Agarose Gel Electrophoresis
Because of its high strength, ability to withstand low percentage, and ability to separate big Deoxyribonucleic Acid (DNA) fragments, Agarose gel electrophoresis has been proven to be an effective and efficient method of isolating nucleic acids. The concept of DNA separation via the gel matrix is determined by the agarose gel's pore size; a higher agarose concentration results in smaller pores (Ylmaz, Cem Ozic, and Elhami, 34). Traditional agarose gel electrophoresis separates DNA fragments between 100 base pairs and 25 kb, whereas pulse field gel electrophoresis uses two distinct alternating current applications to separate DNA bigger than 25 kb. The pulse field gel electrophoresis separates the DNA fragments through the speed at which the fragments of DNA orient to the change in current direction.
Isolating Pure DNA
The DNA isolated from the DNA extraction is "Impure" as it contains the unwanted molecules of lipid, proteins, minerals, and sugars. The unwanted fragments can be eliminated to obtain the pure DNA through various methods and agents. The proteins are removed with the help of protease enzyme that breaks down proteins present. The detergents dissolve the lipids and sugars are broken down by the enzymes such as beta-galactosidase. The processes of digesting and eliminating the unwanted molecules can be sped up through heating and agitation.
Migrating of Charged Molecules
The charged molecules migrate through the matrix of an electric field of the gel electrophoresis medium, where the DNA molecules have negative charge along with their length due to the presence of phosphate part and therefore, move to the positive electrode. The DNA molecule is negatively charged due to the phosphate backbone present which makes the DNA soluble. Also, chloride ions in the solution of sodium chloride are negatively charged moves together with DNA to the positive electrode. The sodium ions are positively charged and therefore will migrate to the negative electrode, its addition neutralizes the negative charge of DNA. The addition of sodium chloride to the harvested cheek cells helps the DNA to come together instead of repelling each other.
Separating DNA Fragments
The Agarose gel separates the DNA molecules conferring to their size during their isolation process where the agarose gel pores allow short DNA fragments to move through quickly but slow down the longer DNA molecules. The DNA fragment which is shorter runs through the pores of the agarose gel easily as compared to the longer DNA fragments. The rate of DNA migration in the agarose is also determined by factors such as the agarose concentration, structure of DNA, the applied voltage, the existence of ethidium bromide to aid visualization, the agarose type used together with the choice of electrophoresis buffer. The small sized DNA moves to the furthest from the base of the Agarose gel contrary to the large molecules that are found near the Base due to the restriction of penetrating through the pore sizes.
Molecules in Cheek Cells
The DNA obtained from the cheek calls contains other cellular molecules such as fats, sugars, proteins and mineral salts which dissolve in the lysis buffer. The iodine test is used to check the presence of sugar by changing from yellowish –brown to black or dark purple. Benedict solution can also be used to test for sugars upon heating, the solution changes to brick red or orange. The proteins can be verified by biuret test where the liquid changes from blue to purple. The fats or lipids can be checked through grease spot test or Sudan red test. For the grease spot, the fats leave a translucent and the Sudan Red test; fats stain red showing the location or places of fats.
Works Cited
Yılmaz, Muhittin, Cem Ozic, and İlhami Gok. “Principles of nucleic acid separation by agarose gel electrophoresis.” Gel Electrophoresis-Principles and Basics. InTech, 2012.
