|Year : 2020 | Volume
| Issue : 3 | Page : 214-219
Determination of serum DNA purity among patients undergoing antiretroviral therapy using NanoDrop-1000 spectrophotometer and polymerase chain reaction
Samuel Jacob Bunu1, Denmo Otele2, Tolulope Alade3, Robinson Tuemi Dodoru3
1 Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, College of Health Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria
2 Department of Community Health Nursing, Faculty of Nursing, College of Health Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria
3 Department of Medical Laboratory Science, College of Health Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria
|Date of Submission||03-May-2020|
|Date of Acceptance||27-Jun-2020|
|Date of Web Publication||12-Sep-2020|
Dr. Samuel Jacob Bunu
Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State
Source of Support: None, Conflict of Interest: None
Background: The study aimed to determine the effects of antiretrovirals on host DNA purity and to qualitatively quantify the DNA primers quality using NanoDrop-1000 spectrophotometer and polymerase chain reaction (PCR). Methods: Serum samples from 50 HIV/AIDS-positive patients who were undergoing antiretroviral therapy were collected at the point of their routine CD4 level checkup. The samples subjected to initial purifications and subsequent qualitative quantification using the NanoDrop-1000 spectrophotometer and PCR analysis. Results: The purity of the DNA from the subject was determined by the NanoDrop-1000 spectrophotometer, and the adequate yield from 1.13 (lowest) to 149 ng/μl (highest) was obtained across the 50 subjects at 260 and 280 nm. The ratio of the absorbance was compared. Thus, DNA products were obtained with high purity, and PCR showed that all patients had the right gene to code for the respective drug-metabolizing enzyme. The NanoDrop-1000 technique and PCR analysis proved to be beneficial in the measurement of both quantity and purity of DNA in the samples. Conclusion: This technique does not only designs better experiments but also ensures improved reporting and significant time- and energy-saving qualitative analysis of DNA and related molecules.
Keywords: DNA, NanoDrop-1000 spectrophotometer, polymerase chain reaction, serum
|How to cite this article:|
Bunu SJ, Otele D, Alade T, Dodoru RT. Determination of serum DNA purity among patients undergoing antiretroviral therapy using NanoDrop-1000 spectrophotometer and polymerase chain reaction. Biomed Biotechnol Res J 2020;4:214-9
|How to cite this URL:|
Bunu SJ, Otele D, Alade T, Dodoru RT. Determination of serum DNA purity among patients undergoing antiretroviral therapy using NanoDrop-1000 spectrophotometer and polymerase chain reaction. Biomed Biotechnol Res J [serial online] 2020 [cited 2021 May 17];4:214-9. Available from: https://www.bmbtrj.org/text.asp?2020/4/3/214/294862
| Introduction|| |
DNA molecules must be extracted from other cellular components before used in different kinds of applications. Cellular components such as membranes, organelles, and proteins that surround and preserve DNA in the biological environment can inhibit the use of DNA molecules in these applications. For this reason, there are several extraction and purification methods developed to separate the cellular components from DNA., DNA molecules usually lost their solubility in organic solvents (such as phenol or chloroform), because of their solubility differences. DNA is often isolated using the solvent extraction method, but the main setbacks of this method are frequent contamination, time-consuming, use of a sophisticated device, low specificity, and toxicity of the solvents used.,
As absorbance measurements will measure any molecules absorbing at a specific wavelength, nucleic acid samples would require purification before measurement to ensure accurate and reliable results. Nucleotides, RNA, ssDNA, mtDNA, and double-stranded DNA (dsDNA) all will absorb at 260 nm and contribute to the total absorbance. The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA. A ratio of approximately 1.8 ng/μl is usually accepted as pure for DNA, while a ratio of approximately 2.0 ng/μl is accepted as pure for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol, or other contaminants that absorb strongly at or near 280 nm. Some researchers had to stumble upon consistent 260/280 ratio change when switching from a standard cuvette spectrophotometer to the NanoDrop-1000 spectrophotometer. This could be due to a change in sample acidity, wavelength accuracy of the spectrophotometer, or the mixture of nucleotide in the sample.
