cocker Spaniels. In spite of that, the FSCN2 gene remains an excellent candidate gene for retinopathies in dogs and the results can contribute to further research in this field.
4

Low incidence of bovine leukocyte adhesion deficiency (BLAD) carriers in Indian cattle and buffalo breeds

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Patel R. K., Singh K. M., Soni K. J., Chauhan J. B., Sambasiva_ R. K. R. S.
Journal of Applied Genetics
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2007
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vol. 48
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issue 2
153-155
EN
BLAD is an autosomal recessive genetic disease that affects Holstein-Friesian (HF) cattle worldwide. It is a disease characterized by a reduced expression of the adhesion molecules on neutrophils. The disease is caused by a mutation that replaces adenine at 383 with guanine, which causes an amino acid change from aspartic acid to glycine. Blood samples and a few semen samples were collected from 1250 phenotypically normal individuals, including HF (N = 377), HF crossbred (N = 334), Jersey (105), other breeds of cattle (N = 160) and water buffalo Bubalus bubalis (N=274) belonging to various artificial insemination stations, bull mother farms (BMFs) and embryo transfer (ET) centres across the country. PCR-RFLP was performed to detect a point mutation in CD18, surface molecules of neutrophils. The results indicate that out of 1250 cattle and buffaloes tested for BLAD, 13 HF purebreds out of 377 and 10 HF crossbreds out of 334 appear to be BLAD carriers. In the HF and HF crossbred population, the percentage of BLAD carriers was estimated as 3.23%. The condition is alarming as the mutant gene has already entered the HF crossbred cattle population and therefore, the population of HF and its crossbreds needs regular screening to avoid the risk of spreading BLAD in the breeding cattle population of India.
5

Identification of gadoid species (Pisces, Gadidae) by PCR-RFLP analysis

100%
Aranishi F., Okimoto T., Izumi S.
Journal of Applied Genetics
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2005
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vol. 46
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issue 1
69-73
EN
A rapid PCR-RFLP analysis was optimized to identify the presence of 3 closely related gadoid fish species: Alaska pollack Theragra chalcogramma, Pacific cod Gadus macrocephalus and Atlantic cod Gadus morhua in commercial seafood products. Gadoid universal primers were designed for PCR amplification of a 558-bp fragment encoding the mitochondrial cytochrome b gene. Without purification of the PCR products, double digestion with Eco32I and Eco105I restriction enzymes generated reproducible species-specific restriction patterns visualizing 3 fragments (106 bp, 161 bp and 291 bp) in Alaska pollack and 2 fragments (106 bp and 452 bp) in Pacific cod, whereas no cleavage was observed in Atlantic cod. This PCR-RFLP analysis is simple, rapid and reliable, and therefore can be routinely applied to discover fraudulent substitution among 3 economically important gadoid species in commercial seafood products.
6

A new PCR-RFLP within the domestic pigeon (Columba livia var. domestica) cytochrome b (MTCYB) gene

100%
Dybus A., Knapik K.
Journal of Applied Genetics
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2005
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vol. 46
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issue 3
315-317
EN
. A total of 244 domestic pigeons (Columba livia var. domestica) were genotyped using the PCR-RFLP method. A 999 bp fragment of the MTCYB gene was amplified. The amplification products were digested with restriction enzymes. PCR-RFLP for MvaI restriction enzyme was observed. Frequencies of alleles were as follows: MTCYBC ? 0.926, MTCYBG ? 0.074. The frequencies of MTCYB/MvaI alleles found in this study for non-homing pigeons considerably deviate from the values found for homing/racing pigeons (allele MTCYBG occurred only in the non-homing breeds).
7

Polymorphism within the LDHA gene in the homing and non-homing pigeons

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Dybus A., Pijanka J., Cheng Y.-H., Sheen F., Grzesiak W., Muszynska M.
Journal of Applied Genetics
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2006
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vol. 47
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issue 1
63-66
EN
A total of 445 domestic pigeons were genotyped for the lactate dehydrogenase (LDHA) gene. Crude DNA was isolated from blood samples and feathers. Two polymorphic sites were identified in intron 6: one near the splice donor site GT is called site H and the other near the splice acceptor site is called site B. Interestingly, the nucleotide changes of both these sites associate perfectly with the A and B alleles of HaeIII polymorphism: the A allele with nucleotide A of site H and nucleotide T of site B; while the B allele with nucleotide G of site H and nucleotide G of site B. In this study, we have identified the molecular difference between alleles A and B of the pigeon LDHA gene. The difference at site H in intron 6 explains the HaeIII polymorphism. The frequencies of LDHAAB and LDHABB genotypes between the analysed groups differ significantly (P < 0.001); the LDHAA allele was more frequent in the groups of pigeons with elevated homing performance (P < 0.001). The functional difference may be due to the change at site B, the potential splice branch site. Since LDHA activity is associated with the homing ability, it is possible that during the process of selection for the homing ability, the LDHAA allele has been selected, and is more prevalent in the top-racing groups.
8
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Lack of association between UBE2E2 gene polymorphism (rs7612463) and type 2 diabetes mellitus in a Saudi population

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Alharbi K., Khan I., Al-Sheikh Y., Alharbi F., Alharbi F., Al-Nbaheen M.
Acta Biochimica Polonica
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2014
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vol. 61
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issue 4
769-772
EN
The ubiquitin-conjugating enzyme E2E 2 (UBE2E2) gene plays an important role in insulin synthe­sis and secretion under conditions in which stress to the endoplasmic reticu­lum is increased in β-cells. In this case-control study, we have selected rs7612462 polymorphism within UBE2E2 gene to identify in a Saudi population the type 2 diabetes mellitus (T2DM) subjects. In total, 376 subjects with T2DM and 380 controls were enrolled in this study. We have collected 5 mL of peripheral blood from each participant for biochemical and molecular analyses. PCR-RFLP was used to generate genotypes at rs7612462 in all of the study subjects. Clinical data and anthropometric measurements of the patients were significantly different from those of the controls (p<0.05). All of the subjects used in this study were non-obese (25
9
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PCR-RFLP detection of point mutations A2143G and A2142G in 23S rRNA gene conferring resistance to clarithromycin in Helicobacter pylori strains

100%
Klesiewicz K., Nowak P., Karczewska E., Skiba I., Wojtas-Bonior I., Sito E., Budak A.
Acta Biochimica Polonica
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2014
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vol. 61
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issue 2
311-315
EN
Background. The occurrence of clarithromycin resistance among Helicobacter pylori strains is a major cause of the treatment failure. Resistance to this drug is conferred by point mutations in 23S rRNA gene and the most prevalent mutations are A2143G and A2142G. The aim of the study was to evaluate the occurrence of A2143G and A2142G mutations in a group of H. pylori strains resistant to clarithromycin. Materials and Methods. The study included 21 clarithromycin-resistant H. pylori strains collected between 2006 and 2009 in southern Poland. Resistance to clarithromycin was quantitatively tested with the E-test to determine the minimal inhibitory concentration (MIC value). The point mutations of H. pylori isolates were detected by PCR followed by RFLP analysis. Results. The MIC values for clarithromycin for the analyzed strains ranged from 1.5 mg/L to 64 mg/L. Nine H. pylori strains exhibited A2143G mutation and A2142G mutation was found in 9 isolates as well. The results of RFLP analysis of 3 clarithromycin-resistant strains were negative for both mutations. The average MIC values for A2143G and A2142G mutants were 6 and 30 mg/L, respectively. Conclusions. Frequencies of A2143G and A2142G mutations were the same in all isolates tested. Strains with A2143G mutation exhibited lower MIC values than A2142G mutants. Application of PCR-RFLP method for detection of clarithromycin resistance allows for better and more efficient management of H. pylori infections.
10

Simultaneous detection of malignant hyperthermia and genetic predisposition for improved litter size in pigs by multiplex PCR-RFLP

100%
Omelka R., Vasicek D., Martiniakova M., Bulla J., Bauerova M.
Folia Biologica
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2004
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vol. 52
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issue 1-2
113-115
EN
The ryanodine receptor gene (RYR1) and the estrogen receptor gene (ESR) are the best commercially used markers for predisposition of stress susceptibility (malignant hyperthermia ? MH) and increased litter size, respectively. A simplified method of simultaneous detection of MH and ESR genotypes has been developed. The method is based on simultaneous amplification of fragments of two genes by multiplex PCR and subsequent digestion of the products with two restriction enzymes. The PCR and the digestion could be performed in a single tube and all genotypes could be detected by electrophoretic separation on the same agarose gel. Thus, the development of the method can decrease the cost of the sample analysis and increase the speed and efficiency of the analysis. In our study, frequencies of mutated T allele of the RYR1 gene in Large White (LW), White Meaty (WM) and Landrace (L) were 0.11, 0.13, and 0.15, respectively. Frequencies of the preferred B allele of the ESR gene in the same breeds were 0.35, 0.26, and 0.06, respectively.
11

Associations between the prolactin receptor gene polymorphism and reproductive traits of boars

100%
Kmiec M., Terman A.
Journal of Applied Genetics
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2006
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vol. 47
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issue 2
139-141
EN
The prolactin receptor gene (PRLR), located on chromosome 16 in pigs, is a candidate gene for reproductive traits. The experiment was aimed to detect the DNA mutations in this gene and to find probable relations between the genotype and some reproductive traits in boars. The polymorphism in the PRLR gene was identified by PCR-RFLP method using specific primers and the restriction enzyme AluI. In total 229 boars of various breeds were genotyped. The frequency of allele A was estimated at 0.62 and allele B at 0.38. Genotype AA was found at a frequency of 0.45, AB at 0.35 and BB at 0.20. We found associations between PRLR genotype and ejaculate volume, sperm concentration, percentage of live sperm, and number of live sperm in the ejaculate (P < 0.01).
12

Molecular identification of hairtail species (Pisces: Trichiuridae) based on PCR-RFLP analysis of the mitochondrial 16S rRNA gene

100%
Chakraborty A., Aranishi F., Iwatsuki Y.
Journal of Applied Genetics
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2005
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vol. 46
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issue 4
381-385
EN
A rapid PCR-RFLP analysis was designed to identify 3 closely related species of hairtails: Trichiurus lepturus, T. japonicus, and Trichiurus sp. 2, basing on partial sequence data (600 bp) of the mitochondrial DNA encoding the 16S ribosomal RNA (16S rRNA) gene. Restriction digestion analysis of the unpurified PCR products of these 3 species, using EcoRI and VspI endonucleases, generated reproducible species-specific restriction patterns showing 2 fragments (250 bp and 350 bp) for T. lepturus in EcoRI digestion and 2 fragments (196 bp and 404 bp) for T. japonicus in VspI digestion, whereas no cleavage was observed for Trichiurus sp. 2 in both EcoRI and VspI digestions. The PCR-RFLP technique developed in this study proved to be a rapid, reliable and simple method that enables easy and accurate identification of these 3 closely related species of the genus Trichiurus.
13

PCR-RFLPs within the lactate dehydrogenase (LDH-A) gene of the domestic pigeon (Columba livia var. domestica)

100%
Dybus A., Kmiec M.
Journal of Applied Genetics
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2002
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vol. 43
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issue 4
501-504
EN
A total of 45 racing pigeons were genotyped using PCR-RFLP method. PCR product of the LDH-A gene was amplified according to the Long-PCR procedure. The amplification products were digested with restriction enzymes. PCR-RFLPs for two restriction enzymes, HaeIII and NlaIV, were observed. Two pairs of alleles LDH-AA and LDH-AB for LDH-A-NlaIV polymorphism and LDH-AC and LDH-AD for LDH-A-HaeIII polymorphism were detected in the homozygous and heterozygous states. Frequencies of alleles were as follows: A ? 0.622, B ? 0.378 and C ? 0.256, D ? 0.744.
14

Use of cytochrome b polymorphism for species identification of biological material derived from cattle, sheep, goats, roe deer and red deer

100%
Natonek-Wisniewska M., Slota E., Kalisz B.
Folia Biologica
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2010
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vol. 58
|
issue 1-2
47-50
EN
The objective of this study was species identification of the following biological trace material: skin, blood stains, meat samples and jawbone with a tooth, which were the subject of expert opinion ordered by a court. The expert appraisement was conducted by an analysis of a cytochrome b fragment. The choice of mtDNA fragment for analysis was based on its conservation in mammals which enabled several farm and wild species to be identified with one pair of primers. The PCR product was differentiated by Tsp509I and AluI enzymes. Due to problems with amplification of roe deer DNA, primers specific to this species only, flanking a cytochrome b fragment (Y14951.1), were designed. On the basis of this analysis, it was concluded that the skin sample was derived from a goat, dried blood from a roe deer, the jawbone fromcattle, and twomeat samples froma roe deer and red deer. od from a roe deer, the jawbone fromcattle, and twomeat samples froma roe deer and red deer. This method allowed rapid and efficient identification of several species of mammals using diverse biological material.
15
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Analysis of Slovak Spotted breed for bovine beta casein A1 variant as risk factor for human health

100%
Miluchová M., Gábor M., Trakovická A.
Acta Biochimica Polonica
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2013
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vol. 60
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issue 4
799-801
EN
The goal of work was identification A1 variant of bovine beta casein which involves ischemic heart disease and diabetes mellitus in human. The digestion of A1beta casein can result in the production of bioactive beta casomorphin-7 (BCM-7); this is not the case with A2. This bioactive peptide has been linked to physiological traits that may elicit effects on components of the vascular and immune systems. The material involved 111 Slovak Spotted breed. Bovine genomic DNA was extracted from whole blood by using commercial kit, and used in order to estimate beta-casein genotypes by means of PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. In the population included in the study were detected all three genotypes, homozygote genotype A1A1 (14 animals), heterozygote genotype A1A2 (37 animals) and homozygote genotype A2A2 (60 animals). In the total population of cattle homozygotes A2A2-0.5405 were the most frequent, while homozygotes A1A1-0.1261 were the least frequent ones. This suggests a superiority of allele A2 (0.7072) which does not produce BCM-7, and thus is safe for human consumption. The expected homozygosity for gene CSN2 is in the population stated a slight increase in homozygosity (0.5858). This caused a slight decrease in the level of possible variability realization (41.80%), which corresponds to the effective number of alleles (1.7071).
16

Restriction fragment length polymorphism of exon 2 Ovar-DRB1 gene in Polish Heath Sheep and Polish Lowland Sheep

88%
Gruszczynska J., Brokowska K., Charon K. M., Swiderek W. P.
Journal of Applied Genetics
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2005
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vol. 46
|
issue 3
311-314
EN
Exon 2 of the Ovar-DR gene is known to encode the MHC outer domain (alpha or beta chain) that forms the binding area to antigens presented. The study was aimed at analysing exon 2 Ovar -DRB1 gene polymorphism in Polish Heath Sheep and Polish Lowland Sheep (Zelazna variety). A total of 101 and 99 ewes of the respective breeds were included in this study. We identified 65 different haplotypes in Polish Heath Sheep and 68 in Polish Lowland Sheep. The PCR-RFLP method and PCR products sequencing made it possible to identify two new sequences of exon 2 Ovar-DRB1 gene (AY230000 and AY248695). A distinct polymorphism in the exon 2 sequence presents possibilities for immune response toward a great variety of pathogens.
17

Restriction fragment length polymorphism of mitochondrial DNA and phylogenetic relationships among five species of Indian freshwater turtles

