Determination of serum DNA purity among patients undergoing antiretroviral therapy using NanoDrop-1000 spectrophotometer and polymerase chain reaction

Bunu, Samuel J.; Otele, Denmo; Tolulope, Alade; Dodoru, Robinson Tuemi

doi:10.4103/bbrj.bbrj_68_20

Cited by 17 publications

(13 citation statements)

References 23 publications

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“…The quality of the extracted DNA was analyzed using Nanodrop 2000c Spectrophotometer (Thermo, USA) to determine the concentration and purity of the DNA to ensure the recommended minimum DNA concentration of 10 ng/μL and a purity level of optical density (OD) 260/280 above 1.8. The OD260/280 > 1.8 was attained [ 16 ]. It was only grab samples 1c and 1b from lagoon 1 and samples 2a and 2c from lagoon 2 that attained the required DNA purity level.…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of microorganisms with industrial potential for methane production in sludge from Kangemi sewage treatment plant, Nyeri county–Kenya

Kimisto

Muia

Ong'ondo

et al. 2023

Heliyon

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Section: Methodsmentioning

confidence: 99%

Molecular characterization of microorganisms with industrial potential for methane production in sludge from Kangemi sewage treatment plant, Nyeri county–Kenya

Kimisto

Muia

Ong'ondo

et al. 2023

Heliyon

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“…DNA quantity was measured using a Qubit® 3.0 Fluorometer and DNA quality evaluated on an Implem Nanophotometer N60 spectrophotometer to obtain the A260/A280 nm ratio. Quality ratios of 1.8–2.0 were considered to be pure DNA ( Bunu et al, 2020 ). DNA extracts were visualised on 1% agarose gels to examine DNA shearing and integrity.…”

Section: Methodsmentioning

confidence: 99%

16S rRNA gene-based microbiota profiles from diverse avian faeces are largely independent of DNA preservation and extraction method

Edwards,

Hoffbeck,

West

et al. 2023

Front. Microbiol.

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The avian gut microbiota has been the subject of considerable recent attention, with potential implications for diverse fields such as the poultry industry, microbial ecology, and conservation. Faecal microbiotas are frequently used as a non-invasive proxy for the gut microbiota, however the extraction of high-quality microbial DNA from avian faeces has often proven challenging. Here we aimed to evaluate the performance of two DNA preservation methods (95% ethanol and RNAlater) and five extraction approaches (IndiSpin Pathogen Kit, QIAamp PowerFecal Pro DNA Kit, MicroGEM PrepGEM Bacteria Kit, ZymoBIOMICS DNA Miniprep Kit, and an in-house phase separation-based method) for studying the avian gut microbiota. Systematic testing of the efficacy of these approaches on faecal samples from an initial three avian species (chicken, ostrich, and the flightless parrot kākāpō) revealed substantial differences in the quality, quantity and integrity of extracted DNA, but negligible influence of applied method on 16S rRNA gene-based microbiota profiles. Subsequent testing with a selected combination of preservation and extraction method on 10 further phylogenetically and ecologically diverse avian species reiterated the efficacy of the chosen approach, with bacterial community structure clustering strongly by technical replicates for a given avian species. Our finding that marked differences in extraction efficacy do not appear to influence 16S rRNA gene-based bacterial community profiles provides an important foundation for ongoing research on the avian gut microbiota.

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“…DNA quantification was done using a Nanodrop-1000 spectrophotometer following the method by Bunu et al 17 The genotyping of CYP2B6 was performed using standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques following the method of Rotger et al 18 validated by Ebeshi et al 9 The sequences for the reverse and forward primers for CYP2B6*6 were 5’-GGTCTGCCCATCTATAAAC-3’ (forward primer) and 5’-CTGATTCTTCACATGTCTGCG-3’ (reverse primer) were used for the gene amplification. The specificity of the primer sequence for each gene studied was confirmed by a BLAST analysis search and comparison of genomic sequences in the National Centre for Biotechnology Information (NCBI) databases ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ).…”

Section: Methodsmentioning

confidence: 99%

The Cyp2b6 Gene Polymorphism and Phenotypic Correlation of Efavirenz-Based Combination Therapy Among the Niger Delta Ethnic Population: Implications in Modern Pharmacogenomics

Bunu¹,

Owaba²,

Vaikosen³

et al. 2022

PGPM

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Purpose DNA polymorphism describes the difference between individuals, groups, or ethnicities, races, etc., in terms of their DNA sequences or phenotypes as relates to drug metabolism. Using predictive genotyping of drug-metabolizing genes, we can develop individuals’ drug therapies that are less toxic and more effective. The main aim of the study was to evaluate genotype–phenotype-based correlation and incidence of genetic polymorphism of efavirenz blood levels among HIV/AIDS patients of the Niger Delta population. Methods A study questionnaire was designed to obtained patients’ data, blood samples were obtained, plasma was separated from the serum using a centrifuge for 5 minutes at 4000 rpm for HPLC analysis, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis was conducted using Bsrl endonuclease enzyme to digest the PCR amplicons. Standard efavirenz was used at 0.5, 1, 2, 4, 16 mg/L to construct a calibration curve. Data were analyzed with SPSS software using chi-square test at p-value ≤0.5 and Microsoft excel 2013, while PCR and RFLP results were obtained after 1% Agarose gel electrophoresis, respectively. Results Phenotypic results showed that the participants had different efavirenz plasma concentrations. Six subjects (12%) had efavirenz plasma levels below 0.10 mg/L, considered ultra-rapid metabolizers (UMs), 22 (44%) 0.10 mg/L to 0.90 mg/L, classified as extensive metabolizers (EMs), 19 (38%) had 1.0 to 3.9 mg/L and were noted as intermediate metabolizers (IM), while 3 (6%) subjects showed efavirenz plasma levels from 4.0 mg/L to 6.0 mg/L, categorized as poor metabolizers (PM). RFLP results showed more than half of the population (56%) with a homozygous wild-type gene with CYP2B6*1*1 allele, 38% were CYP2B6*1*6 (heterozygous mutant) allele and 6% had homozygous mutant gene (CYP2B6*6*6 allele). Out of the 15 male subjects among the 50 patients that participated in the study, 8% were UM, 12% EM, 14% IM while no PM was observed, on the contrary, out of the 35 females participated in the study, 4% were observed as UM, 32% EM, 24% IM, while 6% were PM. Conclusion There was no significant difference (p ≤ 0.05) between genotype and phenotype data for CYP2B6 polymorphism, among the HIV/AIDS patients that participated in this study. Genetic polymorphism of the CYP2B6 gene is prevalent among HIV/AIDs patients in the Niger Delta ethnic population on efavirenz-based HAART treatment, as the population having homozygous mutant gene or PM are >1% (6%).

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Determination of serum DNA purity among patients undergoing antiretroviral therapy using NanoDrop-1000 spectrophotometer and polymerase chain reaction

Cited by 17 publications

References 23 publications

Molecular characterization of microorganisms with industrial potential for methane production in sludge from Kangemi sewage treatment plant, Nyeri county–Kenya

Molecular characterization of microorganisms with industrial potential for methane production in sludge from Kangemi sewage treatment plant, Nyeri county–Kenya

16S rRNA gene-based microbiota profiles from diverse avian faeces are largely independent of DNA preservation and extraction method

The Cyp2b6 Gene Polymorphism and Phenotypic Correlation of Efavirenz-Based Combination Therapy Among the Niger Delta Ethnic Population: Implications in Modern Pharmacogenomics

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