BIOTROPIKA Journal of Tropical Biology
https://biotropika.ub.ac.id/
Vol. 11 | No. 1 | 2023 | DOI: 10.21776/ub.biotropika.2023.011.01.01
Arif et al. 1
COMPARISON OF ATL AND TRITON LYSIS BUFFERS FOR SALIVA DNA EXTRACTION USING MODIFIED MAGNETIC-SILICA NANOPARTICLE BEADS
Muhammad Fauzi Arif1)*, Ahadi Damar Prasetya2), Indra Lesmana3), Niken Satuti Nur Handayani3)
ABSTRACT
Our previous study revealed that modified MAGSi (magnetic-silica nanoparticle beads) had various sizes and shapes that affected DNA extraction. In this study, we combined our MAGSi with two kinds of lysis buffers. Human salivary DNA was extracted using three types of MAGSi combined with triton and ATL as lysis reagents. Each of MAGSi was synthesized differently. The DNA concentration and purity were analyzed using a nanodrop spectrophotometer. Extracted DNA also was used as the template for DNA amplification using the human β-globin gene. The average DNA concentration from the extraction using the combination of MAGSi-1 with the triton buffer, ATL buffer, and without buffer was 42.90 ng/μL, 71.63 ng/μL, and 104.36 ng/μL respectively. The average DNA concentration results from the extraction using the combination of MAGSi-2 with the triton buffer, ATL buffer, and without lysis buffer was 22.43 ng/μL, 30.97 ng/μL, and 71.61 ng/μL. The DNA average concentration from the extraction using a combination of MAGSi-3 with triton buffer, ATL buffer, and without buffer was 22.27 ng/μL, 25.40 ng/μL, and 54.77 ng/μL.
Meanwhile, the DNA purity from triton buffer extraction ranged from 1.43 ± 0.007 to 1.61
± 0.016. The DNA purity from ATL buffer extraction ranged from 1.59 ± 0.031 to 1.88 ± 0.150. The DNA purity from the extraction without lysis ranged from 1.82 ± 0.015 to 1.94
± 0.057. The result indicated that DNA extraction using lysis buffer produced less concentration yield and purity value but was more consistent than the others.
Electrophoresis showed that all extraction results could be used as DNA templates to amplify the human β-globin gene. Further studies are still needed to determine the effectiveness and consistency of the lysis buffer and our MAGSi.
Keywords: beads, DNA concentration, DNA extraction, lysis buffer, nanoparticles
INTRODUCTION
DNA extraction is an essential step in forensic genotyping. Good quality and quantity of DNA lead to optimal amplification and good genotyping products [1]. It is possible to perform DNA extraction using various methods [2]. The selection is suited by the type of sample, the desired target, and contaminants that can contaminate [3, 4].
Samples from saliva, hair, semen, bone, and skin are commonly used for DNA analysis using a molecular approach [5, 6].
DNA extraction from saliva is relatively easy to be executed [7]. In addition, saliva sampling is a non-invasive procedure and does not require special medical skills to be performed. Saliva is stable at room temperature, so it does not need special treatment and storage [8]. Various saliva DNA extraction studies have been successfully performed and given promising results [9, 10, 11, 12]. Problems appear because the number of epithelial cells and leukocytes differs. It makes the saliva DNA extraction result fluctuate. The uncertainty rate increases because saliva contains bacteria that can cause contamination [10, 13, 14].
Several essential points for determining the DNA extraction application have been revealed
[15]. Commercial kits mostly use a spin column for extraction. They provide a clear protocol and good results. The conventional spin column effectively binds nucleic acids. Furthermore, it removes various contaminants. However, this method requires expensive costs, complicated equipment, longer extraction time, skilled personnel, and many single-use disposable laboratory elements [16, 17].
The utilization of centrifugation as the basis for separation will also prolong and complicate the process. The problem will also increase as the samples increase [18].
