Extraction of DNA from Cheek Cells - Research Paper Example

Extraction of DNA from Cheek Cells Name: Institution: Date: Abstract A number of basic procedures are carried out when extracting and purifying DNA from a living cell. Basically, the extraction protocols of DNA form cheek cells involve two parts; a technique for cell lysis, and an enzymatic method to get rid contaminating proteins, macromolecules or RNA. In this experiment, the cell was broken to expose the DNA molecules by blending. The protein and fat in the cell membrane was then emulsified by addition of detergent and salt solutions. The detergent is the lysis buffer that helps to break and expose the cells. The pineapple juice was used as protease for removing protein histones, and the salt was used to remove charge from DNA strands and making DNA clumps. To separate the DNA molecules from the liquid to obtain purified DNA for various applications, chilled alcohol was added to the mixture. From the results of the experiment, it was found that the concentration of DNA was dependent on the volume of saliva. Introduction DeoxyriboNucleic Acid (DNA) is a long and stringy molecule that usually resides in cells, tissues and viruses or any other biological material. The molecule contains the genetic information that is passed from parent to offspring (Butler, 2009). Extraction of DNA is simply the technique or process of removing DNA from a biological sample of cells or viruses using both chemical and physical methods. In 1869, Friedrich Miescher performed the first DNA isolation that remains a routine practice in forensic analysis or molecular biology. Until recently, most techniques for DNA isolation remained complex. Today, it is much easier to extract DNA with the availability of commercial kits and also emergence of automated processes (Carracedo, 2005). Extraction of DNA is a very crucial process in biotechnology. Isolation of DNA is the start point for a number of applications; from routine diagnostic to therapeutic decision making and fundamental research (The Royal Society of New Zealand, 1990). Extraction and purification of DNA are also important in determination of its unique characteristics such as size, shape as well as function (Keer & Birch, 2008). The isolation of DNA is primarily important for the study of genetic causes of various diseases, development of diagnostics and invention of drugs (Roth, 2007). Carrying out forensic science, detection of viruses and bacteria has its basis on DNA extraction. Before a DNA extract can be applied in subsequent experiments, the quality of the extract need to be assessed. Tests such as PCR require good quality of DNA for better performance (Elkins, 2012). Aims i. The first objective of this experiment was to carry out a study on the process and methods of extracting DNA from human cheek cells. ii. The second objective was to determine if equal volumes of saliva have the same concentrations of DNA. Materials and Method Materials 3 glasses, 50Ml, for extraction of DNA 6 cups, 30 mL capacity, for storing saliva and alcohol 30Ml saliva 7.5Ml detergent 15ML pineapple juice 6.40 g of salt 90 ML of alcohol A refrigerator for chilling the alcohol 6 sticks for mixing the solution and collecting clumps of DNA Method Preparation of Materials and apparatus: The alcohol was placed inside the refrigerator and chilled until it could condense the DNA. The saliva was then collected in the cups by spitting. After preparation of all materials and apparatus, 10ml of saliva was poured into a glass. In the next step, 5ml of pineapple juice, 2.5ml of detergent and 2.13 g of salt were added to the saliva in the glass. The mixture was then thoroughly mixed by stirring using a stick for about one minute. 30ml of chilled alcohol was then added into the glass in such a way that it flew down by the glass side, until a layer of the mixture was formed on the surface of the solution. The solution was then left to condense and precipitated by alcohol for about 5 minutes. