Monitoring of Poliovirus Strains Circulating in the Environment and Their Intertypic and Intratypic Differentiation by Rflp Analysis of a PCR Product Derived from the 5’ Non-Coding Region

1994 ◽  
pp. 169-178
Author(s):  
B. Schweiger ◽  
B. Böhtig ◽  
S. Diedrich ◽  
J. M. López-Pila ◽  
E. Schreier
Keyword(s):  
Rflp Analysis ◽  
Coding Region ◽  
Pcr Product

A variable minisatellite sequence in the chloroplast genome of Sorbus L. (Rosaceae: Maloideae)

Genome ◽  
2002 ◽  
Vol 45 (3) ◽  
pp. 570-576 ◽  
Author(s):  
R Andrew King ◽  
Colin Ferris
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Chloroplast Genome ◽  
Length Polymorphism ◽  
Sequence Variation ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Chloroplast Microsatellites ◽  
Sorbus Aucuparia ◽  
Pcr Product ◽  
Restriction Fragment Length

The chloroplast genome is now known to be more variable than was once thought. Reports of RFLP (restriction fragment length polymorphism) and sequence variation, as well as variation in chloroplast microsatellites, are common. Here, data are presented on the variability of a minisatellite sequence in the chloroplast genome of Sorbus species. RFLP analysis of a PCR product comprising the region between the trnM and rbcL genes of nine Sorbus species identified seven size variants. Sequencing revealed the observed size polymorphism to be due to differences in the number of copies of an imperfect 9-bp motif. A more intensive survey of the variability of the minisatellite was undertaken in populations of Sorbus aucuparia. The potential uses of such regions in chloroplast DNA are discussed and a possible mechanism for the evolution of the minisatellite is presented.Key words: atpE, homoplasy, microsatellite, rowan, VNTR.

Classification of wheat PPO genes and effect of non-synonymous cSNP on kernel PPO activity

2008 ◽  
Vol 5 (1) ◽  
pp. 81-86 ◽  
Author(s):  
Wang Xiao-Bo ◽  
Ma Chuan-Xi ◽  
Si Hong-Qi ◽  
He Xian-Fang
Keyword(s):  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Chain Reaction ◽  
Wheat Varieties ◽  
Single Nucleotide ◽  
Pcr Product ◽  
Dnaman Software ◽  
Dna Fragment ◽  
Polymerase Chain

AbstractPolyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products. In this study, wheat PPO sequences (mRNA) were searched/BLASTed in the NCBI database and aligned using DNAMAN software. The results showed that wheat PPO genes could be divided into two clusters (I and II) and that three genes (‘i’) of cluster II seemed not to be located on chromosomes 2A and 2D. Ninety-four single nucleotide polymorphisms (SNPs) were detected between two haplotypes of the PPO gene on chromosome 2D. Eighty of these were found in the coding region (coding (c) SNPs) and 36 were non-synonymous cSNPs, which could affect the PPO amino acid sequence. Primers (STS-H) were designed at some non-synonymous cSNPs sites and were used to investigate the correlations between allelic variants and PPO activity of seeds – a total of 130 common wheat varieties were evaluated in 2 years. The results showed that STS-H could amplify a 460 bp DNA fragment in most cultivars with high PPO activity, while no PCR product was detected in most cultivars with low PPO activity. To improve the selection efficiency of a single dominance molecular marker, the multiplex polymerase chain reaction (PCR) system of STS-H and STS01 markers was also studied, based on the complementary between them.

A Novel Denaturing-High Performance Liquid Chromatography (D-HPLC)-Based Method for Kit Mutation Screening of Patients (pts) with Systemic Mastocytosis (SM) Allows the Identification of Unreported Kit Variants.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4849-4849
Author(s):  
Sabrina Colarossi ◽  
Simona Soverini ◽  
Alessandra Gnani ◽  
Michela Rondoni ◽  
Simona Gatto ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Point Mutation ◽  
Hplc Analysis ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Rflp Analysis ◽  
Rt Pcr ◽  
Kit Mutation ◽  
Pcr Product ◽  
Tm Domain

