Experiment VIII: DNA Isolation And Identification

EXPERIMENT VIII DNA ISOLATION AND IDENTIFICATION 1. DNA Isolation Technique DNA is the genetic material that codes all the information needed for metabolic processes in every organism. This DNA molecule bound to form chromosomes, and are found in the cell nucleus, mitochondria and chloroplast. The DNA that makes up these chromosomes is a polynucleotide forms a double helix, where the two "threads" are polynucleotides pair up with each other in pairs that complement each other through bonds hydrogen, while between one nucleotide and another nucleotide The chains are connected by phosphodiester bonds. Basically DNA isolation can be done from various sources, for example human organs, blood, leaves, fruit flesh, insects, roots, stems, flesh and scales fish. In the same individual, DNA is obtained from different parts will have the same base sequence and size or length. Meanwhile DNA Animal cells contain more protein. One of the difficulties of DNA isolation of higher plants is a strong and frequent process of cell wall destruction in some types of plants, the contamination is difficult to separate from the extract nucleic acid. The presence of the above contamination can inhibit enzyme activity for example, DNA is insensitive to restriction enzymes and interferes with the process DNA amplification using PCR technique. Likewise the special method for destroying the cell until the contents of the cell can escape from the cell. DNA isolation can be carried out at the macro scale and micro scale. On In this practicum, we will carry out simple macro-scale DNA isolation from plants. The part of the plant chosen is to use plant tissue which contain a small amount of the above contaminants, for example fruit flesh or leaves still young. If fruitcsamples are used, determine the water content of each fruit can affect the results of DNA isolation. In principle, DNA isolation can be carried out in three stages, namely, breaking or lysis of cells so that DNA comes out of cells, purifying DNA from cell extracts, and DNA precipitation. The cell lysis process can be carried out mechanically, chemically and enzymatic. Mechanically it can be done by homogenization with blender or by grinding, while chemically it can be done with the addition of detergent, because detergent can damage the nuclear cell membrane. The DNA purification stage in this practicum is carried out in a simple way namely filtering, while in the final stage DNA precipitation is carried out with How to add cold ethanol/alcohol. So you will get DNA in the form of fine threads that appear in the form of a very soft white mist. Material : - Ripe fruit (mango, papaya, or tomato) - Detergent (breaks down cell walls) - Kitchen salt (NaCl) to separate DNA strands - Cold ethanol: precipitates DNA so that the DNA will appear in the form of fine and white threads. - Filter paper or tissue. Tool : - Knife - Spatula - Mortar - Drop pipette - Glass beaker - Test tube. Ways of working: The first step is to peel the fruit and weigh 5 grams part of the fruit flesh. After that, smooth it with mortar, then move it into a glass beaker. Add 50 mL of distilled water, add salt and detergent 2.5 gr each. Next, stir the ingredients until homogeneous, and leave for 10 minute. Detergent is useful for breaking down cell membranes. Once the material is homogenized, Filter the mixture with filter paper/tissue. Next, take 5 mL of the filtered filtrate, then put it in test tube. Then add 0.6 mL of cold ethanol. Cold ethanol used to precipitate DNA. Observe the changes that occur, pay attention color and lumps formed. 2.DNA ANALYSIS Analysis of the qualitative and quantitative presence of nucleic acid molecules (DNA and RNA) can be done using electrophoresis and engineering techniques spectrophotometry. For electrophoresis, agarose gel electrophoresis was used while spectrophotometry is carried out using UV spectrophotometry wavelength 260 nm. Electrophoresis is a method of chemical analysis that is based on movement of charged molecules in an electric field. Molecular movement in an electric field is influenced by shape, size, charge size and chemical properties of molecules. Separation is carried out based on differences in size and molecular weight and the electrical charge contained by these macro-molecules. According to Stenesh in Titrawani (1996) the electrophoresis technique can can be divided into two ways, namely solution electrophoresis (moving boundary electrophoresis) and area