|Year : 2019 | Volume
| Issue : 4 | Page : 595-599
|A crude method of DNA extraction and identification from exfoliated human buccal mucosa cells
Priyanka Kardam1, Monica Mehendiratta2, Shweta Rehani1, Rashi Sharma1, Khushboo Sahay1
1 Department of Oral Pathology and Microbiology, Sudha Rustagi College of Dental Sciences and Research, Faridabad, Haryana, India
2 Department of Oral Pathology and Microbiology, ITS Dental College, Greater Noida, Uttar Pradesh, India
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|Date of Submission||30-Jan-2016|
|Date of Decision||17-Dec-2016|
|Date of Acceptance||16-Nov-2017|
|Date of Web Publication||18-Nov-2019|
| Abstract|| |
Background: DNA analysis has a key role in forensic dentistry. However, techniques of DNA extraction and analysis are far from the reach of majority of medical professionals owing to its expensive set up. Aim: The present study was aimed at formulating a crude method of extracting DNA from human buccal mucosa cells using materials commonly available in the laboratory so that the medical professionals could get more exposure to molecular biology techniques. The objectives were to identify the DNA and to assess its purity. Methods: Buccal mucosa cells from 10 healthy volunteers were taken for DNA extraction following the protocol of cell lysis, purification, and precipitation. DNA was identified using standardized techniques like Diphenylamine test and its purity was assessed using a spectrophotometer. A gel electrophoresis apparatus was also constructed using readily available materials. Results: DNA was extracted from human buccal mucosa cells using a crude method. The standardized tests confirmed the presence of DNA contaminated with proteins. The locally made Gel electrophoresis model exhibited a faint halo around the wells instead of DNA bands. Conclusion: DNA extraction from human buccal mucosa cells was made possible using locally available materials and a crude method, but it was not of high purity.
Keywords: Buccal mucosa, DNA extraction, gel electrophoresis, molecular biology, spectrophotometer
|How to cite this article:|
Kardam P, Mehendiratta M, Rehani S, Sharma R, Sahay K. A crude method of DNA extraction and identification from exfoliated human buccal mucosa cells. Indian J Dent Res 2019;30:595-9
|How to cite this URL:|
Kardam P, Mehendiratta M, Rehani S, Sharma R, Sahay K. A crude method of DNA extraction and identification from exfoliated human buccal mucosa cells. Indian J Dent Res [serial online] 2019 [cited 2019 Dec 14];30:595-9. Available from: http://www.ijdr.in/text.asp?2019/30/4/595/271071
| Introduction|| |
Molecular biology techniques are inaccessible to most medical professionals as these tests are expensive and require specialized equipment available only in some laboratories. These tests also require specially trained scientists and strict quality control. There is a general lack of practical knowledge about the modus operandi.
The process of isolating nucleic acid consists of three steps, namely, cell lysis, purification, precipitation, and concentration. For cell lysis, the cell/nuclear membrane is disrupted to release the DNA. Purification involves removal and denaturation of proteins and inactivation of enzymes capable of degrading DNA. For precipitation and concentration, DNA and water bonds are broken and structural changes are induced to help DNA aggregate. The solution is then centrifuged at high speed to form a pellet.,
The study was aimed at extracting DNA from human buccal mucosa cells using commonly available laboratory materials followed by its identification and assessment of its purity using standardized techniques such as diphenylamine (DPA) test and spectrophotometry. The objectives of the study were to extract DNA using different commonly available cell lysates (such as hand wash and liquid detergent) and purification agents (such as pineapple juice, contact lens solution and heat). Construction of a crude gel electrophoresis apparatus using readily available materials was also attempted.
| Materials and Methods|| |
This prospective study was performed in the Department of Oral Pathology and Microbiology, Sudha Rustagi College of Dental Sciences and Research, Faridabad. The sample group consisted of 10 healthy volunteers in the age group of 20–30 years who were chosen as the source of DNA. An informed consent of the patients and institutional ethical committee clearance were taken before proceeding with the study. The study was carried out in different phases namely DNA extraction, DNA identification, assessment of purity of DNA, and gel electrophoresis.
The first step was to collect the DNA extraction source, i.e. lightly adherent superficial epithelial cells or naturally exfoliated cells of oral cavity which were mainly from buccal mucosa. For the collection, a buffer for swishing the oral cavity was prepared by adding 1.5 g table salt (sodium chloride; commercially available TATA salt) to 5.0 g baking soda (sodium bicarbonate; commercially available TOPS India baking soda) and diluting it to a final volume of 120 ml with water. The volunteers were asked to take 10 ml of buffer solution and swish the oral cavity for 1 min. They were instructed to intentionally bite on the buccal mucosa so that more cells could be exfoliated into the buffer. The swished out buffer was then collected in a test tube.
