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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 214-219

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

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 Submission03-May-2020
Date of Acceptance27-Jun-2020
Date of Web Publication12-Sep-2020

Correspondence Address:
Dr. Samuel Jacob Bunu
Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_68_20

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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 2022 Jan 24];4:214-9. Available from: https://www.bmbtrj.org/text.asp?2020/4/3/214/294862

  Introduction Top

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.[1],[2] 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.[3],[4]

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.[5]

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.[5],[6]

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.[6]

There are different methods available for the quantification of DNA samples.[7] 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.[8],[9] 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.[10]

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.[11] 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.[12] Several studies have demonstrated that overall outcomes in HIV-infected patients are better when care is provided by HIV specialist clinicians,[13],[14],[15] 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.[16],[17]

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.[18] 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.[19],[20],[21],[22],[23]

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.[24] 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.[24] 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 Top

Sample collection

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.

DNA Extraction

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 Quantification

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.[25] 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.[26] 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.[26]

  Results Top

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 Top

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.[27]

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.[28] 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.[29] 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.[30] 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 Top

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.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Rittich B, Spanová A. SPE and purification of DNA using magnetic particles. J Sep Sci 2013;36:2472-85.  Back to cited text no. 1
Sambrook J, Russel DW. Molecular Cloning. 3rd ed. NY, USA: Cold Spring Harbor Laboratory Press; 2001.  Back to cited text no. 2
Wink M. An Introduction to Molecular Biotechnology. 2nd ed. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co, KGaA; 2011.  Back to cited text no. 3
Deng M, Jiang C, Jia L. N-methylimidazolium modified magnetic particles as adsorbents for solid phase extraction of genomic deoxyribonucleic acid from genetically modified soybeans. Anal Chim Acta 2013;771:31-6.  Back to cited text no. 4
Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 1997;22:474-6, 478-81.  Back to cited text no. 5
NanoDrop and Design are Registered Trademarks of NanoDrop Technologies Wilmington, Delaware USA ©2007 NanoDrop Technologies, Inc. Available from: http://www.nanodrop.com. [Last accessed on 2020 Apr 03].  Back to cited text no. 6
Sheikh SN, Lazarus P. Re-usable DNA template for the polymerase chain reaction. Nucleic Acids Res 1997;25:3537-42.  Back to cited text no. 7
Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93.  Back to cited text no. 8
Siqueira JF, Jung IY, Rôças IN, Lee CY. Differences in prevalence of selected bacterial species in primary endodontic infections from two distinct geographic locations. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:641-7.  Back to cited text no. 9
DNA Quantification: Comparison of UV Spectrophotometry and PicoGreen Analysis. LGC Limited; 2014. G2/CS/1214. Available from: http://www.lgcgroup.com/genomics. [Last accessed on 2020 Apr 03].  Back to cited text no. 10
Cohen MS, Chen YQ, McCauley M, Gamble T, Hosseinipour MC, Kumarasamy N, et al. Prevention of HIV-1 infection with early antiretroviral therapy. N Engl J Med 2011;365:493-505.  Back to cited text no. 11
