BIOL2151 Genetics

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BIOL2151 Genetics

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BIOL2151 Genetics

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Course Code: BIOL2151
University: Australian National University

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Country: Australia

Question:
Describe the Genetics For Deoxyribonucleic Acid Extraction. 
Answer:
Introduction
Deoxyribonucleic acid (DNA) extraction or isolation is process through which DNA is segregated or separated from the membranes, proteins and other cellular contents of a cell. The DNA extraction is one of the complex and labor intensive steps of analysis of DNA. The DNA extraction employs methods that can take overnight incubation, or the extraction procedure can be completed within hours and minutes if the right reagents are used in the extraction process to quicken the process. The extraction of the DNA requires careful and cautious handling of the sample to reduce and minimize crossover and contamination (Yuan et al., 2012). This report deals with the various types, methods and materials employed in the extraction of DNA from biological samples.
The extraction DNA from a biological sample involves three basic steps (figure 1)-
Break open or Lyse of the cells
 
Separation of DNA from other cellular components
 
Isolation of DNA
Materials and Methods
Lyse of cells- For the purpose of extraction of DNA from a biological sample, the cell must be lysed. The cellular components of a living cell consist of plasma membrane, cell membrane, cytoplasm, nucleus, endoplasmic reticulum, ribosomes, lysosomes, mitochondria, vacuoles, plastids (plant cell), cell wall (plant cell). However, the genetic material is only contained in the nucleus and to effectively separate the nuclear material from the other components involve certain methods and materials (Claassen et al., 2013). The different methods of cell lysis include (figure 2)
Physical methods- the 4 main physical methods are manual grinding, sonication, freeze thaw method and liquid homogenization.
 
Chemical methods- In the chemical method the detergents that are used include Triton X-100, Tween 80, Tween 20, Sodium Dodecyl Sulfate (SDS), CHAPS
 
