RANELLE JANINE L. ASI BIO 120 S-5L EXERCISE 9. Genomic DNA Isolation & Exercise 10. Polymerase Chain Reaction (PCR) Cell and molecular biology is a science based on the various systems of a cell resulting to its regulation, maintenance and function. Many of these systems involve genetic information hence the study of the DNA is an essential part of this field. To able to analyze DNA, it must first be isolated and purified from its natural environment filled with biological molecules and compounds that cause physical or chemical interference in an experimental set-up. Several protocols have been established to efficiently extract DNA, one of which, the CTAB method, was performed and studied in this exercise. (De la Parte and Dita, 2014) As in all biological studies, the analysis of DNA requires samples in high enough amounts. A method called Polymerase Chain Reaction (PCR), which is significantly dependent on the size distribution and quality of the extraction of DNA, has been developed to produce exponential copies of —or simply, to ‘amplify’—DNA. PCR follows basic elements of natural DNA synthesis and replication processes and uses them in a more simplified manner easily duplicated in the laboratory such as denaturation by heating and exposure of DNA to polymerases in a reaction mix. The introduction of PCR has revolutionized and accelerated the field of molecular biology and has expanded the applications of DNA analysis to various biological and medical procedures (McPherson and Moller, 2006). In the CTAB method for DNA extraction, the buffer used to suspend the DNA contains the following: (1) hexadecyl trimethyl ammonium bromide (CTAB, from which the method is named after), a detergent which disrupts fluid membranes, (2) EDTA, a chelating agent that acts on magnesium ions required for DNase activity, (3) Beta-mercaptoethanol, which helps in denaturing proteins by breaking disulfide bonds and removing tannins and polyphenols present in the crude extract, (4) PVP, which removes phenolic compounds in plant DNA extracts, and (5) Tris-HCl at pH 8 and NaCl, which aids in precipitation by neutralizing the negative charges on the DNA. (Gaikwad, n. d.) In this experminent, PVP and Beta-mercaptoethanol was not used as the chemicals are too dangerous for undergraduate students to handle. A classic method for DNA purification is phenol-chloroform extraction in which the nucleic acid solution is extracted by successively washing with a volume of chloroform:isoamyl alcohol. The phenol removes protein contaminants by denaturing them as they accumulate in the organic phase during intermittent centrifugation. DNA is non-soluble in alcohol and is hence precipitated upon addition of 100% isopropanol or ethanol. The DNA solution is finally washed using 70% of the alcohol used to remove salt and alcohol remnants. (Gaikwad, n. d.) The quality of extracted DNA can be enhanced by following some additional steps: grinding of leaf samples in liquid nitrogen to lyophilise them as dry tissue can be efficiently disrupted while DNA is unhydrated, taking care to only collect the aqueous phase of the solution after addition of chloroform:isoamyl as it contains all the DNA and the less noticable interphase underneath it contains contaminants, and the use of fresh sterile tips and tubes for every steps to minimize re-entry of previous contaminants in the solution. (Zidani et. al, 2005) In the PCR method, a reaction mix is prepared using the following reactants: (1) PCR buffer containing the dipolar Tris-HCl which optimizes the pH in the mix for the action of DNA polymerases, KCl, which assists in primer-template annealing and most importantly, MgCl 2, which affects the specificity and efficiency of the reaction as it is a cofactor required for the action of Taq DNA polymerase, (2) dNTP mix, a stock solution containing equimolar concentrations of the four types of dNTPs to be used as the building blocks of new DNA strands to be primed by the polymerases, (3) sense and antisense primers, which are complementary to the ends of the two strands of the DNA target to signal where DNA priming should begin, (4) Taq polymerase, a thermostable DNA polymerase
able to withstand high temperatures that the mix will be subjected to during the main steps, and (5) genomic DNA from which the target DNA fragment to amplified will be replicated from (McPherson & Moller, 2006). Sterile nanopure water was used as a blank for comparison with the reactions happening in the tube containing genomic DNA. PCR proceeds in three main steps: denaturation, annealing and extension or synthesis. Denaturation is carried on by heating the double-stranded DNA at 94°C to separate the complementary strands that will serve as template in further cyclings. Pre-denaturation is sometimes done at the same temperature to ensure complete separation of strands. Annealing then occurs upon rapid cooling of the solution, allowing oligonucleotide primers to hybridize to the template. In this phase, however, the single strands of the template are too long and complex to be able to completely reanneal spontaneously. The gene fragment to be amplified will completely form double-stranded fragments upon further cycling of this step and the extension step. The extension step involves heating of the reannealed DNA to 72°C, the temperature at which the thermostable DNA polymerase in the mix will operate most efficiently in synthesizing new DNA strands.
