Jeremy Joe Magauay Biology 120 S-5L Genomic DNA Isolation and Polymerase Chain Reaction Cell and Molecular Biology aims to better understand the biological processes happening at the molecular level using concepts and techniques in Chemistry and Biology. By doing so, information about the different series of interactions among different biological molecules inside a cell can be generated (www.scripps.edu). DNA isolation and analysis techniques are, therefore, essential in studying these cellular processes as important biological molecules are encoded in the DNA. To date, different DNA isolation techniques are being used by researchers and new ones are also being developed for better DNA extraction and preservation. In studies involving DNA, the extracted sample must be of high purity, yield, and concentration. However, some studies only require a part or a series of sequences in the whole genome when studying certain biological pathways. To amplify these target sequences or gene fragments for further analyses, Polymerase Chain Reaction [PCR] can be employed. PCR is based on the concept of DNA denaturation and renaturation, and on the binding specificity of nucleotides. Since different tissue samples also require certain steps for successful DNA extraction, specific DNA isolation protocols can be used. In this experiment, the CTAB method was used to isolate the genomic DNA from Papaya [Carica papaya] leaf. A gram of papaya leaf tissue was first pulverized in 3 mL of 2x CTAB buffer in a pre-cooled mortar and pestle. The CTAB buffer was added to aid the lysis of the cells. EDTA was added in the buffer to inhibit the activity of DNase enzymes that degrade the DNA (www.nbpgr.ernet.in). Polyvinylpyrrolidone, another component of the isolation buffer, eliminates polyphenols from the extract by precipitating these compounds out of the solution (www.biotecharticles.com). A volume of 1 mL of the liquid part of the homogenate was then transferred into the microcentrifuge tubes. The tubes were later incubated and spun at 10,000 rpm for 15 minutes. The supernatant was collected and added with an equal volume of chloroform:isoamyl alcohol to separate the DNA dissolved in the aqueous layer from other organic compounds dissolved in the organic layer (www.nbpgr.ernet.in) and was centrifuged again at 10,000 rpm for 15 minutes. When an interface formed, the aqueous phase was transferred to another microcentrifuge tube and an equal volume of ice-cold propanol was added to precipitate the nucleic acids. The tubes were spun at 13,000 rpm for 15 minutes. The supernatant was discarded and a volume of 1 mL of ice-cold 70% ethanol was added to wash the pellet. DNA is known to be insoluble in ethanol so it will just precipitate out of the solution while salts and other contaminants soluble in ethanol will remain dissolved
(info.gbiosciences.com). The tubes were centrifuged at 13,000 rpm again for 2 minutes, twice. The washing solution was then poured out and the pellets were allowed to dry. The use of fresh sterile tips and tubes was needed in every step so as to prevent the contamination of the DNA extract. To assess the purity of the DNA sample, spectrophotometry was employed. A volume of 5 microliters of DNA sample was diluted first with distilled water to a final volume of 1000 microliters. The absorbance of the diluted sample was measured at 260 nm and at 280 nm. The absorbance ratio was then determined by dividing the reading at 260 nm with the reading at 280 nm. This was done to compare the concentration of nucleic acids and proteins; nucleic acids have a higher absorbance at 260nm while proteins have a higher absorbance at 280 nm (www.biotek.com). At a range of 1.8 - 2.0, the diluted sample is expected to be of high purity. Polymerase Chain Reaction [PCR] was then done to amplify the extracted DNA. A reaction mix or PCR cocktail is needed for the amplification of the gene of interest. A buffer is added to keep the DNA polymerase’s optimal activity (www.babec.org). 2'deoxynucleoside 5'-triphosphates or dNTP’s were added to supply the needed nucleotides for DNA elongation. A sense primer and an antisense primer were also added to the PCR cocktail. These primers will be recognized by the DNA polymerase to initiate the DNA elongation. The DNA polymerase commonly used in PCR is the Taq polymerase from the Thermophilus aquaticus bacterium because it is able to withstand the high temperatures (bioinfo.bact.wisc.edu). Thus, ensuring that DNA elongation would still be completed in PCR cycles. The genomic DNA was then added to serve as the template for amplification. Sterile nanopure water was a component of the PCR mix to dilute the concentrated salts added (www.babec.org). The diluent must be sterile to prevent contamination of the solution. The first step in PCR is the denaturation of the double stranded DNA [predenaturation of the dsDNA at 94C for 3 minutes can also be done prior to this step] to unwind the dsDNA. For 1 minute, the temperature was set to 94C for the complementary DNA strands to separate. Then the annealing of the sense and antisense primers to the single-stranded DNA templates was carried out at 59C. These primers anneal to complementary sequences of the DNA templates that flank the region to be amplified (www.babec.org). The extension of the newly synthesized DNA by the Taq DNA polymerase takes place at 72C. The Taq polymerase extends the primers by adding nucleotides complementary to the DNA template. The last step in PCR would be the final extension of the newly synthesized DNA strands at 72C for 10 minutes. This step allows the complete replication of the nascent DNA (www.neb.com). DNA electrophoresis was also done by loading DNA samples in agarose gel wells and subjecting the gel in an electric field. Since DNA is a negatively charged molecule, it will be pulled in the direction of the cathode (biotechlearn.org.nz). Base on the bands on the gel, one can infer the sizes of the DNA fragments amplified by PCR.
Samples A, B, C, D, F, and G have the same banding patterns, suggesting that they have the same lengths of DNA fragments. Sample E, on the other hand, showed a faint band signifying that the sample loaded contains less amounts of DNA fragments (www.bio.davidson.edu). To check for the purity of the isolated DNA, samples were subjected to spectrophotometry. The absorbance readings of the samples fell to the ideal 260/280 absorbance ratio values. Therefore, the isolated DNA was of high purity and concentration. In doing PCR, a negative control is needed so as to make sure that the PCR cocktail used for PCR is not contaminated with the DNA template (www.qiagen.com). Comparing the agarose gel prepared by the class with that of the hypothetical PCR gel, the class’ agarose gel contained bigger fragments. Also seen in the hypothetical gel are bands from the patients’ DNA that resemble that of the Dengue serotypes. The virus might have already hijacked numerous cells as the virus’ genetic material is already integrated into the patient’s genome, leading to the production of more viruses (www.nature.com). PCR is important to be done on samples from the patients so that right treatments specific for targeting the virus with the certain strain can be prescribed to them.
