Background of the Study:
DNA is the building blog and blueprints for life to exist, all living organisms have DNA. The Separation of DNA is the most needed and vital technique used to study DNA. The separation of DNA from its cells and its sanitization is of key significance in the biotechnology field and forensics. The Separation and purification of DNA are the primary phases in the study and analyzing of DNA that let researchers identify genetic-based disorders, make DNA fingerprints of any persons, and also make genetically engineered organisms that could make favorable products for example antibiotics, hormones, and insulin.
The separation of DNA is a basic procedure in order to collect the DNA for succeeding molecular or for forensic studies. DNA can be collected from different types of cells. The primary step is to break or lyse the cell. This could be achieved by crushing a portion of tissue in the blender. After the breakdown of cells, a salt solution for example NaCl and a solution of detergent that contains the compound SDS (sodium dodecyl sulfate) are mixed in it. These solutions collapse and blend all the fat & proteins that make up the cell membrane. In the final stage, ethanol is mixed in it as DNA is solvable in water. The ethanol lets DNA precipitate, and settled-out from the solution, hence it leaves all the components of the cell that are not solvable in ethanol. The DNA could be wound on a rod and pulled off from the solution at this stage. The Strawberries are very soft and easily pulverize. Strawberries have big genomes and they are octoploids which means strawberries have eight chromosomes in all of its cells. Hence, strawberries are a special fruit that is used widely in labs for DNA extraction. The soap assists in dissolving the phospholipid bilayers of the organelles and cell membrane. The main purpose of using salt is to break up all the protein chains that are bounded all over the nucleic acids. DNA cannot be dissolved in alcohol (ethanol). The colder alcohol is, the less solvable the DNA would be. Therefore make it sure that ethanol is in an ice state or cold (Christou, A., Georgiadou, E. C., 2014).
Method of Extracting DNA of Strawberry Using Salting Out Method
- Kiwi and Strawberry fruit tissues
- 5M NaCl
- Proteinase K
- TNES buffer solution
- Auto pipette
- Loading dye
- Micro-centrifuge tubes
- Ethanol 100%
- Razor blade
- Ethanol 70%
- Agarose Gel
In the first, fruit samples are labeled with K and S for Kiwi and Strawberry. These samples are already prepared in such a manner that fruit tissues were chopped with the help of razor blades and then they have placed in 1.5ML micro-centrifuge tubes. Now in order to break down the proteins of the tissues, 10 microliters of proteinase are added to the tube and well slightly. Then 600 microliter of TNES buffer is mixed in the tube to attain a stable pH which is vital for the survival of the tissues. Moreover, the buffer solution also helps the cell lysis. After that, the centrifuge tube is incubated at around 37oC for one night (Sreenivasaprasad, S., Brown, A. E., & Mills, P. R., 1992).
In the centrifuge tube sample, 170 microliters of NaCl are added and shake for around 10 seconds to get mixed. The main purpose of adding NaCl is to precipitate all the proteins from that of the sample. After that, the centrifuge tube is centrifuged for around 5 minutes at 12,000 revolutions per minute, and then separate the precipitated proteins from the solution in the centrifuge tube.
The other sterilized microcentrifuge tubes are labeled and then added 300 microliters of supernatant. The supernatant liquid contains DNA material. 300 microliter of 100% ethanol is then auto-pipetted and mixed with the supernatant in a new microcentrifuge tube. The tube is then gently inverted three to four-time so that its solution get mixed. The main purpose of adding 100 ethanol (alcohol) is to precipitate the DNA. After that, the solution is incubated for around ten minutes at room temperature. After the process of incubation, the tubes are centrifuged for around five minutes at 12,000 revolutions per minute so that the precipitated DNA can be settled at the bottom of the tube (Degani, C., Rowland, L. J., Levi, 1998).