Enzymatic methods- enzymatic methods are easy to perform. However, the cost incurred by employing these methods are very high. Lysozymes are used for the lysis of bacterial cells and Chitinase is used in the lysis of Yeast cells. Pectinase is used in the lysis of plant cells for the extraction of genetic material.
The traditional methods have some inherent disadvantages. Thus, there are cell lysis buffers, cell disruptors and enzymes that are commercially available like the Qsonica Ultrasonicator, Cell Lytic- extraction reagent by Sigma Aldrich, Glo-lysis buffer by Promega. 
Separation of DNA from other cellular components- After the lysis of the cell, the solution contains not only the genetic material but also the other unwanted constituents like reagents, salts, proteins, detergents. Thus, a variety of steps can be employed to remove the various contaminants (Innis et al., 2012), which will leave the DNA in a pure form (figure 3). The various common steps that are used for this procedure include the following-
Ethanol precipitation- this precipitation process is commonly done using isopropanol or ice cold ethanol. DNA is insoluble in alcohol solution and ethanol acts a s an anti-solvent, thus it aggregates together when the solution is centrifuged. DNA precipitation can be increased by elevating the ionic strength, by adding sodium acetate. Due to the presence of monovalent cations like Na+, ethanol acts to precipitate the nucleic acids. The precipitation of the nucleic acid form can be collected after a centrifugation.
Chloroform-phenol extraction- this procedure is employed to denature the protein preset in the sample. After centrifuging the sample, denatured and broken protein residues remain in the sample in the organic phase. Thus, in the aqueous solution that contains the nucleic material is mixed with chloroform to remove the phenol residues which was previously used to denature the proteins (Rubin et al., 2014).
Minicolumn purification- the main fact behind this step is that nucleic acids often adsorb to the silica, which depends on the salt concentration in the buffer solution and the pH of the solution.
Isolation of DNA- After the effective separation of the nuclear material from the other contaminants. The prime and vital variables of a pure DNA, are concentration, molecular weight and the rate at which the nuclear material gets precipitated. DNA concentration of 20ng/ml is considered to the lowest threshold of getting a DNA precipitate, which will be viable to recover. The most common and frequent method employed in the final concentration of the DNA is the ethanol precipitation. Specifically, two volumes of ice cold ethanol are used to precipitate the nuclear material (Min et al., 2014). At a temperature of around -20 degree Celsius or less, in the presence of salt, absolute ethanol effectively precipitates the chains of nucleic acids. However, DNA molecules that are shorter than 200 base pair cannot be efficiently precipitated by ethanol. Thus, the optimum levels for pellet formation depend on the concentration of the DNA (figure 4). The various centrifugation steps include centrifugation at a rate of 1200 rpm for 15 minutes to minimize and reduce the levels of DNA loss from samples that only a few micrograms of sample. Isopropanol solution of 1 volume can be used in place of 2 volumes of ethanol for DNA precipitation. The step is advantageous as less volume of liquid used for centrifugation. However, isopropanol is more volatile in comparison to ethanol. Even isopropanol is difficult to remove from the remaining traces of the DNA molecules. Another disadvantage is that certain solutes like sodium chloride also precipitate along with the other DNA molecules when isopropanol is used (Sam et al., 2012).
Determination of DNA concentration and purity of DNA samples- DNA molecules obtained after the concentration of DNA can be further assessed to verify the concentration and purity of the sample. The various methods include are absorbance methods by measuring the optical density of the sample. Agarose gel electrophoresis, and usage of fluorescent DNA- binding dyes (Yuan et al., 2012).
Conclusion
Therefore, from the above report it can be concluded that for the effective extraction of the DNA molecules from the biological samples (plant or animal) requires lysis of the cellular sample, separation of the DNA molecules and finally extraction of the DNA material. There are both the conventional and modern day techniques that enhance the extraction of the DNA material. However, they are costly equipment and requires following of special handling procedures. The successful extraction of the DNA molecule also employs complex procedures and methods for the quantification and estimation of the DNA material. 
Reference
Adams, M. D., Fields, C., & Venter, J. C. (Eds.). (2012). Automated DNA sequencing and analysis. Elsevier.
Bloom, B. R., & Glade, P. R. (Eds.). (2014). In vitro methods in cell-mediated immunity. Academic Press.
Claassen, S., du Toit, E., Kaba, M., Moodley, C., Zar, H. J., & Nicol, M. P. (2013). A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples. Journal of microbiological methods, 94(2), 103-110.
Innis, M. A., Gelfand, D. H., Sninsky, J. J., & White, T. J. (Eds.). (2012). PCR protocols: a guide to methods and applications. Academic press.
Karp, A., Ingram, D. S., & Isaac, P. G. (Eds.). (2012). Molecular tools for screening biodiversity: plants and animals. Springer Science & Business Media.
Min, J. H., Woo, M. K., Yoon, H. Y., Jang, J. W., Wu, J. H., Lim, C. S., & Kim, Y. K. (2014). Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid. Analytical biochemistry, 447, 114-118.
Rubin, B. E., Sanders, J. G., Hampton?Marcell, J., Owens, S. M., Gilbert, J. A., & Moreau, C. S. (2014). DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure. Microbiologyopen, 3(6), 910-921.
Sam, S. S., Lebel, K. A., Bissaillon, C. L., Tafe, L. J., Tsongalis, G. J., & Lefferts, J. A. (2012). Automation of genomic DNA isolation from formalin-fixed, paraffin-embedded tissues. Pathology-Research and Practice, 208(12), 705-707.
Westermeier, R. (2016). Electrophoresis in practice: a guide to methods and applications of DNA and protein separations. John Wiley & Sons.
Yuan, S., Cohen, D. B., Ravel, J., Abdo, Z., & Forney, L. J. (2012). Evaluation of methods for the extraction and purification of DNA from the human microbiome. PloS one, 7(3), e33865.

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