Centers for Disease Control and Prevention. HIV in the United States: The Stages of Care-CDC Fact Sheet; 2014. Available from: http://www.cdc.gov/nchhstp/newsroom/docs/HIV-Stages-ofCare-Factshett-508.pdf. [Last accessed on 2020 Apr 03].  Back to cited text no. 12
Kitahata MM, van Rompaey SE, Shields AW. Physician experience in the care of HIV-infected persons is associated with earlier adoption of new antiretroviral therapy. J Acquir Immune Defic Syndr 2000;24:106-14.  Back to cited text no. 13
Landon BE, Wilson IB, McInnes K, Landrum MB, Hirschhorn LR, Marsden PV, et al. Physician specialization and the quality of care for human immunodeficiency virus infection. Arch Intern Med 2005;165:1133-9.  Back to cited text no. 14
Kitahata MM, van Rompaey SE, Dillingham PW, Koepsell TD, Deyo RA, Dodge W, et al. Primary care delivery is associated with greater physician experience and improved survival among persons with AIDS. J Gen Intern Med 2003;18:95-103.  Back to cited text no. 15
Delgado J, Heath KV, Yip B, Marion S, Alfonso V, Montaner JS, et al. Highly active antiretroviral therapy: Physician experience and enhanced adherence to prescription refill. Antivir Ther 2003;8:471-8.  Back to cited text no. 16
O'Neill M, Karelas GD, Feller DJ, Knudsen-Strong E, Lajeunesse D, Tsui D, et al. The HIV workforce in New York State: Does patient volume correlate with quality? Clin Infect Dis 2015;61:1871-7.  Back to cited text no. 17
Hirsch MS, Günthard HF, Schapiro JM, Brun-Vézinet F, Clotet B, Hammer SM, et al. Antiretroviral drug resistance testing in adult HIV-1 infection: 2008 Recommendations of an International AIDS Society-USA panel. Clin Infect Dis 2008;47:266-85.  Back to cited text no. 18
Tural C, Ruiz L, Holtzer C, Schapiro J, Viciana P, González J, et al. Clinical utility of HIV-1 genotyping and expert advice: The Havana trial. AIDS 2002;16:209-18.  Back to cited text no. 19
Cingolani A, Antinori A, Rizzo MG, Murri R, Ammassari A, Baldini F, et al. Usefulness of monitoring HIV drug resistance and adherence in individuals failing highly active antiretroviral therapy: A randomized study (ARGENTA). AIDS 2002;16:369-79.  Back to cited text no. 20
Durant J, Clevenbergh P, Halfon P, Delgiudice P, Porsin S, Simonet P, et al. Drug-resistance genotyping in HIV-1 therapy: The VIRADAPT randomised controlled trial. Lancet 1999;353:2195-9.  Back to cited text no. 21
Baxter JD, Mayers DL, Wentworth DN, Neaton JD, Hoover ML, Winters MA, et al. A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS. AIDS 2000;14:F83-93.  Back to cited text no. 22
Cohen CJ, Hunt S, Sension M, Farthing C, Conant M, Jacobson S, et al. A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy. AIDS 2002;16:579-88.  Back to cited text no. 23
Brescia P, Banks P. Multi-Volume Analysis of Nucleic Acids Using the Epoch/Take3 Spectrophotometer System. Winooski, (VT): BioTek Instruments, Inc.; 2009.  Back to cited text no. 24
Lo J. Dyslipidemia and lipid management in HIV-infected patients. Curr Opin Endocrinol Diabetes Obes 2011;18:144-7.  Back to cited text no. 25
Aryal S. Polymerase Chain Reaction (PCR), Principle, Procedure, Types, Applications, and Animation. Available from: microbiologyinfo.com. Last updated on 2018 July 06, Last accessed on 2020 Apr 03.  Back to cited text no. 26
Masyeni S, Sintya E, Megawati D, Sukmawati NM, Budiyasa DG, Aryastuti SA, et al. Evaluation of antiretroviral effect on mitochondrial DNA depletion among HIV-infected patients in Bali. HIV AIDS(Auckl)2018;10:145-150.  Back to cited text no. 27
Selvaraj S, Ghebremichael M, Li M, Foli Y, Langs-Barlow A, Ogbuagu A, et al. Antiretroviral therapy-induced mitochondrial toxicity: Potential mechanisms beyond polymerase-γ inhibition. Clin Pharmacol Ther 2014;96:110-20.  Back to cited text no. 28
Côté HC, Brumme ZL, Craib KJ, Alexander CS, Wynhoven B, Ting L, et al. Changes in mitochondrial DNA as a marker of nucleoside toxicity in HIV-infected patients. N Engl J Med 2002;346:811-20.  Back to cited text no. 29
Gingelmaier A, Grubert TA, Kost BP, Setzer B, Lebrecht D, Mylonas I, et al. Mitochondrial toxicity in HIV type-1-exposed pregnancies in the era of highly active antiretroviral therapy. Antivir Ther 2009;14:331-8.  Back to cited text no. 30


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]

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