Abstract SM is characterized by activating mutations of the Kit tyrosine kinase receptor. While the so-called ‘enzimatic site’ (ES) type mutation D816V renders Kit resistant to imatinib, ‘regulatory’ type mutations are sensitive to inhibition. Kit mutation screening with sensitive methods is important for appropriate therapeutic management of SM. Our aims were: to set up and optimize a D-HPLC-based screening method for mutations in different critical regions of the kit receptor; to assess the sensitivity and reliability of our D-HPLC assay as compared to RFLP analysis; to characterize additional mutations/polymorphisms. The analysis was performed on 37 SM pts. For each sample, a RT-PCR product spanning the catalytic and activation loops in the ES was screened in parallel by D-HPLC, followed by sequencing of D-HPLC-positive cases, and by RFLP according to an already reported method for the detection of the D816V. By RFLP analysis, 24/37 (65%) pts were positive for the D816V. By D-HPLC analysis, an abnormal eluition profile was seen in 26/37 (70%) pts - all the 24 pts scored as mutated by RFLP as well as two additional pts. Direct sequencing confirmed the presence of the D816V in all the 24 RFLP-positive cases, and showed that in two of these cases a I798I polymorphism was also present. The two pts scored positive by D-HPLC but negative by RFLP were found to have the same I798I polymorphism. The 11 pts who did not harbour ES type mutations were further investigated by D-HPLC analysis of a RT-PCR product corresponding to the transmembrane (TM) and juxtamembrane (JM) domains. D-HPLC showed an abnormal elution profile in 4 pts. Direct sequencing confirmed the presence of a point mutation in all cases. One patient showed a silent mutation at codon 546. Three pts showed a novel point mutation at codon 541 in the TM domain, resulting in a Met to Leu amino acid substitution. This is the second Kit TM domain mutation reported in a human disease and further supports the hypothesis of a role of the TM domain in regulating the enzymatic activity of an otherwise normal catalytic site. Further characterization of this novel mutant is ongoing. Morphologic and cytofluorimetric analyses of bone marrow biopsies and aspirates will be compared in order to assess whether the pts share any peculiar pathologic feature. Cos-7 cells are currently being transfected with M541L, D816V and wild-type kit in order to evaluate the effects of the M541L on kit enzymatic activity by western blot analysis of total and phosphorylated kit. Patient mast cells will be cultured in the presence or absence of kit ligand or imatinib, dasatinib and nilotinib in order to assess the sensitivity of the M541L to different kit inhibitors. Our novel D-HPLC-based assay proved a straightforward, reliable and sensitive method for kit mutation analysis. It also highlighted the importance of screening for mutations other than the D816V. D-HPLC analysis allowed us to find a novel M541L mutation which is now under characterization.

scholarly journals First detection and molecular characterization of kobuvirus from a dog in Turkey

2019 ◽  
Vol 75 (05) ◽  
pp. 6248-2019
Author(s):  
ZEYNEP AKKUTAY-YOLDAR ◽  
TAYLAN KOÇ B. ◽  
ÇIĞDEM OĞUZOĞLU T.
Keyword(s):  
Molecular Characterization ◽  
Rectal Swab ◽  
Clinical Symptoms ◽  
Phylogenetic Analyses ◽  
Amino Acid Sequences ◽  
Coding Region ◽  
Gastrointestinal Infections ◽  
Protein Coding ◽  
Pcr Product ◽  
Domestic Carnivores

Canine kobuvirus (CaKVs) is a newly emerging virus detected in dogs in several countries. However, kobuvirus infection has not yet been described in domestic carnivores in Turkey. In this study, we tested blood and rectal swab samples to determine the presence of kobuvirus in a dog with clinical symptoms by reverse transcription-polymerase chain reaction (RT-PCR), using 3D (RNA polymerase) region primers of canine kobuviruses. To provide molecular characterization data, the Maximum Likelihood (ML) method was used for the phylogenetic analyses. The PCR product of the partial protein-coding region of the 3D protein gene from the rectal swab was amplified, purified, and sequenced. Phylogenetic analysis of amino acid sequences suggests that our CaKV strain was closely related to US-CaKVs,and placed on a monophyletic clade as a sister branch localized in the CaKV cluster. These results indicate that CaKV exists in dogs in Turkey. With a similarity of 94.2–96.1%, it is like other CaKVs. To our knowledge, this is the first report of CaKV infection of a dog by in Turkey. Further studies are needed to determine its role in dog gastrointestinal infections.

