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Deoxyribonucleic acid (DNA) is a very vital component of human biology which contains genetic instructions essential for development and functioning of body. Extraction of DNA is a process of isolating DNA from rest of the cellular components for molecular and forensic analysis. This process is carried out by cell disruption and proteolysis by adding AL buffer and protease solution. The AL buffer, which consists of Guanidium thiocynate acts as a 'cell lytic' agent. Similarly protease solution removes protein by proteolysis. The membrane lipids are removed by using detergents. Ethanol is used as a dehydrating and precipitating agent for DNA extraction. Being insoluble in alcohol, DNA aggregates and gives pellets upon centrifugation when it is added to ethanol. On this process, the alcohol soluble salts are also removed. Finally, the DNA attached on membrane of spin column is resolubilized and eluted by the addition of AE buffer which is alkaline in nature.
The genomic DNA extracted above can be run on agrose gel by electrophoresis. Agrose gel electrophoresis is a technique to separate DNA or RNA molecules by size. It is carried out by the movement of negatively charged nucleic acid molecules through the matrix of agrose gel within an electric field. The size of the DNA molecule can be estimated by use of DNA ladder or a "marker" in agrose gel electrophoresis. A DNA ladder is a solution of DNA molecules that give a band of particular size on agrose gel electrophoresis run. DNA ladders of different sizes like 1 kb, 10 kb etc are commercially available on market.
Ethidium bromide is the most commonly used dye that fluoresces under UV light when intercalated into DNA. For this purpose, the DNA suspension is incorporated with this dye prior to running in the gel. During the process of electrophoresis, shorter DNA molecules move faster and migrate farther away than the longer molecules. (Sambrook J, Russel DW, 2001) By comparison of the DNA band with the DNA ladder, the length of the DNA can be estimated.
The yield and purity of the extracted genomic DNA can also be determined by UV spectrophotometry, which involves the measurement of absorbance of the DNA solution by using UV spectrophotometer.DNA yield can be determined by measuring the concentration of DNA in the original extract by its absorbance at 260 nm. "Ideally, a suspension of 50 microgram DNA in 1 ml of water gives the absorbance of 1.0 at 260nm wavelength when light travels through a path length of 1cm." This statement can be used to calculate the concentration of genomic DNA in the prepared sample.
Similarly, the ratio of the absorbance readings at 260 nm and 280 nm provides an estimate of the purity of the DNA with respect to the contaminants like Protein and RNA that apparently alter the actual DNA concentration. Generally, the presence of the protein will lower the ratio and presence of RNA will make the ratio higher. To ensure that the extracted DNA sample is pure, the ratio of absorbance at 260nm to the absorbance 280 nm should be in the range of 1.8 to 2.0
Aim of Practical
(i)To extract DNA from own buccal cells
(ii) To verify DNA in extracted sample by agrose gel electrophoresis
(iii) To quantify the concentration of DNA in the extracted sample
(iv) To quantify and test the purity of the extracted DNA sample
Materials and Methods
Step I: Extraction of DNA
"Omni-Swab package" was used to scrape the buccal cells from mouth by rubbing the collection pad firmly against the inside of cheek. The pad was ejected on a labelled micro centrifuge tube by pressing the handle towards the collection pad. After that 400 ml of phosphate buffered saline (PBS), followed by 14 micro litres QUIAGEN protease solution and 400 micro litre of AL buffer was added to the sample tube. This preparation was mixed by vortexing for 15 seconds, and then incubated at 56 degree Celsius for 10 minutes. After a brief centrifuge to remove the drops from inside the lid, 400 micro litre of ethanol was added to the sample tube and mixed by vortexing for 15 seconds. This was followed by brief centrifuge to remove the drops from inside the lid. After that, 700 micro litre of the above preparation was carefully pipetted in a QIAmp spin column and then capped properly and centrifuged at 8000 rpm for 1 minute. The filtrate fluid obtained in the collection tube was discarded. After this, the QIAmp spin column was palced in a new collection tube and 500 micro litre of AW1 buffer was added to the column. After capping the column, the preparation was centrifuged at 8000 rpm for 1 minute. Again the filtrate fluid in the collection tube was discarded and new collection tube was used to keep the QIAmp spin column. At this stage, 500 micro litre of AW2 buffer was added to the column and centrifuged at 13000 rpm for 4 minutes. Again the filtrate fluid in the collection tube was discarded, and new collection tube was used to keep the column. Finally, the column was added with 150 micro litre of AE buffer, incubated at room temperature for 1 minute, and centrifuged at 8000 rpm for 1 minute. This final step eluted the DNA in the collection tube, which was then lebelled and stored inside the refrigerator at -70 degree Celsius.