One of the DNA extraction methods that could be the answer to the spin column extraction problem is DNA extraction using beads. Beads can be made by reacting magnetic nanoparticles with silica [17]. The result is magnetic-silica nanoparticles. The size and morphology of the particles affect their ability to bind nucleic acid molecules. Several studies have also revealed that morphology and particle size are influenced by various factors such as modified magnetic-silica nanoparticle preparation techniques, reaction conditions, reaction concentrations, and mixing methods [19]. Multiple approaches have been developed to adjust the size and morphology of the beads [20, 21, 22, 23]. The identical and smaller
Submitted : December, 24 2022 Accepted : May, 2 2023
Authors affiliation:
1) Laboratory of Microbiology and Molecular Genetics, Department of Biology, Faculty of Mathematics and Life Sciences, Mulawarman University.
2) Research Center for Radiation Detection
and Nuclear Analysis Technology, Research Organization for Nuclear Energy, National Research and Innovation Agency.
3)Laboratory of Genetics and Breeding, Deparment of Tropical Biology, Faculty of Biology, Universitas Gadjah Mada.
Correspondence email:
How to cite:
Arif, MF, Prasetya AD, Lesmana I, Handayanti NSN. 2023. Comparison of ATL and triton lysis buffers for saliva DNA extraction using modified magnetic- silica nanoparticle beads. Journal of Tropical Biology 11 (1): 1-8.
particle size is expected to expand the binding area of nucleic acids.
The lysis buffer also plays a vital role in the DNA extraction process. Various lysis buffers can be applied for DNA extraction [24]. Ordinarily, lysis buffers contain compounds that can dissolve cell plasma membranes, degrade proteins and inactivate nuclease enzymes [25, 26]. The result of the process is cell lysis. Incomplete lysis causes a low extraction yield.
In a previous study, we analyzed our modified magnetic-silica nanoparticle’s structure, size, and morphology. The study gave promising results. In this study, we researched and combined our modified magnetic-silica nanoparticles with several lysis buffers to extract DNA from saliva.
METHODS
In this study, DNA was extracted from salivas using a combination of three types of MAGSi (magnetic-silica nanoparticle beads) synthesized differently with two different types of lysis buffer.
After that, the extracted DNA quality was analyzed by UV-Vis spectrophotometry and agarose gel electrophoresis with a concentration of 0.8%.
Extracted DNA was also used as a template for human β-globin gene amplification. The result of the amplification was visualized in 2% agarose gel electrophoresis.
Sample collection. Saliva from subjects was collected for DNA extraction. A total of 300 µL of saliva samples were taken from Central Java. All procedures for the study were approved by the Ethics Commission (Ethical Clearance Code, Ref.
No.:KE/FK/1399/EC/2021 and Ref. No.:
KE/FK/1400/EC/2021). Subjects also have agreed to give informed consent.
Synthesis of MAGSi. MAGSi-1 Conventional Method. MAGSi-1 was synthesized by hydrolysis of TEOS (tetraethyl orthosilicate) according to the Stober method [27] (Figure 1a). A total of 0.3 g
magnetic nanoparticles was rinsed using ethanol.
After rinsing, the magnetic nanoparticles were dispersed in 180 mL ethanol. The synthesis was performed by stirring the solution using a magnetic stirrer at 400 rpm and 75°C of temperature (Figure 1b). A total of 4 mL of TEOS was added to the solution. The stirring process was continued for 30 mins. A total of 10 mL of NH4OH and DI water were added to the solution. The solution was reacted for 4 hours at 50°C and aged overnight at room temperature. The reaction was performed under inert N2 atmosphere conditions. The precipitate product was separated from the solution using an external magnet and was washed using DI water and ethanol sequentially twice. The magnetic-silica nanoparticle beads were rewashed using DI water until pH 7.
MAGSi-2 - Ultrasonic Horn Method. MAGSi-2 was synthesized using the ultrasonic horn. The synthesis process was done with the same amount and reaction with MAGSi-1 (Figure 1d).
Sonication was carried out using an ultrasonic liquid processor at 20 kHz (Sonic-Vibra Cell VCX 500, Sonics & Materials, Inc). Sonication was done during the magnetic nanoparticles dispersion process in 180 mL ethanol for 5 mins, TEOS addition for 5 mins, and 10 mL NH4OH 25 % and DI-water addition for 2 mins. The mixture was stirred for 6 hours and left overnight for 24 h. The precipitate-washing process is the same as MAGSi-1.
