scholarly journals Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

2016 ◽  
Vol 2016 ◽  
pp. 1-4
Author(s):  
Chun Wu
Keyword(s):  
Dna Polymerase ◽  
Click Reaction ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sequencing Analysis ◽  
Abasic Site ◽  
Nylon Membrane ◽  
Abasic Sites ◽  
Polymerase Chain ◽  
Dna Sequencing Analysis ◽  
Endonuclease Digestion

Biotinylation of deoxyguanosine at an abasic site in double-stranded oligodeoxynucleotides was studied. The biotinylation of deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal. The biotinylation enables visualization of the biotinylated oligodeoxynucleotides by chemiluminescence on a nylon membrane. In order to investigate the biotinylated site, the biotinylated oligodeoxynucleotides were amplified by the DNA polymerase chain reaction. Replacement of guanine opposing the abasic site with adenine generated by the activity of the terminal deoxynucleotidyl transferase of DNA polymerase was detected by DNA sequencing analysis and restriction endonuclease digestion. This study suggests that 2-oxohex-5-ynal may be useful for the detection of the unpaired deoxyguanosine endogenously generated at abasic sites in genomic DNA.

scholarly journals RANCANGAN PRIMER SPESIFIK GEN MACROPHAGE MANNOSE RECEPTOR (MMR) UNTUK POLYMERASE CHAIN REACTION (PCR) DAN SEKUENSING DEOXYRIBO NUCLEIC ACID (DNA)

2018 ◽  
Vol 22 (2) ◽  
pp. 158
Author(s):  
Yani Triyani ◽  
Nurizzatun Nafsi ◽  
Lelly Yuniarti ◽  
Nanan Sekarwana ◽  
Endang Sutedja ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequencing ◽  
Mannose Receptor ◽  
Primer Design ◽  
Sequencing Analysis ◽  
Nucleotide Polymorphisms ◽  
Chain Reaction ◽  
Specific Pcr ◽  
Polymerase Chain ◽  
Dna Sequencing Analysis

The order (sequencing) determinationof DeoxyribonucleicAcid (DNA) bases is the gene’s most basic information, using the methodof Polymerase Chain Reaction (PCR) as its stage. A key factor of successful detection by PCR is specific PCR primer design choice. Thedetection of diversity of Mycobacterium Mannose Receptor (MMR) gene, responsible for recognizing mannosylated antigen structureof Mycobacterium tuberculosis (M.tb) by DNA sequencing of exon 7 chromosome 10p12, related to susceptiblity for PulmonaryTuberculosis(TB), was first performed in China in 2012. The purpose of this study was to find specific primerfromboth design originatedfrom the research in China/primer I and my own design/primer IIby using Primer3 software. This study was based on 10 healthy subjectsand was a preliminary study of a research titled. The Relationship of Single Nucleotide Polymorphisms (SNPs) of Macrophage MannoseReceptor Gene to Pulmonary Tuberculosis Cases. The examination materials consist of 3 mL of EDTA blood and DNA extraction from itsbuffy coat. The resulting DNA was processed by PCR to amplify MMR gene with primer I and II. The primer I successfully amplified DNAfragments up to 780bp while primer II only 329 bp. The MMR gene DNA sequencing analysis was performed on the amplification resultof both kinds primers by using DNA Baser and Ensembl−BLAST software. The results were different, DNA sequencing result by using theprimer I was found in several chromosomes and also in several loci. Whereas, by using the primer II, it was only found in chromosome10 and in the same locus. Based on this study, it can be concluded that the specific primer design is one of the most important factorsin the success of DNA sequencing.

scholarly journals DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

2015 ◽  
Vol 112 (27) ◽  
pp. E3476-E3484 ◽  
Author(s):  
Mónica Berjón-Otero ◽  
Laurentino Villar ◽  
Miguel de Vega ◽  
Margarita Salas ◽  
Modesto Redrejo-Rodríguez
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Multiple Displacement Amplification ◽  
Translesion Synthesis ◽  
Mobile Element ◽  
Genome Replication ◽  
Abasic Site ◽  
Abasic Sites

DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very low-fidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage Φ29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites.

scholarly journals Identification of Cryptosporidiumspecies and genotypes in dairy cattle in Brazil

2013 ◽  
Vol 22 (1) ◽  
pp. 22-28 ◽  
Author(s):  
Flavio Medeiros Paz e Silva ◽  
Raimundo Souza Lopes ◽  
João Pessoa Araújo-Junior
Keyword(s):  
Dairy Cattle ◽  
Fragment Length Polymorphism ◽  
Dairy Calves ◽  
Sequencing Analysis ◽  
Chain Reaction ◽  
Paulo State ◽  
Potential Source ◽  
High Prevalence ◽  
Polymerase Chain ◽  
Dna Sequencing Analysis

In this study, we identified Cryptosporidium species and genotypes present in dairy cattle in the central region of São Paulo state, Brazil. Fecal specimens were collected from 200 animals (100 calves and 100 cows) in ten dairy farms. Fecal samples were examined using microscopic examination (ME), enzyme immunoassay (EIA) and polymerase chain reaction (PCR). Cryptosporidiumspecies and genotypes were determined by restriction fragment length polymorphism (RFLP) or DNA sequencing analysis of the SSU-rRNA and GP60 genes. The occurrence of Cryptosporidium spp. infection was 14% (28/200). The occurrence in calves (26%) was significantly higher than in cows (2%). Of the 27 Cryptosporidium-positive specimens submitted to genotyping, C. andersoni was identified in 23 (85.1%), C. bovis in three (11.1%), and the zoonotic C. parvum subtype IIaA15G2R1 in one (3.7%). The study demonstrates thatCryptosporidium spp. infection was common and widespread in dairy cattle in this region and that calves have a high prevalence of C. andersoni. Furthermore, the presence of C. parvumsubtype IIaA15G2R1 indicates that dairy calves from this region should be considered a potential source of zoonotic Cryptosporidiumoocysts.

Human c-fms proto-oncogene: comparative analysis with an abnormal allele

1985 ◽  
Vol 5 (2) ◽  
pp. 422-426
Author(s):  
J S Verbeek ◽  
A J Roebroek ◽  
A M van den Ouweland ◽  
H P Bloemers ◽  
W J Van de Ven
Keyword(s):  
Comparative Analysis ◽  
Dna Sequencing ◽  
Sequencing Analysis ◽  
Base Pairs ◽  
Small Deletion ◽  
Viral Oncogene ◽  
Close Proximity ◽  
Genetic Sequences ◽  
Dna Sequencing Analysis

The organization of the human c-fms proto-oncogene has been determined and compared with an abnormal allele. The human v-fms homologous genetic sequences are dispersed discontinuously and colinearly with the viral oncogene over a DNA region of ca. 32 kilobase pairs. The abnormal c-fms locus contains a small deletion in its 3' portion. DNA sequencing analysis indicated that it was 426 base pairs in size and located in close proximity to a putative c-fms exon.

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