After that, the supernatant is removed very carefully with the help of an auto pipette. All the remaining traces of ethanol were drained by putting the tube upside down. Then the remaining DNA pellets were washed in 300 microliter of cold 70% ethanol which removes all the lingering protein components. Afterward, the tube is micro-centrifuged for around 5 minutes at 12,000 revolutions per minute in order to ensure that the DNA is completely pilled. The 70% ethanol is then removed with the help of a micropipette (Congiu, L., 2000). The centrifuge tube is then opened and heated on a heated block at 30C for around 20 minutes to make sure that all the ethanol is evaporated. The traces of ethanol could prevent the dissolution of DNA so it is very important that ethanol is completely evaporated (Mercado, J. A., El Mansouri, I., Jiménez-Bermúdez, S., Pliego-Alfaro, F., & Quesada, M. A.,1999).
The DNA present in the tube is then dissolved by adding 20 microliters of water in the tube and mixing it gently. The in a clean tube, two microliters of loading dye are added in 10 microliters of sample solution. This solution is then loaded on the agarose gel. The gel will run and generate the image as shown in figure 1.
By adding 170 milliliters of 5M solution of NaCl in the sample, all the protein in the solution precipitates. The solution becomes murky. After the process of centrifugation, two-layer are formed. In the bottom layer, the solid protein particle is gathered whereas the supernatant contains dissolved DNA. Now by adding 100% ethanol to the supernatant, precipitates are formed. The addition of 100% ethanol makes the DNA precipitate and makes it very easy to separate it from other components of the cell. The DNA pellets are then compared with the YouTube method. The DNA pellet that is obtained by using the salt out method is found to be large, opaque, and white. The salt out method is more effective than any other DNA extraction method. Figure 1 is showing the results of agarose gel and figure 2 is the results obtained by the YouTube method.
In the process of agarose Gel electrophoresis, there is an inverse proportionality in between the distance between the gel and base pairs in DNA. The DNA that has a short strand moves more distance through the gel as compared to that of short strand DNA. YouTube method produces long strands of DNA whereas the salting-out method produces short strands of DNA. The DNA Extracted from salting-out method has wide applications in laboratory and forensic studies.
The salting-out method has its own limitations, the results are only applicable when the conditions and reagents are maintained. Any slight variation in the experimental conditions could cause huge differences in the result. Moreover, it is quite tricky and difficult to amplify the DNA by using the salt out method.
Discussion of Results
There are two main aim of this experiment.
- To extract the DNA from strawberry fruit
- Compare the Salt Out method with the YouTube Method.
In my experiment, both of the aims are achieved, a sufficient quantity of DNA is extracted from strawberry and kiwi fruits and demonstrated the validity of salting-out method. The results from the YouTube method is also analyzed.
DNA Extraction methods are one of the major discovery in the scientific method. DNA extraction has so many application in the field of treatment and diagnostics of diseases, forensic studies and genetic engineering. There are number of methods discovered to extract DNA as disused in this report. Scientists are searching for more easy and cost effective ways of DNA extraction. Many scientist and researchers consider salting out method and as straightforward and easy method however the amplification of DNA is pretty hard and difficult. It makes shorter DNA strands as compare to other method. There is a need to discover an easy and reliable method for DNA Amplification and minimizing afore highlighted limitations in it.
Christou, A., Georgiadou, E. C., Filippou, P., Manganaris, G. A., & Fotopoulos, V. (2014). Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops. Gene, 537(1), 169-173.
Sreenivasaprasad, S., Brown, A. E., & Mills, P. R. (1992). DNA sequence variation and interrelationships among Colletotrichum species causing strawberry anthracnose. Physiological and Molecular Plant Pathology, 41(4), 265-281.
Mercado, J. A., El Mansouri, I., Jiménez-Bermúdez, S., Pliego-Alfaro, F., & Quesada, M. A. (1999). A convenient protocol for extraction and purification of DNA from Fragaria. In Vitro Cellular & Developmental Biology-Plant, 35(2), 152-153.
Congiu, L., Chicca, M., Cella, R., Rossi, R., & Bernacchia, G. (2000). The use of random amplified polymorphic DNA (RAPD) markers to identify strawberry varieties: a forensic application. Molecular Ecology, 9(2), 229-232.
Degani, C., Rowland, L. J., Levi, A., Hortynski, J. A., & Galletta, G. J. (1998). DNA fingerprinting of strawberry (Fragaria× ananassa) cultivars using randomly amplified polymorphic DNA (RAPD) markers. Euphytica, 102(2), 247-253.