MAGSi-3 - Ultrasonic Bath Method. MAGSi-3 was synthesized using an ultrasonic cleaner bath (Branson 1510, 40 kHz, Branson Ultrasonics Corp) (Figure 1c). Magnetic nanoparticles were dispersed into 180 mL ethanol and spread for 10 mins, then 4 mL TEOS was dripped for 30 mins, continued with 10 mL of NH4OH 25% and 10 mL of DI water.
Then, the sonication of the reactant was continued for 3 hours bath at 50°C, followed by overnight aging at room temperature. The residue was washed as the previous method.
Figure 1. Schematic illustration of synthesis MAGSi (magnetic-silica nanoparticle beads) process (a).
Magnetic particle synthesis, (b). Magnetic stirring, (c). Bath sonication, and (d). Probe sonication
Arif et al. 3 Saliva DNA extraction. Three types of MAGSi
were homogenized using vortex as pre-treatment.
A total of 50 μL MAGSi were prepared from each type. Beads were separated from DI water using a magnet. A 500 μL lysis buffer and 10 μL proteinase K were added. The mixture was placed into a 2 mL microtube. After the solution was ready, 300 μL of saliva was added. The solution was then incubated at 65°C for 10 mins. After that, the solution was homogenized using a shaker at 1050 rpm for 10 mins. Beads were separated from the solution using a magnet. The solution was discarded, and 1000 μL absolute ethanol was added for washing. The solution was homogenized using a shaker at 1050 rpm for 10 mins. The magnetic beads were separated from the solution, and the step was repeated. After that, the beads were incubated at 65°C for 10 mins. A total of 100 μL of nuclease- free water was added to dissolve the DNA bound to the beads. The solution was homogenized and the beads were separated from the nuclease-free water using a magnet. Nuclease-free water containing DNA was placed into a 1.5 mL tube.
The product was stored at a temperature of -20°C.
DNA quantification. The extracted DNA was measured for its concentration and purity.
Measurements were performed using nanodrop spectrophotometry UV lights. The A260 nm and A280 nm were used for the measurement. A total of 2 μL of nuclease-free water was used as Blanco.
The solution containing DNA was homogenized using a vortex as pre-treatment. For quantification, 2 μL DNA solutions were put into the nanodrop (MaestroNano Pro, MaestroGen). Each calculation was repeated three times as a replication.
DNA amplification of β-globin gene. DNA was amplified using PCR (Polymerase Chain Reaction). The universal β-globin gene was targeted. The primers used were designed by Gupta and Agrawal, 2003 [28] named Primer-1 ('5- CCAAGGACAGGTACGGCTGTCATC-3') and
Primer-5 (‘5-
CCTTCCTATGACATGAACTTAACATT-3’).
DNA amplification components such as DNA polymerase enzyme, dNTPs, loading dye, and cofactors were provided by MyTaqTM Red Mix (Bioline, Meridian Bioscience). The mix ratio was calculated according to the original protocol. DNA amplification was carried out in a thermal cycler (T100, Bio-Rad). The reaction was done using protocols from Hernaningsih et al. 2022 [29].
Amplification was started by a pre-denaturation reaction at 95°C for 2 mins. The reaction was followed by denaturation at 95°C for 15 s, annealing at 54°C for 15 s, and extension at 72°C for 2 mins, with 34 repetitions of the cycle. The reaction was terminated with a final extension at 72°C for 10 mins. The length of the amplified band
is 704 bp which will be visible when visualized by agarose gel electrophoresis.
DNA visualization. Genomic DNA and amplicons were visualized using agarose gel electrophoresis. Agarose was prepared by mixing agarose powder, TBE buffer, and fluorescence (FloroSafe DNA Stain, 1st Base). The gel concentration was 0.8% for genomic DNA electrophoresis and 2% for amplification result electrophoresis. A total of 5 μL of the mixing product was used. The electrophoresis process was carried out with a 50-volt for 30 mins. The electrophoresis results were visualized under UV light.
RESULTS AND DISCUSSION
DNA extraction was conducted in this study using magnetic-silica nanoparticle beads made from three different methods. The three magnetic- silica nanoparticle beads were named MAGSi-1, MAGSi-2, and MAGSi-3. DNA extraction was performed by combining three MAGSi with two lysis buffers and without a lysis buffer. The DNA yields were measured for their concentration and purity. The DNA yields were measured for their concentration and purity. Each combination was analyzed with three repetitions. All the combinations have succeeded in extracting human saliva DNA.