The next step was to extract DNA from the collected epithelial cells present in buffer following the standard protocol of cell lysis, purification and precipitation., For this study, we specifically used commercially available liquid hand wash (Lifebuoy total 10) and liquid detergent (Vim dishwash liquid). Using a pipette, 2 ml of cell lysate was added to swish out buffer in the test tube. The test tube was gently inverted 10–12 times, taking care to avoid foam formation. A similar procedure was performed using different varieties of cell lysates.
For the next step, i.e. purification, various purification enzymes such as bromelain present in pineapple juice (undiluted and freshly prepared), papain, pancreatin and subtilisin in contact lens solution (Bausch + Lomb, Renu fresh multipurpose) and heat were used. Purification was done by adding 2 ml of purification agent using a pipette to the test tube already containing buffer solution and cell lysate solution. The test tube was left untouched for 2 min [Table 1].
|Table 1: A260/A280 of deoxyribonucleic acid samplesusing various combinations of cell lysates and purification enzymes|
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Ethanol (100%) was cooled (−20°C) by keeping overnight in the freezer, and it was used as the precipitating solution. Cooled alcohol of double the volume of solution in the test tube was carefully poured over it. The test tube was kept at an angle of 45° using a custom made stand in the entire process to ensure layer by layer deposition and no disturbance in the solution underneath. The test tube was again left undisturbed for 10 min at room temperature which was maintained at 20°C–23°C.
DPA test was used as a test for confirming the presence of deoxyribose (sugar moiety of DNA) in the spooled out strands from the sample. DPA stock solution was prepared by adding 15 g DPA to acids (1000 ml glacial acetic acid and 15 ml sulfuric acid). The working solution was freshly prepared before use by the addition of 5 ml of 2% acetaldehyde (100 μl acetaldehyde and 5 ml water) for each 1000 ml of the stock solution. Solution was kept in dark glass to avoid its darkening by light. To 1 ml of DNA sample, 2 ml of DPA working solution was added and the test tube was kept in boiling water for 15 min.
Assessment of purity of DNA
Spectrophotometer was used to measure the absorbance (wavelength at which the light is absorbed) which helped in the assessment of purity of DNA in the sample obtained. The spectrophotometer (HITACHI U-2900) was switched on for 10 min for the lamp to warm up followed by calibration of the instrument according to the standard protocol. DNA sample was taken in a quartz cuvette, and absorbance was measured at 260 and 280 nm for different combinations of cell lysates and purification enzymes. A260/A280 ratio was calculated to estimate the purity of the sample.
To construct a working model of gel electrophoresis apparatus, commonly available laboratory and household materials were used as a replacement of the expensive gel electrophoresis apparatus available in the market [Table 2]. A plastic box measuring 15 cm × 10 cm was used as the gel electrophoresis chamber. A glass slab measuring 7 cm × 4 cm was taken to cast the gel and copper wires of 0.1 cm diameter were tied to both ends of the glass slab as a substitute of electrodes. A cheaper and inferior quality replacement of agarose, Agar (1.0%–1.2% w/v) was poured over the glass plate to around 1 mm thickness. The glass slab was then kept to cool in a refrigerator for 25–30 min at 4 degrees Celsius. Next, an autoclaved plastic cap of 0.8 cm diameter was used to gently cut out wells in the agar and wells were then completely dried using paper points (routinely used in root canal procedure). The wells were at a distance of 1 cm from the wire connected to the negative terminal of the battery pack and 1.5 cm from each other [Figure 1].
|Table 2: Materials used for constructing a working model of gel electrophoresis apparatus|
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|Figure 1: Construction of gel electrophoresis apparatus. (a) Two metal wires tied to the ends of a glass slab; (b) Liquid agar being poured over the glass slab using a pipette; (c) Plastic cap being used to cut out the wells in solidified agar gel; (d) Complete gel electrophoresis working model|
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A running buffer was prepared by adding 0.05 g of table salt (Sodium chloride), and 2 g of baking soda (Sodium bicarbonate) to 1 L distilled water. The pH was brought to 7.5 using a pH meter. Next, the cooled glass plate with agar gel was placed into the plastic box (electrophoresis chamber) and the ends of copper wires tied to glass plate were then connected to a series of 9 volts batteries. Running buffer was poured into the plastic box until the agar gel was immersed up to 3 mm in it.
Loading buffer consisted of 0.5 ml glycerine mixed with 0.1 ml distilled water. This loading buffer can be used as ×5 to ×10 dilution for the DNA sample, i.e. for 0.5 ml of DNA sample, 0.05–0.1 ml of loading buffer was used. After this, DNA sample with loading buffer was poured into the well cut in the agar plate using a micropipette. In another well, orange G (negatively charged) dye was added as control. This setup was left undisturbed for 45 min.