electrophoresis. On technique solution electrophoresis, a buffer solution containing macro-molecules placed in a closed room and electrified. Migration speed of macro-molecules is measured by observing the separation of the molecules (looks like a ribbon) in the solvent. Meanwhile, regional electrophoresis technique is to use a solid material that functions as a supporting medium which contains (is given) a buffer solution. Supporting media commonly used are agarose gel, starch gel, gel polyacrylamide and cellulose polyacetate paper. DNA electrophoresis is a technique to separate DNA samples based on size (molecular weight) or structure the physics of the molecule so that DNA molecules are of different sizes but have the same base composition can be separated. The gel commonly used is Agarose is a polysaccharide extracted from seaweed So this electrophoresis is known as agarose gel electrophoresis. This technique simple, quickly formed, and able to separate DNA fragments according to the size. Agarose gel electrophoresis can separate DNA samples by size several hundred to 20,000 base pairs (pb). Agarose gel is used as NA movement (running) medium. Parameters that can influence the rate of DNA migration are size DNA molecule. Larger molecules will move more slowly, for example DNA Linear DNA will move faster than circular DNA. Besides that, DNA conformation, applied voltage, concentration of agarose and DNA dye used also influences the rate of migration. The working principle of electrophoresis is to separate molecules based on electrical charge. Positive electric charges will attract negative charges. On the contrary Negative charges will attract positive charges and will repel each other the load is the same. The DNA molecule is negatively charged (phosphate sugar skeleton) so in an electric field it will migrate through the gel matrix towards the positive pole (anode). The larger the molecular size, the lower the migration rate. Agarose gel electrophoresis consists of several components, including: DNA samples which are the result of multiplication using PCR techniques, agorasa gel (concentration 0.5 – 2%), EtBr (DNA chelating agent) which will fluoresce when exposed to UV light, loading buffer, running buffer (facilitates delivery current, usually TAE or TBE), marker (DNA marker) which is a series Standard DNA fragments are useful for estimating fragment sizes varies on the DNA template that is electrophoresed, as well as the UV lamp transilluminator which helps in visualization of DNA 3. Agarose Gel Electrophoresis This stage is carried out to determine the DNA that has been isolated as well The quality of the DNA obtained can be seen, the 1% agarose gel used for Electrophoresis was made by dissolving 0.4 grams of agarose in 40 ml of TAE buffer (Tris-acetate 40 mM and Na2EDTA 1mM pH 8) with heating. After agarose dissolved and cooled to approximately 45oC, then agarose gel poured into molds and allowed to solidify. DNA samples to be electrophoresis mixed with 1X loading buffer (6x loading buffer: bromophenol blue 0.25% (w/v) and sucrose 40% (w/v). Electrophoresis is performed in the TAE buffer at a voltage of 70-100 Volts. Electrophoresis is stopped when bromophenol blue had migrated approximately 2/3 of the gel length. Agarose gel then soaked in 250 ug/mL EtBr solution for 3 – 5 minutes, next soaked in TAE buffer for 5 – 10 minutes or distilled water. DNA bands can observed with UV light using a UV Transilluminator. 4. UV-VIS Spectrophotometry The UV spectrophotometric method is used for quantitative DNA testing based on the principle that ultraviolet radiation will be absorbed by nucleotides. The solution resulting from total DNA isolation was dissolved in TE buffer pH 8 with DNA-TE buffer ratio (3 : 7 v/v). Next, pour it into a cuvette, Then the total DNA was analyzed for purity at wavelengths 230, 260 and 280 nm using a UV-VIS spectrophotometer. Absorbance was read at three wavelength to detect protein contamination (280 nm) and contamination of polysaccharides and phenolic compounds and humic acid (230 nm). Purity DNA is determined by the ratio value of absorbance 260 nm/absorbance 280 nm and absorbance 230 nm/absorbance 260 nm. Maximum absorption of UV radiation by DNA occurs at a wavelength of 260 nm. If the abortion obtained is 1,000, then the DNA concentration is equivalent to 50 µg/mL. This value is a conversion factor, so a formula is obtained to calculate it DNA concentration as follows : DNA (µg/mL) = A 260 x 50µg/mL x dilution factor.