Genomic DNA electrophoresis in 0.8% agarose gel successfully detected the DNA bands (Figure 3). The DNA bands were characterized by their size. Genomic DNA is composed of large size making its movement slow. A slight smear indicates a small degraded component or other smaller molecule, such as RNA.
DNA extraction using MAGSi-1 gives a higher concentration than the others. The combination of triton buffer and ATL buffer resulted in lower concentrations when compared to the extraction without lysis buffer (Figure 2). DNA extraction using triton buffer is highest when combined with MAGSi-1, and the concentration value is 42.90 ng/μL. The highest concentration using ATL buffer is 71.63 ng/μL, and the result was given when combined with MAGSi-1. The extraction of MAGSi-1 without lysis buffer resulted in the highest DNA concentration, 104.36 ng/μL.
All extraction results using MAGSi showed that the extraction without lysis buffer gives the best value (Table 1). The purity of MAGSi-1 without lysis buffer was 1.85 ± 0.045. The purity of MAGSI-2 without lysis buffer was 1.94 ± 0.057.
The purity obtained by extraction of MAGSi-3 without lysis buffer was 1.82 ± 0.015. An exemplary DNA extraction result is closest to 1.8.
The DNA was used to amplify one of the human β-globin genes. Based on amplification result
electrophoresis, it is known that all of the DNA results can be used as the template for amplification (Figure 4). All the bands glow brightly, indicating that the amplification is going
well. The size of the partial β-globin gene was 704 bp. This result shows that the combination of lysis buffer and MAGSi can be used as a reference for future extraction.
Table 1. DNA concentration purity from extraction using a combination of MAGSi and lysis buffer
Beads Sampl es
Triton Buffer ATL Buffer Without buffer
DNA Concentration
(ng/µL)
Purity (A260/A28
0)
DNA Concentration
(ng/µL)
Purity (A260/A28
0)
DNA Concentration
(ng/µL)
Purity (A260/A280) MAGSi
-1
1 42.80 1.63 67.90 1.59 104.40 1.84
2 44.10 1.60 73.20 1.62 109.40 1.82
3 41.80 1.60 73.80 1.56 99.27 1.90
Average 42.90 ± 1.15 1.61 ± 0.02 71.63 ± 3.25 1.59 ± 0.03 104.36 ± 5.07 1.85 ± 0.04 MAGSi
-2
1 23.80 1.51 29.50 1.73 75.02 1.87
2 22.40 1.50 34.20 1.74 72.22 1.98
3 21.10 1.45 29.20 1.75 67.59 1.96
Average 22.43 ± 1.35 1.49 ± 0.03 30.97 ± 2.80 1.74 ± 0.01 71.61 ± 3.75 1.94 ± 0.06 MAGSi
-3
1 21.80 1.43 25.30 1.90 48.24 1.82
2 21.30 1.44 26.20 1.73 57.47 1.81
3 23.70 1.44 24.70 2.02 58.59 1.84
Average 22.27 ± 1.27 1.43 ± 0.01 25.40 ± 0.75 1.88 ± 0.15 54.77 ± 5.68 1.82 ± 0.02
Figure 2. Measurement of DNA concentration from extraction using a combination of MAGSi and lysis buffer
42.90
71.36
104.36
22.43 30.97
71.61
22.27 25.40
54.77
0,00 20,00 40,00 60,00 80,00 100,00 120,00
Triton Buffer ATL Buffer Without Buffer
DNA Concentration (ng/μL)
MAGSi-1 MAGSi-2 MAGSi-3
Figure 3. Genomic DNA electrophoresis from extraction using a combination of lysis buffer and MAGSi in 0,8% agarose gel. M: 1 kb DNA ladder (GeneRuler 1 kb plus, Thermo Fisher), 1: MAGSi- 1 and triton buffer, 2: MAGSi-2 and triton buffer, 3: MAGSi-3 and triton buffer, 4: MAGSi-1 and ATL buffer, 5: MAGSi-2 and ATL buffer, 6:
MAGSi-3 and ATL buffer, 7: MAGSi-1 and without lysis buffer, 8: MAGSi-2 and without lysis buffer, 9: MAGSi-3 and without lysis buffer.
