Development of an Efficient Inverse PCR Method for Isolating Gene Tags from T-DNA Insertional Mutants in Rice

2010 ◽  
pp. 139-146 ◽  
Author(s):  
Sung-Ryul Kim ◽  
Jong-Seong Jeon ◽  
Gynheung An
Keyword(s):  
Inverse Pcr ◽  
Insertional Mutants ◽  
Pcr Method

Using an Inverse PCR Method to Clone the Wheat Cytokinin Oxidase/Dehydrogenase Gene TaCKX1

2008 ◽  
Vol 26 (3) ◽  
pp. 143-155 ◽  
Author(s):  
De-Shun Feng ◽  
Hong-Gang Wang ◽  
Xian-Sheng Zhang ◽  
Ling-Rang Kong ◽  
Ji-Chun Tian ◽  
...  
Keyword(s):  
Inverse Pcr ◽  
Cytokinin Oxidase ◽  
Dehydrogenase Gene ◽  
Pcr Method

The Evolution of Techniques Used for the Detection of the F8 IVS 22 Inversion Mutation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4057-4057
Author(s):  
Jacky A. Cutler ◽  
Louise Bourdon ◽  
Geoffrey F. Savidge ◽  
Mike J. Mitchell
Keyword(s):  
Southern Blot ◽  
Southern Blotting ◽  
Poor Quality ◽  
Inverse Pcr ◽  
Causative Mutation ◽  
Long Pcr ◽  
Archived Samples ◽  
Number Of Patients ◽  
Pcr Method ◽  
Long Range Pcr

Abstract Almost half of patients with severe hemophilia A have a genomic rearrangement of the F8 gene, resulting in the separation of exons 1–22 from exons 23–26. This disruption is due to a recombination hotspot involving a 9.5kb region within intron 22 (int22h-1) and one of at least two extragenic copies (int22h-2 and inth22-3) located telomeric to the F8 gene. The 3 copies of int22h are estimated to be 99.9% homologous, with consistent differences between them confined to only 3 nucleotides. The more distal Int22h-3 lies in opposite orientation to the intragenic copy, and recombination between these two results in an inversion mutation, and a total lack of F8 protein. It was recently shown that int22h-2 is oriented in the same direction as int22h-1, and recombination events between these will result in either deletion or duplication rather than inversion. Three techniques have been published for the detection of the IVS22 recombination mutations: Southern blot, Long-PCR and Inverse PCR. We present data on a significant number of patients tested by more than one technique, and highlight difficulties in the detection/positive identification of mutations by each. Southern blotting (the only available method for many years) requires a large amount of DNA, not readily available from immuno-compromised patients, relies on radioisotope activity, and takes several days to obtain a result. All forms of recombination can be distinguished, but polymorphic variants can be misinterpreted as rare inversions resulting in patients being incorrectly assigned as inversion positive. In 1998, a long range PCR method was published for the analysis of IVS22 inversions. This technique, more rapid and less hazardous than Southern blotting, has proved to be problematic in many laboratories. The primers degrade rapidly, do not consistently permit multiplexing, and amplification is highly dependant on freshly extracted, high purity DNA. In 2005, a technique based on genomic digestion, followed by self ligation and PCR was published. This inverse PCR methodology has proved to be robust and can be used for fresh or archived samples, generating reproducible results within 36 hours. Proximal and distal recombinations are indistinguishable, and non-causative polymorphisms are not detected. During evaluation of the emerging technologies this laboratory has parallel tested a significant number of samples. 36 patients, including 6 carrier females, gave concordant results with Southern blot and inverse PCR. 12 patients with polymorphic, non-haemophilia associated, banding patterns gave a normal result by inverse PCR. 3/12 had been incorrectly assigned as mutation positive by southern blot; with 1 discovered only by the subsequent detection of non-linkage in the family. 25 patients gave identical results by long PCR and inverse PCR, including 4 carrier females. 2 patients were wrongly defined as positive by long PCR due to poor quality DNA. A result was unobtainable for 23 patients by southern blot, and for 6 patients by long PCR, due to insufficient or poor quality DNA. Of these, 8/23 and 6/6 were subsequently resolved by inverse PCR. Of the remaining 15/23 failures, the causative mutation has since been identified elsewhere in the F8 gene in 8 patients, while 7 require repeat bleeding. In conclusion, the inverse PCR technique provides a fast, reliable, reproducible and safe method for the detection of IVS 22 recombination mutations, and is recommended as the method of choice for diagnostic laboratories.

scholarly journals Identification of a novel polyomavirus from vervet monkeys in Zambia