Small changes in the pH of of the solution would cause the 260/280 to vary because acidic solutions will underrepresent the 260/280 ratio by 0.2–0.3 ng/μl, while a basic solution will overrepresent the ratio by 0.2–0.3 ng/μl. Although the absorbance of a nucleic acid at 260 nm is commonly on a plateau, the absorbance curve at 280 nm is moderately sharply sloped. A slight shift in wavelength accuracy will have a large effect on 260/280 ratios. The resultant 260/280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260/280 ratios for the four nucleotides present. The normally accepted ratios of 1.8 and 2.0 ng/μl for DNA and RNA, respectively, are rules of thumb. The actual ratio will depend on the nucleotide composition of the nucleic acid.,
Furthermore, a 260/230 ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for pure nucleic acid are often higher than the respective 260/280 ratio values. Expected 260/230 values are ordinarily in the range of 2.0–2.2 ng/μl. If the ratio is significantly lower than expected, it may indicate the presence of contaminants which absorb at 230 nm.
There are different methods available for the quantification of DNA samples. A greater absorbance value relates to greater quantities of nucleic acids. A wide range of ultraviolet (UV) spectrophotometers are available, varying from traditional instruments that quantify DNA in plates or cuvettes, to instruments such as the NanoDrop-1000 spectrophotometer that are designed to purify and quantify DNA using micro-volumes of the given sample., Another method is the PicoGreen® technique, where a fluorescent nucleic acid stain binds to dsDNA. A higher level of the fluorescent signal indicates a greater concentration or amount of DNA.
Antiretroviral therapy (ART) employed in the treatment of HIV infection has improved steadily since the advent of potent combination therapy in 1996. ART has dramatically reduced HIV-associated morbidity and mortality and has transformed HIV infection into a manageable chronic condition. In addition, ART combination is highly effective at preventing HIV transmission across the globe. However, less than one-third of HIV-infected individuals in the developed world have suppressed viral loads, mostly resulting from undiagnosed HIV infection and failure to link or retain diagnosed patients in contemporary medical care. Several studies have demonstrated that overall outcomes in HIV-infected patients are better when care is provided by HIV specialist clinicians,,, reflecting the complexity of HIV infection and its treatment. Appropriate training, continuing education, and clinical experience are all components of optimal patient care. Providers who do not have this requisite training and experience should consult HIV experts when needed to improve the quality of life among infected patients.,
Genotypic and phenotypic resistance assays are used to assess the viral strains and select the treatment strategies and the polymerase chain reaction (PCR) technique found useful application in this field. These assays provide information on resistance to various classes of antiretroviral agents such as fusion inhibitors, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, protease inhibitors, and integrase strand transfer inhibitors. Most genotypic assays involve sequencing the reverse transcriptase, protease, and integrase enzymatic genes to detect mutations that are known to confer viral drug resistance. A genotypic assay that assesses mutations in the gp41 (envelope) gene associated with resistance to the fusion inhibitor (enfuvirtide) is also commercially available. Resistance assays are important tools to inform treatment decisions for patients who experience pharmacotherapeutic failure while on ART. Several prospective studies assessed the utility of resistance testing to guide antiretroviral drug selection in patients with therapeutic failure.,,,,
The assessment of the purity of a nucleic acid sample is often performed by a procedure commonly referred to as the A260/A280 ratio which refers to two spectrophotometric measurements made at these definite wavelengths. Nucleic acid samples would be expected to have a higher absorbance at 260 nm than at 280 nm, while for a protein sample, the reverse is the case. Using these extinction coefficients, pure nucleic acid samples would have an A260/A280 ratio of 2.0 ng/μl, while protein would be 0.57 ng/μl. Samples that contain a mixture of protein and DNA would, of course, be influenced by both macromolecules. According to the Beer-Lambert law, the total absorbance of a solution is the sum of the absorbance of the components comprising the solution; thus, since a ratio is taken of absorbance, the path length variable is canceled out, provided the same vessel is used for both measurements. Therefore, the purpose of this study was to determine the effects of antiretrovirals on host DNA purity and to qualitatively quantify the DNA primers quality using NanoDrop-1000 spectrophotometer and PCR.
