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

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.

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 Survey of genomic repeat sequence-PCRs that detect differences between inbred mouse strains

1995 ◽  
Vol 65 (2) ◽  
pp. 151-155 ◽  
Author(s):  
Philip A. Wood ◽  
Doug A. Hamm
Keyword(s):  
Genomic Dna ◽  
Inbred Mouse ◽  
Pcr Amplification ◽  
Repeat Sequence ◽  
Mouse Strains ◽  
Repeat Sequences ◽  
Dna Repeat ◽  
Polymerase Chain ◽  
Dna Repeat Sequences ◽  
Congenic Mouse Strains

SummaryWe have developed molecular markers that distinguish between several inbred and congenic mouse strains using polymerase chain reaction (PCR) amplification of genomic DNA repeat sequences. Mouse genomic DNA, digested with four base recognition site-restriction endonucleases, was amplified by PCR using primers for the following repeat sequences: Bl (Alu homolog), LINE, LLR3, IAP, human Alu and myoglobin. Amplification products analysed by agarose gel electrophoresis and stained with ethidium bromide produced unique DNA fragments, some of which are specific for each of 12 strains tested. This method can be used for molecular analysis of the mouse genome, including genetic monitoring.

A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different phosphoglucomutase-specific probes

2000 ◽  
Vol 78 (6) ◽  
pp. 683-690 ◽  
Author(s):  
Elzbieta Wyroba ◽  
Birgit H Satir
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Molecular Size ◽  
Molecular Probes ◽  
Hybridization Analysis ◽  
Yeast Genome ◽  
Specific Primers ◽  
Genome Database

Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phospho glyco protein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes. These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2. Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3 – I-3) not found in other species. PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe. A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.Key words: parafusin, phosphoglucomutase, yeast, hybridization, PCR.

scholarly journals Molecular Cloning of Cyanobacterial Pteridine Glycosyltransferases That Catalyze the Transfer of either Glucose or Xylose to Tetrahydrobiopterin

2010 ◽  
Vol 76 (22) ◽  
pp. 7658-7661 ◽  
Author(s):  
Yeol Gyun Lee ◽  
Ae Hyun Kim ◽  
Mi Bi Park ◽  
Hye-Lim Kim ◽  
Kon Ho Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Cloning ◽  
Environmental Samples ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Activity Assay ◽  
Genes Encoding ◽  
Cyanobacterial Genes

ABSTRACT Here, we report cloning of cyanobacterial genes encoding pteridine glycosyltransferases that catalyze glucosyl or xylosyl transfer from UDP-sugars to tetrahydrobiopterin. The genes were cloned by PCR amplification from genomic DNA which was isolated from culture and environmental samples and overexpressed in Escherichia coli for an in vitro activity assay.

Exposure to low Earth orbit of an extreme-tolerant cyanobacterium as a contribution to lunar astrobiology activities

2019 ◽  
Vol 19 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Daniela Billi ◽  
Claudia Mosca ◽  
Claudia Fagliarone ◽  
Alessandro Napoli ◽  
Cyprien Verseux ◽  
...  
Keyword(s):  
Uv Radiation ◽  
Genomic Dna ◽  
Lunar Regolith ◽  
Pcr Amplification ◽  
Low Earth Orbit ◽  
The Moon ◽  
Test Bed ◽  
Planetary Protection ◽  
Lunar Regolith Simulant ◽  
Space Conditions

AbstractBy investigating the survival and the biomarker detectability of a rock-inhabiting cyanobacterium, Chroococcidiopsis sp. CCMEE 029, the BIOMEX space experiment might contribute to a future exploitation of the Moon as a test-bed for key astrobiology tasks such as the testing of life-detection technologies and the study of life in space. Post-flight analyses demonstrated that the mixing of dried cells with sandstone and a lunar regolith simulant provided protection against space UV radiation. During the space exposure, dried cells not mixed with minerals were killed by 2.05 × 102 kJ m−2 of UV radiation, while cells mixed with sandstone or lunar regolith survived 1.59 × 102 and 1.79 × 102 kJ m−2, respectively. No differences in survival occurred among cells mixed and not mixed with minerals and exposed to space conditions in the dark; this finding suggests that space vacuum and 0.5 Gy of ionizing radiation did not impair the cells’ presence in space. The genomic DNA of dead cells was severely damaged but still detectable with PCR amplification of a short target, thus suggesting that short sequences should be targeted in a PCR-based approach when searching for traces of life. The enhanced stability of genomic DNA of dried cells mixed with minerals and exposed to space indicates that DNA might still be detectable after prolonged periods, possibly up to millions of years in microbes shielded by minerals. Overall, the BIOMEX results contribute to future experiments regarding the exposure of cells and their biomarkers to deep space conditions in order to further test the lithopanspermia hypothesis, the biomarker stability and the microbial endurance, with implications for planetary protection and to determine if the Moon has been contaminated during past human missions.

Transient injury to rat lung mitochondrial DNA after exposure to hyperoxia and inhaled nitric oxide

2004 ◽  
Vol 286 (1) ◽  
pp. L23-L29 ◽  
Author(s):  
Richard T. Lightfoot ◽  
Steven Khov ◽  
Harry Ischiropoulos
Keyword(s):  
Nitric Oxide ◽  
Mitochondrial Dna ◽  
Genomic Dna ◽  
Cellular Proliferation ◽  
Pcr Amplification ◽  
Inhaled Nitric Oxide ◽  
Treatment Groups ◽  
Fischer 344 ◽  
Mitochondrial Number

The effect of hyperoxia alone and in combination with inhaled nitric oxide (NO) on the integrity of lung mitochondrial DNA (mtDNA) in vivo was evaluated in Fischer 344 rats. PCR amplification of lung mtDNA using two sets of primers spanning 10.1 kb of the mtDNA revealed that inhalation of 20 ppm of NO in conjunction with hyperoxia (>95% O2) reduced the amplification of mtDNA templates by 10 ± 1% and 26 ± 3% after 24 h of exposure. The ability of mtDNA to amplify was not compromised in rats exposed to 80% O2, even in the presence of 20 ppm of inhaled NO. Surprisingly, exposure to >95% O2 alone for either 24 or 48 h did not compromise the integrity of mtDNA templates compared with air-exposed controls, despite evidence of genomic DNA injury. Interestingly, inhaling NO alone for 48 h increased mtDNA amplification by 12 ± 2% to 21 ± 7%. Injury to the lung mtDNA after exposure to >95% O2 plus 20 ppm of NO was transient as rats allowed to recover in room air after exposure displayed increased amplification, with levels exceeding controls by 20 ± 3% to 29 ± 4%. Increased amplification was not due to cellular proliferation or increased mitochondrial number. Moreover, the ratio of pulmonary mtDNA to genomic DNA remained the same between treatment groups. The results indicate that hyperoxia fails to induce significant injury to mtDNA, and whereas inhalation of NO with hyperoxia results in mtDNA damage, the lesions are rapidly repaired during recovery.

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