Genetic Polymorphisms of Chicken Antiviral Mx1 and TVB Genes in Indigenous and Giriraja Chicken

2021 ◽  
Author(s):  
S. Nihar ◽  
S. Naveen Kumar ◽  
Wilfred Ruban ◽  
H.M. Yathish ◽  
R. Nagaraja ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Dna Isolation ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Isoamyl Alcohol ◽  
Gene Frequencies ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Recent Developments ◽  
Mx1 Gene

Background: Recent developments in molecular genetics lead to addressing certain poultry diseases via breeding for disease resistance. The present study was carried to identify and compare the genetic polymorphism in Chicken Mx1 and TVB genes among the indigenous and Giriraja chicken using PCR-RFLP technique. Methods: Blood samples were collected from 50 indigenous and 50 Giriraja birds and DNA isolation was done by Phenol: Chloroform: Isoamyl alcohol method. PCR amplification of Chicken Mx1 (exon 14) and Chicken TVB (exon 3) genes was carried out followed by RFLP analysis. Result: PCR product sizes of 301 bp and 303 bp of Mx1 and TVB genes, respectively were successfully amplified. RFLP analysis of Mx1 gene with Hyp8I restriction enzyme revealed three genotypes AA, AB and BB. In indigenous birds genotypic frequencies of AA, AB and BB were 0.314, 0.493 and 0.194, respectively and gene frequencies were 0.56 and 0.44 for alleles A and B, respectively. In Giriraja birds, genotypic frequencies for AA, AB and BB were 0.27, 0.499 and 0.23, respectively and gene frequencies were 0.52 and 0.48 for alleles A and B, respectively. RFLP analysis of TVB gene with NlaIII restriction enzyme revealed two genotypes viz., AA and AB. In indigenous birds genotypic frequencies of AA and AB were 0.81 and 0.18, respectively and gene frequencies were 0.9 and 0.1 for alleles A and B, respectively. In Giriraja birds genotypic frequencies for AA and AB were 0.774 and 0.211, respectively and gene frequencies were 0.88 and 0.12 for alleles A and B, respectively.

CD24 Gene Polymorphism - A Novel Prognostic Factor in Esophageal Cancer

2014 ◽  
Vol 29 (1) ◽  
pp. e49-e54 ◽  
Author(s):  
Eran Sadot ◽  
Sarah Kraus ◽  
Michael Stein ◽  
Ilana Naboishchikov ◽  
Ohad Toledano ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Cancer Patients ◽  
Poor Prognosis ◽  
Statistical Significance ◽  
Rflp Analysis ◽  
Tumor Stage ◽  
Control Group ◽  
Coding Region ◽  
Regional Lymph Nodes ◽  
Significant Difference

Background The CD24 gene has been correlated with poor prognosis of various malignancies. The significance of CD24 in esophageal cancer remains unknown. Our aim was to evaluate the association between CD24 genetic polymorphism and esophageal cancer. Materials and Methods Between June 2011 and May 2012 patients with esophageal cancer and healthy controls were prospectively enrolled and clinicopathological data were collected. Genomic DNA was extracted and restriction fragment length polymorphism (RFLP) analysis was performed to determine CD24 polymorphism at the coding region of CD24, which results in a substitution of the amino acid Ala by Val. Statistical significance was determined by unpaired t-test, χ2-test, and Fisher's exact test. Results A total of 102 patients were included, of whom 51 had esophageal cancer and the rest comprised a healthy control group. The incidence of the polymorphism variant (Val/Val) among the healthy subjects and the esophageal cancer cohort was 6% in both groups. The incidence of N3 (metastasis in 7 or more regional lymph nodes) was markedly higher in those esophageal cancer patients who carried the polymorphism variant compared with those who did not carry it (66% and 2%, respectively, p=0.007). No significant difference was found between the groups with regard to age, gender, histology type, tumor location, tumor stage, and other histological characteristics of the tumor. Conclusions This CD24 polymorphism may serve as a novel prognostic marker identifying esophageal cancer patients with poor prognosis. Further studies are warranted to evaluate CD24 function and to validate its predictive potential with regard to esophageal cancer.