| Methods|| |
Full serum samples were collected from 50 HIV-positive unrelated patients who were undergoing treatment at a public tertiary health facility and were routinely checking their CD4 level. The patients who were receiving efavirenz, lamivudine, and tenofovir combination highly active ART were randomly selected to participate in the study. Serum samples were collected from participants and were refrigerated at below − 20°C before biochemical analysis. The study was approved by the Ethics and Research Committee of the Federal Medical Centre, Yenagoa, Bayelsa State, Nigeria.
The DNA was extracted using Quick-DNA Blood Miniprep Kits (obtained from Iqaba Biotec, West Africa). Each frozen blood sample was thawed at normal room temperature (20°C), and 100 μl of serum (whole blood) was transferred to a clean polypropylene tube. 650 μl of genomic lysis buffer was added into the tube, mixed completely by vortexing for 4–6 s, and allowed to stand 6–10 min at 20°C. The mixture was transferred to a Zymo-Spin Column in a collection tube and was centrifuged at 10,000 × g for about 1 min. The collection tube was discarded with the flow-through. The Zymo-Spin Column was transferred into a new collection tube; 200 μl of DNA Pre-Wash Buffer was added to the spin column and was centrifuged at 10,000 × g for 1 min. 500 μl of gDNA (genomic DNA) wash buffer was added to the spin column and centrifuged at 10,000 × g for another 1 min and then transferred to a clean microcentrifuge tube, and 50 μl DNA elution buffer was added to the spin column. The tube was incubated at room temperature for about 2–5 min and centrifuged at top speed for 30 s to elute the DNA. The eluted DNA purity, yield, UV/Visible absorbance, and absorbance ratio were quantified using the NanoDrop-1000 Spectrophotometer.
DNA was quantified using the NanoDrop-1000 spectrophotometer. The DNA concentrations were determined by measuring the absorbance at 260 nm wavelength (A260) and 280 nm wavelength (A280). Purity was determined by calculating the ratio of absorbance at 260 nm and the absorbance at 280 nm (A260/A280) [Figure 1] and [Figure 2]. The spectrophotometer was connected to a software installation system. The machine was initialized, blanked by using DNA nuclease-free water, elution buffer using two microlitres each respectively, the measurement of the gDNA was read using the same volume and the pedestal was cleaned after each reading to prevent cross-contamination of the DNA products [Figure 2].
|Figure 1: The ratio of DNA 260/280 ratio absorbance after NanoDrop-1000 spectrophotometric analysis|
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|Figure 2: The ratio of DNA 260/230 ratio absorbance after NanoDrop-1000 spectrophotometric analysis|
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Polymerase chain reaction - PCR
The validity of PCR assay is one that has been assessed for the optimal range of primer annealing temperatures, reaction efficiency, and specificity using a standard set of samples. The presence of mt-DNA and n-DNA was performed using a standard PCR. The forward and reverse primers were 5'-GGTCTGCCCATCTATAAAC-3' and 5'-CTGATTCTTCACATGTCTGCG-3', respectively. The PCR reaction was carried out in a total of 25 μL reaction mixture containing, 200 μM of deoxynucleoside triphosphate mixture, 2.0 mM of MgCl2, 1 × PCR buffer, and 1 Unit of Taq polymerase; 0.5 μM of each primer, 5 ng genomic DNA, and nuclease-free water. All PCR reactions were performed on GeneAmp® PCR System 9700 programmable thermal cycler. For 1 min, a final extension at 72°C for 6 min, and the product mixture held at 10°C until further use. The PCR products base pair were confirmed gel electrophoresis and Gel Photosystem (blue light) – P1-1002.
| Results|| |
The results from the DNA determination and polymerase chain reaction of analyzed serum samples are expressed in tables and charts:
DNA Quantification result is shown in [Table 1].
|Table 1: Extraction and quantification of DNA purity with NanoDrop-1000 spectrophotometer|
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Polymerase chain reaction result
PCR results showed that all the subjects' DNA appeared at 500 bp DNA molecular ladder.