Figure 4. Partial β-globin gene electrophoresis in 2% agarose gel. M: 1 kb DNA ladder (GeneRuler 1 kb plus, Thermo Fisher), 1:
MAGSi-1 and triton buffer, 2: MAGSi-2 and triton buffer, 3: MAGSi-3 and triton buffer, 4:
MAGSi-1 and ATL buffer, 5: MAGSi-2 and ATL buffer, 6: MAGSi-3 and ATL buffer, 7:
MAGSi-1 and without lysis buffer, 8: MAGSi- 2 and without lysis buffer, 9: MAGSi-3 and without lysis buffer.
Arif et al. 5 The DNA extraction process plays a vital role
in molecular analysis. This process is expected to generate a sufficient amount of DNA for the amplification process by PCR. The yield can be known from the DNA concentration value calculated after the extraction. In addition, the DNA purity value is also essential to be noticed.
DNA purity describes the level of clarity of extraction product from contaminants. In this study, DNA was extracted from a small amount of saliva. DNA extraction using a small amount of sample provides some advantages, such as fewer reagents used, so it reduces costs, reduces the risk of sample degradation, simplifies the treatment, and reduces processing time [30].
The DNA concentration value describes the amount of DNA successfully extracted in specific solvent volumes. Various spectrophotometric techniques have been developed to measure the number of DNA concentrations, such as ultraviolet absorption, fluorescence measurement, and diphenylamine reactions [32]. Each measurement has a different basic principle. The fluorescent uses the measurement of the luminescence excitation between the fluorescents dye and double-stranded DNA [32]. The basic principle of diphenylamine measurement is measuring the analysis result reaction between x-hydroxy-c-keto pentose and diphenylamine produced after the glycosidic bond between purine and deoxyribose bases is broken [33]. The most commonly used technique is the measurement of ultraviolet absorption. This technique utilizes ultraviolet absorption by DNA hydrogen bonds at a wavelength of 260 nm [34]. In this study, Concentration was measured by nanodrop spectrophotometry which was classified as an ultraviolet absorption technique. The wavelengths used for spectrophotometry were A260 and A280. The increased amount of DNA dissolved in the solvent could make the concentration value captured by the nanodrops higher. However, the PCR amplification process can run and be executed with a small number of
DNA concentrations [35]. All extracted DNA successfully amplified the β-globin gene.
Aside from concentration, DNA purity is also needed to be considered. Pure DNA has a value of 1.8 or close to 1.8. The presence of contaminants such as polysaccharides, proteins, and residual reagents such as ethanol, 2-propanol, or lysis buffer interfere with the DNA amplification process by PCR. These contaminants make the purity value of A260/A280 away from 1.8. The purity value accepted for molecular forensic analysis is 1.6 – 2.0 [36]. A purity ratio below 1.6 on the reading indicates significant protein contamination [34].
That can happen because A260/A280 ratio is less sensitive to protein contamination, so a slight decrease in value indicates the presence of high protein contaminants [37]. Besides protein, other contaminants such as ethanol, sodium acetate, and ammonium acetate cause reading errors and invalid values [30].
In this study, we used a combination of lysis buffer and modified magnetic-silica beads for DNA extraction from human saliva. Data showed that MAGSi-1 succeeded in extracting DNA with the highest concentration value. Our previous study showed that MAGSi-1 had a heterogeneous particle distribution with sizes ranging from 160 nm to 500 nm. The study revealed that the different MAGSi synthesis techniques affected the particle distribution, where MAGSi-2 and MAGSi-3 were more homogeneous than MAGSi-1. The result followed the research conducted by Edrissi et al.
2011 [19]. Our previous study also analyzed the morphology of each MAGSi using SEM and TEM (Figure 5, Figure 6). The data showed that the MAGSi-1 particles were spherical shape agglomerated while MAGSi-2 particles were clustered spherical, and MAGSi-3 particles were smaller clustered spherical. The magnetic-silica nanoparticle beads affect their affinity with silica to bind the extracted DNA [20, 21, 38]. This made each MAGSi produce a different concentration value.
Figure 5. SEM of (a) MAGSi-1, (b) MAGSi-2, and (c) MAGSi-3 in 20.000x magnification (a) and 30.000x magnification (b and c). Bar: 0.5 µm.