75%
Rohilla M. S., Tiwari P. K.
Journal of Applied Genetics
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2008
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vol. 49
|
issue 2
167-182
EN
DNA-based identification of species for phylogenetic analysis as well as forensic identification is widely being carried out with the help of polymerase chain reaction (PCR). In this study, a successful effort has been made to identify 5 species of Indian freshwater turtles, including 3 hard-shell turtles (Geoemydidae), i.e. Kachuga dhongoka, K. kachuga and Geoclemys hamiltoni, and 2 species of soft-shell turtles (Trionychidae), i.e. Aspideretes gangeticus and Lissemys punctata punctata, by using a well-optimized PCR-RFLP method. The analysis of nucleotide sequence variations in the PCR-amplified mitochondrial cyt-b genes (encoding cytochrome b) from the 5 species revealed its usefulness in the taxonomic differentiation of these species. On the basis of cyt-b sequence data and the PCR-RFLP pattern, a phylogeny was developed to resolve the genetic relationships between these species, living in the same habitat type. In comparison, the PCR-RFLP of mitochondrial 16S rDNA genes appeared less decisive in analysing phylogenetic relationships or even in species differentiation. Further, the molecular method (PCR-RFLP) developed here is simple, rapid, reliable and reproducible; hence it can be routinely applied for species identification, essential for conservation and management of endangered chelonian species.
18

Simultaneous identification of ryanodine receptor 1 (RYR1) and estrogen receptor (ESR) genotypes with the multiplex PCR-RFLP method in Polish Large White and Polish Landrace pigs

75%
Kaminska S., Rusc A., Wojtasik K.
Journal of Applied Genetics
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2002
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vol. 43
|
issue 3
331-335
EN
A method allowing simultaneous genotyping of two loci: ryanodine receptor 1 (RYR1) and estrogen receptor (ESR) is presented. In multiplex PCR amplification, two amplicons were simultaneously produced: a 272 bp fragment of RYR1 gene and a 185 bp fragment of ESR gene and were then subjected to ?one-tube? restriction enzyme digestion with Hin6 I and Ava I, respectively. A total of 122 Polish Large White and Polish Landrace pigs were genotyped by this method, demonstrating its reliability, convenience and lower costs. This method may be useful in the wide-scale genotyping of both loci in pig breeding programmes.
19
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Metody oparte o amplifikację DNA techniką PCR wykorzystywane w ocenie bioróżnorodności mikroorganizmów glebowych

71%
Łyszcz M., Gałązka A.
Kosmos
|
2017
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vol. 66
|
issue 2
193-206
PL
Mikroorganizmy glebowe, pod względem cech genomowych i fenotypowych, stanowią wysoce zróżnicowaną grupę organizmów żywych. Z powodu tak dużej różnorodności ważne jest dobranie odpowiednich metod, dających największy stopień różnicowania mikroorganizmów. Narzędziami umożliwiającym analizę zmienności genetycznej mikroorganizmów są techniki genetyczne, a wśród nich jedną z najważniejszych jest łańcuchowa reakcja polimerazy, czyli PCR (Polymerase Chain Reaction), technika opracowana w latach 1980. Niniejsza praca stanowi przegląd podstawowych zagadnień dotyczących badania zmienności genetycznej mikroorganizmów glebowych w oparciu o markery molekularne z wykorzystaniem technik bazujących na reakcji PCR tj. PCR-RFLP, TRFLP, ARDRA, RAPD.
EN
Soil microorganisms represent a highly diverse group of living organisms in terms of genomic and phenotypic characteristics. Due to such a large diversity, it is important to select appropriate identification methods which would secure its most complete determination. Genetic techniques are proper tools of choice for analyzing genetic variability of microorganism, the most important of which is the polymerase chain reaction (PCR), developed in the 1980s. This work presents an overview of the basic issues concerning studies on genetic variability of soil microorganisms with help of molecular markers and application of PCR techniques such as PCR-RFLP, TRFLP, ARDRA, RAPD.
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