The results showed that all the subjects' DNA were amplified at 500 bp DNA aligning with DNA molecular ladder. This is presented in [Figure 3].
|Figure 3: Representative of the PCR product after agarose gel electrophoresis (Sample NN11–NN30) and DNA molecular ladder|
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| Discussion|| |
The ratio of 260/280 value gives an estimated purity of the DNA product, its effect on PCR, and the specific PCR system used for the amplification. The purity of the DNA from the subject was determined by NanoDrop-1000 spectrophotometer, and the adequate yield was obtained from 1.13 (lowest) to 149 ng/μl (highest) at 260 nm and 280 nm, respectively. Hence, the DNA products were obtained in high purity except for 11 participants, whose DNA were below 4.0 ng/μl. The absorbance at 260 nm ranges from 0.023 to 2.991 ng/μl, while at 280 nm from 0.009 to 1.579 ng/μl, respectively.
A lower ratio indicates that the DNA sample does not only contain DNA (more precisely, nucleic acids) but also impurities that affect the absorption maximum above 260 nm. Typical suspects areproteins and phenolic molecules. Phenol, when present in a reaction medium, is likely to give false results since because inhibits most polymerase enzymes. Therefore, the DNA sample should be free of contaminations of phenol and other phenolic components.
DNA quantification is an important role in the detection of the gene responsible in coding most metabolizing enzymes,, especially in immunocompromised patients on antiretrovirals using PCR. DNA concentration and purity are determined by measuring the ratio of UV absorbance at 260 and 280 nm. In this study, we extracted DNA from 50 different HIV-positive unrelated individuals and the value of A260/A280 showed the variation on the quantity of DNA and/or gene present. Samples NN34, 15, 43, 36, 11, 28, 9, 19, 40, 13, 10, 5, 22, 18, 48, 16, 14, 17, 18, 20, and 12 showed high DNA purity with sample NN12 having the highest purity (149.57 ng/μl), samples NN1, 7, 2, 4, 45, 33, 31, 6, 38, 32, 37, 47, 27, 30, 21, 42, 46, and 25 had moderate purity in this order, and finally, samples 39, 50, 23, 41, 29, 44, 24, 49 3, 35, and 36 were least pure with less than 5 ng/μl purity level. This correlate with a report that the ratio of mt-DNA to n-DNA was significantly lower in ART-treated HIV patients. Further, another study that was aimed to determine the effect of HIV infection and ART on placental mitochondria with qRT-PCR also found the ratio to be significantly reduced in ART and HIV-1 exposed placentas in comparison with uninfected controls. For amplification of DNA, we optimize the primer and annealing temperatures to determine the specificity using a standard set of samples by gradient temperature after proper calculations using the nucleotide present in the DNA sequence used for the analysis. In this study, the gradient temperature was determined by the forward and reverse primers. PCR amplification efficiency was close to to 100%, as all the samples appeared at 500 molecular base pairs (bp), which is regarded as the best parameter assay. In a preliminary study, one would require an amplification efficiency of 90%–105%. Low reactions' efficiency may be caused by poor primer design or by suboptimal reaction conditions associated with the components of the PCR. The presence of an inhibitor or external contaminant can also result in an apparent increase of PCR inefficiency, while high concentration and purity of DNA can increase the level of inhibitor. Therefore, the higher the purity, the more efficient the gene present in the patient DNA to code for drug-metabolizing enzymes.
| Conclusion|| |
NanoDrop-1000 spectrophotometer and PCR are very accurate methods for the quantification of mitochondrial and genomic DNA. DNA purity directly affects the qualitative Polymerase Chain Reaction amplification process. The higher the purity, the more efficient the amplification procedure, and the quantity of gene present to code for drug-metabolizing enzymes.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]