Staining the gel
To visualize the DNA bands, staining was done by pouring methylene blue solution (0.1%) over the agar gel. The setup was left undisturbed for the whole night.
| Results|| |
After a time interval of 10 min DNA strands were seen rising from the interface of alcohol and aqueous layers during DNA extraction which were spooled/pulled out using a metal hook [Figure 2]. A green-blue color was obtained after performing the DPA test on extracted DNA, which indicated the presence of deoxyribose (sugar moiety of DNA) [Figure 3]. On performing spectrophotometry, the purest DNA sample (A260/A280 = 1.22) was obtained using hand wash as the cell lysate and pineapple juice as the purification enzyme. The results for all other combinations of lysates and purification enzymes have been listed in [Table 1]. In gel electrophoresis, the control sample exhibited movement toward the positive end as expected. The movement was visible in the form of a tongue-shaped orange colored streak. A halo was seen around the well which indicated that there was some movement of the sample [Figure 3].
|Figure 2: Results of DNA extraction from various sources. Strands of DNA strands rising from the solution of: (a) Banana; (b) Onion; (c) Buccal mucosa; (d) Kiwi|
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|Figure 3: Results of gel electrophoresis and diphenylamine test. (a) Movement of control toward the positive end in the form of a tongue shaped orange colored streak; (b) A halo seen around the well which contained the extracted DNA sample; (c) A green-blue color obtained after performing the diphenylamine test on extracted DNA|
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| Discussion|| |
DNA (Deoxyribonucleic acid) provides a unique identity to every human being. It is an integral component of all tissues of body such as hair, nails, biopsy sample, saliva, blood, and buccal mucosa. These sources show variations only in quantity and quality of the DNA.
There has been a great progress in the field of forensic science owing to the development of newer and more reliable techniques of DNA extraction and typing.,, However, the molecular biology is still at the grass-root level owing to the expensive equipment and specialization it demands with regard to technology. This study was conducted with an aim to bring the usually unreachable molecular biology. A pilot run based on MacGyver's experiments to extract DNA was carried out on different fruits and vegetables such as onion, kiwi, and banana following which the study was performed on human buccal mucosa cells.
On the cells of human buccal mucosa, the study was carried out in four stages, namely, DNA extraction, DNA identification, assessment of purity of DNA, and gel electrophoresis. A successful DNA extraction was carried out following the routine protocol of cell lysis, purification and precipitation using cheaper and easily available substitutes of expensive chemicals and equipment. The next step was to confirm the presence of DNA in the solution. For this purpose, the biochemical test for the presence of deoxyribose was used. DPA reagent confirmed the presence of DNA by reacting with deoxyribose (sugar moiety of DNA) in an acidic medium to produce a blue colored solution.
To assess the purity of DNA extracted, spectrophotometry was used. Spectrophotometry is based on the principle of Beer-Lambert law, which states that when a sample is placed in the beam of a spectrophotometer, there is a direct linear relationship between the amount (concentration) of its constituent (s) and the amount of energy it absorbs. The wavelength at which the light is absorbed (absorbance) is a function of molecular structure of the compound. The nitrogenous bases present in the DNA, allows it to absorb light in the UV range (260 nm). Proteins are the most common contaminants extracted along with DNA and they also absorb in the ultraviolet range (280 nm). To determine the purity of DNA sample extracted, a ratio of absorbance at A260 nm and A280 nm is used. A good quality DNA sample should have A260/A280 ratio of 1.7–2.0. If the A260/A280 ratio is more than 1.75, DNA is pure enough to proceed. If the ratio is >2.1, it is not DNA and if it is <1.75, the DNA is highly contaminated with protein.
In the present study, an attempt was also made to construct a working model of gel electrophoresis apparatus. The movement of charged particles through an electrolyte when subjected to an electric field resulting in their migration towards the oppositely charged electrode is known as electrophoresis. Electrophoresis is affected by various factors such as net charge on the particles, mass and shape of the particles, pH of medium, and strength of electrical field. Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA. Gel electrophoresis separates DNA fragments by molecular weight in a solid support medium (agarose gel). DNA samples are poured into the sample wells using a pipette and application of an electric current at the anodal end (negative) causes the negatively-charged DNA to migrate (electrophorese) toward the cathodal end (positive). The rate of migration is proportional to size: smaller fragments move more quickly and move farther away from the anodal end. In our study, this model was not fully successful; this could probably be due to the use of an inferior quality replacement of agarose.
For better results the authors suggest the use of better/known substitutes available in the market, for example, agarose instead of agar, proteinase K instead of pineapple juice and voltage regulator instead of transistor batteries. However, more sampling sites could also be tried for DNA extraction, for example, blood, exfoliated cells from different sites of the oral cavity.
Hence, to conclude we would like to say that molecular biology is still at grass-root level in India which leads to lack of knowledge amongst the health-care professionals and students. However, if the researchers pour in the efforts to devise cost economic techniques for performing molecular biology, it could lead to an increase in exposure and thus awareness of these professionals.
The authors would like to acknowledge Late. Dr. P. K. Jain without whose guidance this project would not have been possible. We would also like to acknowledge Dr. T. K. Mishra whose help was very fruitful for the project.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Priyanka Kardam
D-81, Ground Floor, Saket, New Delhi - 110 017
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]
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