Figure 6. TEM of (a) MAGSi-1, (b) MAGSi-2, and (c) MAGSi-3. Bar: 50 nm
This research studied the effect of using different lysis buffers on DNA extraction using MAGSi. Presented data revealed that MAGSi could extract salivary DNA with the best concentration and purity on the combination of MAGSi-1 and without lysis buffer. That is an advantage because it will reduce the costs of the lysis buffer preparation. Further and in-depth study must be executed to get more accurate results.
Using triton buffer and ATL buffer as lysis buffer resulted in lower concentrations, but it was always more stable than the process without lysis buffer.
This consistency is an excellent aspect to consider in extraction material [39]. Rather than a technique for obtaining high concentrations, consistency is preferable because it provides certainty for users who perform DNA extraction. The lysis buffer works to dissolve the cell membrane [40]. It is a commonly combined proteinase K enzyme to degrade proteins and inactivate cellular enzymes such as nuclease, RNAse, and DNAse [41].
The extracted DNA was used as a template to amplify the human β-globin gene, and it was successfully amplified. The amplification target was 704 bp (Figure 4). It indicated that DNA extracted from saliva using MAGSi combined with lysis buffer and without lysis buffer could amplify specific genes. The human β-globin gene was used because this single target gene is often used for genotyping of thalassemia mutation [29]. In addition, the amplicon was of medium length, so it did not need extended reaction time [28, 42].
CONCLUSION
Salivary DNA extraction using the combination of lysis buffer and our modified magnetic-silica nanoparticle beads provided promising results. The results gave a good amount of concentration and purity. The extraction product could be used as a DNA template for DNA amplification. The concentrations and purity values were different but consistent. All extraction results can amplify the human β-globin gene. Further studies indeed are still needed to determine the effectiveness and consistency.
ACKNOWLEDGMENT
The authors would like to thank the subjects who participated in the testing. The authors also thank Laboratory Genetics and Breeding, Faculty of Biology, Universitas Gadjah Mada, and Research Organization for Nuclear Energy, National Research and Innovation Agency for providing facilities and reagents.
REFERENCES
[1] Lee SB, Shewale JG (2017) DNA extraction methods in forensic analysis. In:
Encyclopedia of Analytical Chemistry. John Wiley & Sons, Ltd. pp 1–18.
[2] Carpi FM, Di Pietro F, Vincenzetti S, Mignini F, Napolioni V (2011) Human DNA extraction methods: Patents and applications.
Recent Patents on DNA & Gene Sequences 5 1–7.
[3] Mi Y, Vanderpuye O (2013) Comparison of different DNA extraction methods for forensic samples. Journal of Natural Sciences Research 3 (11): 32–39.
[4] Nielsen K, Mogensen HS, Hedman J, Niederstätter H, Parson W, Morling N (2008) Comparison of five DNA quantification methods. Forensic Science International:
Genetics 2 (3): 226–230. doi:
10.1016/j.fsigen.2008.02.008.
[5] Chong KWY, Thong Z, Syn CK (2020) Recent trends and developments in forensic DNA extraction. WIREs Forensic Science 3 (2): 1–23. doi: 10.1002/wfs2.1395.
[6] Ghatak S, Muthukumaran RB, Nachimuthu SK (2013) A simple method of genomic DNA extraction from human samples for PCR- RFLP analysis. Journal of Biomolecular Techniques 24 (4): 224–231. doi:
10.7171/jbt.13-2404-001.
[7] Quinque D, Kittler R, Kayser M, Stoneking M, Nasidze I (2006) Evaluation of saliva as a source of human DNA for population and association studies. Analytical Biochemistry
353 (2): 272–277. doi:
10.1016/j.ab.2006.03.021.
Arif et al. 7 [8] Dumache R, Ciocan V, Muresan C,
Parvanescu R, Enache A (2019) Advantages of salivary DNA in human identification. In:
Saliva and Salivary Diagnostics. IntechOpen.
pp 1–8.
[9] Kim Y-W, Kim Y-K (2006) The effects of storage of human saliva on DNA isolation and stability. ORAL MEDICINE 31 (1): 1–16.
[10] Lazarevic V, Gaïa N, Girard M, François P, Schrenzel J (2013) Comparison of DNA extraction methods in analysis of salivary bacterial communities. PLoS ONE 8 (7):
e67699. doi: 10.1371/journal.pone.0067699.