Gel Electrophoresis exposes the molecular sizes of different DNA fragments as the lightest or shortest fragments travel fastest down the gel and the heaviest or largest fragments travel most slowly and are left near the top part of the gel. In this run, samples A-F show almost identical bands, indicating that all six samples are the same DNA. Two bands are found in each well which implies that each sample has two differently sized DNA fragments. The higher bands are most likely genomic DNA and the lower, larger bands are DNA in similarly sized fragments in higher concentrations. (Magdeldin, 2012)
The result of spectrophotometry using BioTek Gen5 indicates raw absorbance values, meaning they were read without subtracting the blank values, absorbance corrected to 1 cm pathlength and subtracted with blank values, the 260/280 ratio used to identify the type of sample, and calculated DNA concentration to quantify the amount of the DNA present in the sample. (Brescia & Banks, 2012) Nucleic acids absorb maximally at 260 nm, the report indicated values on the 260 column to be higher than readings at other wavelengths, hence initially confirming the sample as nucleic acid.
According to Brescia and Banks (2012), the 260/280 ratio for pure DNA is 1.8 to which all of the values in the column are near, further confirming the identities of all the samples in the wells to be nucleic acids. Elevated ratios usually indicate presence of RNA, while ratios below 1.8 signal presence of contaminating protein or phenol; hence most of the samples were not pure. Concentrations of dsDNA are computed by multiplying the 260 value by the sample’s dilution factor then by dsDNA’s correction factor, 50. The results show high concentration of DNA in the samples as dilution was kept to a minimum during preparation.
In this sample run, the band in the second lane indicates that it is longer than 1,500 kbp as it is genomic DNA that was not fragmented. However, the DNA from the third to the seventh lane travelled down the gel as they were smaller due to fragmentation during amplification. The bands on the five lanes also travelled different distances indicating that fragment lengths vary as the DNA polymerase replicated (and eventually fragmented) the DNA strands at different loci where the primer attached on each species’ genome. The site where the primer attached for Species A was around 700 kb from the tip of the segment, and hence resulted to a fragment that is 700kbp long. Species B’s fragment size was about 550 kbp, Species C and D’s are the same at around 300 kbp (hence, they have same gene loci), and Species E’s was about 250 kbp. Negative controls in gel electrophoresis serve the purpose of indicating the presence of contaminants in the reaction mix used in the PCR or in the gel itself. In this run, there is no indication of any contaminants in the samples.
REFERENCES: De la Parte, E. M. & Dita, M. (2014) Basic Aspects of Molecular Biology and DNA Extraction [Lecture slides]. Retrieved from http://www.fao.org/fileadmin/templates/agphome/documents/Pests_Pesticides/caribbeantr4/10Molecula rBiology.pdf Gaikwad, A. B. (n. d.) DNA extraction: Comparison of Methodologies. Retrieved from http://www.nbprgr.ernet.in McPherson, M. J. & Moller, G. S. (2006) PCR (2nd ed.) New York, NY: Taylor & Frnacis Group Schleif, R. (1993) Genetics and Molecular Biology (2nd ed) London: The Johns Hopkins University Press.
Zidani, S., Ferchichi, A. and Chaieb, M. (2005) Genomic DNA extraction method from pearl millet (Pennisetum glaucum) leaves. African Journal of Biotechnology Vol. 4. Retrieved from http://www.academicjournals.org/AJB Brescia, P. J. & Banks, P. (2012). BioTek Tech Note: DNA Quantification using Gen5TM. Winooski, VT: BioTek Instruments, Inc. Magdeldin, S. (ed.) (2012) Gel Electrophoresis: Principles and Basics. Rejika, Croatia: InTech Books and Journals.