2013 ◽  
Vol 94 (6) ◽  
pp. 1357-1364 ◽  
Author(s):  
Hiroki Yamaguchi ◽  
Shintaro Kobayashi ◽  
Akihiro Ishii ◽  
Hirohito Ogawa ◽  
Ichiro Nakamura ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Nucleotide Sequence Identity ◽  
Cultured Cells ◽  
Vervet Monkey ◽  
Inverse Pcr ◽  
Vervet Monkeys ◽  
Merkel Cell ◽  
Entire Genome ◽  
Sequence Identity ◽  
Pcr Method

To examine polyomavirus (PyV) infection in wildlife, we investigated the presence of PyVs in Zambia with permission from the Zambia Wildlife Authority. We analysed 200 DNA samples from the spleens and kidneys (n = 100 each) of yellow baboons and vervet monkeys (VMs) (n = 50 each). We detected seven PyV genome fragments in 200 DNA samples using a nested broad-spectrum PCR method, and identified five full-length viral genomes using an inverse PCR method. Phylogenetic analysis of virally encoded proteins revealed that four PyVs were closely related to either African green monkey PyV or simian agent 12. Only one virus detected from a VM spleen was found to be related, with relatively low nucleotide sequence identity (74 %), to the chimpanzee PyV, which shares 48 % nucleotide sequence identity with the human Merkel cell PyV identified from Merkel cell carcinoma. The obtained entire genome of this virus was 5157 bp and had large T- and small t-antigens, and VP1 and VP2 ORFs. This virus was tentatively named vervet monkey PyV 1 (VmPyV1) as a novel PyV. Comparison with other PyVs revealed that VmPyV1, like chimpanzee PyV, had a longer VP1 ORF. To examine whether the VmPyV1 genome could produce viral proteins in cultured cells, the whole genome was transfected into HEK293T cells. We detected VP1 protein expression in the transfected HEK293T cells by immunocytochemical and immunoblot analyses. Thus, we identified a novel PyV genome from VM spleen.

Construction of Genome Library for the Toxic Gene of Alexandium minutum

2013 ◽  
Vol 779-780 ◽  
pp. 235-238
Author(s):  
Li Wang ◽  
Jia Yuan Li ◽  
Qi Xu ◽  
Qing Ping Zhong ◽  
Zhen Lin Liao
Keyword(s):  
Nucleotide Sequence ◽  
Stop Codon ◽  
Inverse Pcr ◽  
Methionine Aminopeptidase ◽  
Coding Region ◽  
Genome Library ◽  
Toxic Gene ◽  
Pcr Method ◽  
Dna Fragment ◽  
Growth Metabolism

Genome library of toxic Alexandium minutum were constructed. A 1014bp DNA reconstruction fragment was obtained and PCR method testified that this fragment had strong amplification signal in toxic strains while no signal in non-toxic strains. The external sequence of the DNA fragment was analyzed by inverse PCR method, and a 240bp nucleotide sequence which translated into proteins aminophenol was Methionine Aminopeptidase (MAP). The sequence of aminophenol was BLAST in NCBI, and found that it had 97% similarity with Alexandium fundyense. This sequence was exact uniform with the reported sequence in the coding region, but there were three bases mutated in non-coding region behind stop codon. Probably, in the course of growth metabolism of Alexandium minutum, MAP executed different physiological functions and controlled the production of the toxin. In the meantime, map gene induced encoding products of the related poisonous gene, and enabled it has active expressions to starting toxin synthesis.

scholarly journals Modified Inverse PCR Method for Cloning the Flanking Sequences from Human Cell Pools

BioTechniques ◽  
1999 ◽  
Vol 27 (4) ◽  
pp. 660-662 ◽  
Author(s):  
Zhu-Hong Li ◽  
De-Pei Liu ◽  
Chih-Chuan Liang
Keyword(s):  
Human Cell ◽  
Inverse Pcr ◽  
Flanking Sequences ◽  
Pcr Method

Comparison of two molecular diagnostic methods for identifying Beijing genotype of Mycobacterium tuberculosis

2020 ◽  
Author(s):  
Ghorban Ali Mahghani ◽  
Mohammad Kargar ◽  
Farshid Kafilzadeh ◽  
Homa Davoodi ◽  
Ezzat Allah Ghaemi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Clinical Isolates ◽  
Inverse Pcr ◽  
Beijing Genotype ◽  
Molecular Diagnostic ◽  
Beijing Family ◽  
Pcr Method

Background and Objectives: The Beijing family of Mycobacterium tuberculosis has been identified as a severe pathogen among this species and found in many clinical isolates during the last decade. Early identification of such genotype is import- ant for better prevention and treatment of tuberculosis. The present study performed to compare the efficiency of Real-Time PCR and IS6110-Based Inverse PCR methods to identify the Beijing family. Materials and Methods: This study was carried out on 173 clinical isolates of Mycobacterium tuberculosis complex in Golestan Province, northern Iran. DNA extraction performed by boiling and determining the Beijing and non-Beijing strains carried out using Real-Time PCR and IS6110-Based Inverse PCR. Results: In both Real-Time PCR and IS6110-Based Inverse PCR method, 24 specimens (13.9%) of the Beijing family were identified and the result of the IS6110-Based Inverse PCR method showed that all the Beijing strains in this region belonged to the Ancient Beijing sub-lineage. Conclusion: Although the efficacy of the two methods in the diagnosis of the Beijing family is similar, the IS6110-Based Inverse PCR is more applicable to the ability to detect new and old Beijing family.  