[11] Goode MR, Cheong SY, Li N, Ray WC, Bartlett CW (2014) Collection and extraction of saliva DNA for next generation sequencing.
Journal of Visualized Experiments (90):
e51697. doi: 10.3791/51697.
[12] Smith AK, Kilaru V, Klengel T, Mercer KB, Bradley B, Conneely KN, Ressler KJ, Binder EB (2015) DNA extracted from saliva for methylation studies of psychiatric traits:
Evidence tissue specificity and relatedness to brain. American Journal of Medical Genetics, Part B: Neuropsychiatric Genetics 168 (1):
36–44. doi: 10.1002/ajmg.b.32278.
[13] Philibert RA, Zadorozhnyaya O, Beach SRH, Brody GH (2008) Comparison of the genotyping results using DNA obtained from blood and saliva. Psychiatric Genetics 18 (6):
275–281. doi:
10.1097/YPG.0b013e3283060f81.
[14] Abraham JE, Maranian MJ, Spiteri I, Russell R, Ingle S, Luccarini C, Earl HM, Pharoah PPD, Dunning AM, Caldas C (2012) Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping. BMC Medical Genomics 5 (19): 1–6.
[15] Alaeddini R, Walsh SJ, Abbas A (2010) Forensic implications of genetic analyses from degraded DNA-A review. Forensic Science International: Genetics 4 (3): 148–
157. doi: 10.1016/j.fsigen.2009.09.007.
[16] Bordelon H, Russ PK, Wright DW, Haselton FR (2013) A magnetic bead-based method for concentrating DNA from human urine for downstream detection. PLoS ONE 8 (7):
e68369. doi: 10.1371/journal.pone.0068369.
[17] Oberacker P, Stepper P, Bond DM, Höhn S, Focken J, Meyer V, Schelle L, Sugrue VJ, Jeunen G-J, Moser T, Hore SR, von Meyenn F, Hipp K, Hore TA, Jurkowski TP (2019) Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation. PLoS Biology
17 (1): e3000107. doi:
10.1371/journal.pbio.3000107.
[18] Noh GS, Liu H, Kim MG, Qiao Z, Jang YO, Shin Y (2020) Multi-sample preparation assay for isolation of nucleic acids using bio-silica with syringe filters. Micromachines 11 (9):
823. doi: 10.3390/mi11090823.
[19] Edrissi M, Soleymani M, Adinehnia M (2011) Synthesis of silica nanoparticles by ultrasound-assisted sol-gel method:
Optimized by Taguchi robust design.
Chemical Engineering and Technology 34
(11): 1813–1819. doi:
10.1002/ceat.201100195.
[20] Morel AL, Nikitenko SI, Gionnet K, Wattiaux A, Lai-Kee-Him J, Labrugère C, Chevalier B, Deleris G, Petibois C, Brisson A, Simonoff M (2008) Sonochemical approach to the synthesis of Fe3O4@SiO2 core - Shell nanoparticles with tunable properties. ACS Nano 2 (5): 847–856. doi:
10.1021/nn800091q.
[21] Villa S, Riani P, Locardi F, Canepa F (2016) Functionalization of Fe3O4 NPs by silanization: Use of amine (APTES) and thiol (MPTMS) silanes and their physical characterization. Materials 9 (10): 826. doi:
10.3390/ma9100826.
[22] Fuentes-García JA, Alavarse AC, Maldonado ACM, Toro-Córdova A, Ibarra MR, Goya GF (2020) Simple sonochemical method to optimize the heating efficiency of magnetic nanoparticles for magnetic fluid hyperthermia. ACS Omega 5 (41): 26357–
26364. doi: 10.1021/acsomega.0c02212.
[23] Dheyab MA, Aziz AA, Jameel MS (2021) Recent advances in inorganic nanomaterials synthesis using sonochemistry: A comprehensive review on iron oxide, gold and iron oxide coated gold nanoparticles.
Molecules 26 (9): 2453. doi:
10.3390/molecules26092453.
[24] Lever MA, Torti A, Eickenbusch P, Michaud AB, Šantl-Temkiv T, Jørgensen BB (2015) A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types.