Multiple displacement amplification of DNA for ITS-RFLP analysis of individual juveniles of Bursaphelenchus

Nematology ◽  
2005 ◽  
Vol 7 (2) ◽  
pp. 253-257 ◽  
Author(s):  
Wolfgang Burgermeister ◽  
Kai Metge
Keyword(s):  
Multiple Displacement Amplification ◽  
Diagnostic Testing ◽  
Rflp Analysis ◽  
Bursaphelenchus Xylophilus ◽  
Pinewood Nematode ◽  
Closely Related Species ◽  
Amplification Method ◽  
Pcr Product ◽  
Template Dna ◽  
Identification Analysis

AbstractDifferentiation of the plant-pathogenic pinewood nematode, Bursaphelenchus xylophilus, from non-pathogenic Bursaphelenchus species is difficult because of high morphological similarities among closely related species. In recent years, ITS-RFLP analysis has become a useful tool for Bursaphelenchus species identification. Analysis of individual nematodes is hampered by the fact that sufficient template DNA for ITS-PCR cannot be extracted reliably. We have employed a whole genome amplification method, termed multiple displacement amplification (MDA), to 26 DNA extracts from individual juveniles to increase the amount of template DNA. Preamplification of the whole genomic DNA by MDA provided sufficient amounts of PCR product for ITS-RFLP analysis with 12 out of 20 B. xylophilus, two B. mucronatus, two B. fraudulentus and two B. eggersi samples tested. The introduction of MDA to ITS-RFLP analysis of nematodes may improve the reliability of diagnostic testing for limited samples and permit verification of analytical results.

scholarly journals Membrane Adsorption with Direct Cell Culture Combined with Reverse Transcription-PCR as a Fast Method for Identifying Enteroviruses from Sewage

2005 ◽  
Vol 71 (1) ◽  
pp. 72-79 ◽  
Author(s):  
D. Papaventsis ◽  
N. Siafakas ◽  
P. Markoulatos ◽  
G. T. Papageorgiou ◽  
C. Kourtis ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Sewage Treatment ◽  
Rflp Analysis ◽  
Fast Method ◽  
Coding Region ◽  
Protein Coding ◽  
Membrane Filters ◽  
Reverse Transcription Pcr ◽  
Detection And Identification ◽  
Study Sites

ABSTRACT We present a new approach for the detection and identification of enteroviruses concentrated and isolated from sewage. Samples were collected from two study sites located at Nicosia and Limassol sewage treatment plants in Cyprus. Viruses were adsorbed to cellulose nitrate membrane filters, cultured directly from the membrane filters by using the VIRADEN method, and identified by reverse transcription-PCR, followed by 5′ untranslated region (5′-UTR) restriction fragment length polymorphism (RFLP) analysis and partial sequencing of the VP1 protein coding region. Initial subgrouping based on the HpaII restriction profile showed that all of the isolates except one belonged to the same genetic subcluster. Partial VP1 sequencing revealed that most isolates belonged to serotypes coxsackie B4 (42.5%) and coxsackie Α9 (30%), whereas coxsackie B2 (17.5%) and coxsackie B1 (3%) isolates were less frequently observed. One poliovirus type 2 isolate (2.5%) of vaccine origin was also found. The HpaII digests predicted the genetic subcluster for all isolates. They also accurately differentiated the isolates as nonpolio or polio isolates. This approach seems to be very promising for environmental surveillance of enterovirus circulation and epidemiology, with all of the significant effects that this entails for public health. Partial VP1 sequencing is efficient for molecular serotyping of enteroviruses, while 5′-UTR RFLP analysis with HpaII can also be considered an asset for the initial subclassification of enterovirus isolates.

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