Investigation of duck plague virus in hoar areas of Bangladesh

2020 ◽  
Vol 26 (1-2) ◽  
pp. 73-78
Author(s):  
A Hossen ◽  
MH Rahman ◽  
MZ Ali ◽  
MA Yousuf ◽  
MZ Hassan ◽  
...  
Keyword(s):  
Cytopathic Effect ◽  
Clinical Sample ◽  
Chorioallantoic Membrane ◽  
Clinical Samples ◽  
Isolation And Identification ◽  
Duck Plague Virus ◽  
Pcr Method ◽  
Polymerase Chain ◽  
The Individual ◽  
Free Ranging

Duck plague (DP) is the most important infectious disease of geese, ducks and free-ranging water birds. The present study was conducted to determine the prevalence of duck plague virus followed by isolation and identification. For these purposes, a total of 155 cloacal swabs samples were collected randomly from duck of different haor areas of Bangladesh including 45 (41 surveillance and 4 clinical) samples from Netrokona; 42 (40 surveillance and 2 clinical) samples from Kishoregonj; 30 samples from Brahmanbaria and 38 samples from Sunamganj. The samples were processed and pooled (1:5 ratio) for initial screening of target polymerase gene of duck plague virus by polymerase chain reaction (PCR) method. All the samples of a positive pool were then tested individually for identifying the individual positive samples. The result showed that out of 155 samples, 41 (26.45%) were found positive in which 17 were from Netrokona, where 15 (36.58%) were from surveillance samples and 2 (50%) were from clinical sample; 16 were from Kishoregonj, where 14 (35%) were from surveillance samples and 2 (100%) were from clinical sample; 2 (6.6%) were from Brahmanbaria and 5 (13.15%) were from Sunamganj. These positive samples were inoculated into 9-10 days embryonated duck eggs (EDE) through chorioallantoic membrane (CAM) route for the isolation of virus. The EDE died earlier was also chilled, and in a similar way, the CAMs were collected and again performed PCR for id entification of virus. Out of 41 PCR positive samples, 26 samples were isolated and reconfirmed by PCR. Subsequently, DPV was isolated in primary duck embryo fibroblasts cell culture and confirmed by observing cytopathic effect (CPE). Bang. J. Livs. Res. Vol. 26 (1&2), 2019: P. 73-78

USING DNA MARKERS ENCODING P24 AND GP51 PROTEINS IN PCR-RT TO INCREASE SENSITIVITY AND SPECIFICITY OF THE PCR METHOD FOR INDICATION OF BLV

2019 ◽  
pp. 8-14
Author(s):  
R.F. Safina ◽  
◽  
G.R. Lukmanova ◽  
K.V. Usoltsev ◽  
N.I. Khammadov ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Dna Markers ◽  
Pcr Method

Diagnosis of helicobacter pylori infection: problems and prospects

2020 ◽  
pp. 54-59
Author(s):  
A. S. Molostova ◽  
N. S. Gladyshev ◽  
A. V. Svarval ◽  
R. S. Ferman ◽  
A. B. Karasyova ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Urease Activity ◽  
Diagnostic Methods ◽  
Study Group ◽  
Biochemical Method ◽  
Antimicrobial Drugs ◽  
Non Invasive ◽  
Number Of Patients ◽  
Pcr Method ◽  
Positive Results

(HP) infection was performed using invasive and non-invasive methods. The study group consisted of 95 patients with dyspepsia. HP infection was detected in 47 patients (49.4 %). The expediency of using a set of diagnostic methods for detecting HP (PCR, immunochromatographic, bacteriological and method for determining urease activity) is proved. Most often (100 %) in patients HP infection was detected in biopsies using the PCR method. Somewhat less frequently it was detected when examining biopsies with an invasive biochemical method (AMA RUT Reader) (82 %) and fecal immunochromatographic method (83 %). Despite the fact that helicobacteriosis was detected bacteriologically in a small number of patients (24 %), this method is of particular value, since it allows you to assess the sensitivity to antimicrobial drugs and probiotics, and does not give false positive results.

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