Step II: Verification of Extracted DNA sample from agrose gel electrophoresis
On the next week, DNA sample prepared above was taken out of refrigerator and thoroughly defrosted. On another hand 0.8% agrose gel in Tris Boric acid EDTA (TBE) buffer was kept stand by. After that, 10 micro litre of extracted DNA preparation (from step I) and 2 micro litre of loading buffer was taken in a small micro centrifuge tube and mixed well. 10 micro litre of this mixture was then loaded carefully on well number 11 of the agrose gel by ejecting 10 micro litre of the solution with the help of a micropipette tip. The first well was already loaded with 10 micro litre of DNA ladder of 10kb size. After loading the samples in all the wells of the gel, electrophoresis was started by applying 70 V voltage across the gel for one and half hour. This process resulted in the separation of DNA components which was then viewed under UV light .
Step III: Determination of Yield and Purity of Extracted DNA from spectrophotometer
The DNA sample obtained in step I was diluted 1: 20 by mixing 50 micro litre genomic DNA with 950 micro litre water. The absorbance of the diluted genomic DNA sample was measured at 260nm and 280nm using UV spectrophotometer and UV cuvette.
Results of Agrose gel electrophoresis:
10 kB Marker 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig I. Output of agrose gel electrophoresis seen under UV light (Left). Lane 11 was the DNA from my buccal cells
Fig 2. Hyper ladder
Results of spectrophotometer:
Absorbance at 260 nm = 0.22
Absorbance at 280 nm = 0.14
Total Volume of extracted DNA sample = 0.2 ml
Dilution = 50 micro litre DNA sample + 950 micro litre distilled water
Thus, dilution factor = 1:20
Absorbance reading of 1:20 diluted sample at 260 nm = 0.22
Concentration of DNA sample = Absorbance x dilution factor x 50 microgram/ml
= 0.22 x 20 x 50
= 220 microgram per ml
Total amount of extracted DNA = concentration x total sample volume
= 220 x 0.2 = 44 micro gram of DNA
Purity of DNA:
Ratio of A260/A280 = 0.22/0.14 = 1.57
Range of purity = 1.57/1.8 to 1.57/2.0
= 0.78 to 0.87
That is, 78% to 87% pure DNA
The genomic DNA sample extracted from own buccal cell was verified, quantified and tested for its purity by agrose gel electrophoresis and UV spectrophotometry. The agrose gel showed different lanes of DNA with different bands. The Agrose Gel electrophoresis output seen under UV light showed that majority of DNA in lane 11 (i.e. my sample) of figure 1 was found to be concentrated on the top region making a thick band at the level of 10 Kb DNA ladder. This indicates that majority of the DNA on my sample was high molecular weight DNA. However some faint bands are present at the lower zone of the lane indicating the presence of trace amount of apoptotic DNA as well.
Fig 3. Nucleosomal structure
In human genome, DNA remains associated with histone protein.During the process of extraction, CAD (Caspase Activated DNAse) cuts the DNA into fragments, forming different bands giving the shape of ladder. In the process high molecular weight DNA remains at top region of the lane and apoptotic DNA gets confined at the bottom. (Herrmann M et al, 1994).
The spectrometric analysis revealed that the DNA sample extracted by myself was found to be 78% to 87% pure. The ratio of purity was 1.57 which is slightly lower than the lower limit of the purity range (1.8 to 2.0). It is very vital to get a good quality of DNA without contamination, to get good results. (Tan SC, and Yiap BC, 2009)The possible source of compromise in purity might be due to protein contamination. Incomplete proteolysis sometimes gives reduced ratio of purity. Another source of error might during mixing after dilution of the sample. The improperly mixed sample gives unstable absorbance value. Spectrophotometer with poor reproducibility might also cause fluctuation in the absorbance value, giving unsatisfactory results. Nevertheless, the purity of the DNA sample extracted by myself was not grossly compromised. However, efforts must be taken to get absolutely pure DNA sample in future days.
In this laboratory session, a volume of 0.2 ml of DNA sample containing total amount 44 micro gram of DNA was extracted from own buccal cells. Thus the concentration of DNA in the extracted sample was found to be 220 micro gram per ml. Agrose gel electrophoresis result revealed that the extracted sample contained majority of high molecular weight DNA and trace amount of apoptotic DNA. Regarding purity, the extracted DNA sample was found to be 78% to 87% pure.