Front Microbiol. doi:
10.3389/fmicb.2015.00476
[25] Watson JD, Baker TA, Bell SP, Gann A, Levine M, Losick RM (2004) Molecular Biology of the Gene. 5th ed. USA, Benjamin Cummings.
[26] Doyle K (1996) The source of discovery:
protocols and applications guide. USA, Promega.
[27] Sharafi Z, Bakhshi B, Javidi J, Adrangi S (2018) Synthesis of silica-coated iron oxide nanoparticles: Preventing aggregation without using additives or seed pretreatment. Iranian
Journal of Pharmaceutical Research 17 (1):
386–395.
[28] Gupta A, Agarwal S (2003) Efficiency and cost effectiveness: PAGE-SSCP versus MDE and Phast gels for the identification of unknown β thalassaemia mutations. Journal of Clinical Pathology: Molecular Pathology 56 237–239.
[29] Hernaningsih Y, Syafitri Y, Indrasari YN, Rahmawan PA, Andarsini MR, Lesmana I, Moses EJ, Rahim NAD, Yusoff NM (2022) Analysis of common beta-thalassemia (β- Thalassemia) mutations in East Java, Indonesia. Frontiers in Pediatrics 10 925599.
doi: 10.3389/fped.2022.925599.
[30] Khosravinia H, Murthy HNN, Parasad DT, Pirany N (2007) Optimizing factors influencing DNA extraction from fresh whole avian blood. African Journal of Biotechnology 6 (4): 481–486.
[31] Li X, Wu Y, Zhang L, Cao Y, Li Y, Li J, Zhu L, Wu G (2014) Comparison of three common DNA concentration measurement methods.
Analytical Biochemistry 451 (1): 18–24. doi:
10.1016/j.ab.2014.01.016.
[32] Ahn SJ, Costa J, Emanuel JR (1996) PicoGreen quantitation of DNA: effective evaluation of samples pre-or post-PCR.
Nucleic Acids Research 24 (13): 2623–2625.
[33] Croft DN, Lubrant M (1965) The estimation of deoxyribonucleic acid in the presence of sialic acid: Application to analysis of human gastric washings. Biochemical Journal 95 612–620.
[34] Bunu SJ, Otele D, Alade T, Dodoru R (2020) Determination of serum DNA purity among patients undergoing antiretroviral therapy using NanoDrop-1000 spectrophotometer and polymerase chain reaction. Biomedical and Biotechnology Research Journal 4 (3): 214–
219. doi: 10.4103/bbrj.bbrj_68_20.
[35] Lorenz TC (2012) Polymerase chain reaction:
Basic protocol plus troubleshooting and optimization strategies. Journal of Visualized Experiments (63): e3998. doi: 10.3791/3998.
[36] Khare P, Raj V, Chandra S, Agarwal S (2014) Quantitative and qualitative assessment of DNA extracted from saliva for its use in forensic identification. Journal of Forensic Dental Sciences 6 (2): 81–85. doi:
10.4103/0975-1475.132529.
[37] Glasel GA (1995) Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques 18 (1): 62–
63.
[38] Riani P, Lucchini MA, Thea S, Alloisio M, Bertoni G, Canepa F (2014) New approach for the step by step control of magnetic nanostructure functionalization. Inorganic Chemistry 53 (17): 9166–9173. doi:
10.1021/ic501194n.
[39] Griffiths L, Chacon-Cortes D (2014) Methods for extracting genomic DNA from whole blood samples: current perspectives. Journal of Biorepository Science for Applied Medicine 2 1–9. doi: 10.2147/bsam.s46573.
[40] Price CW, Leslie DC, Landers JP (2009) Nucleic acid extraction techniques and application to the microchip. Lab on a Chip 9 (17): 2484–2494. doi: 10.1039/b907652m.
[41] Herzer S (2001) DNA Purification. In:
Molecular Biology Problem Solver : A Laboratory Guide. USA, John Wiley & Sons, Ltd. p 575.
[42] Mubarak SMH, Al-Koofee DAF, Radhi OA, Ismael J, Al-Zubaidi ZF (2020) An optimization and common troubleshooting solving in polymerase chain reaction technique. Systematic Reviews in Pharmacy 11 (2): 427–436. doi: 10.5530/srp.2020.2.63.
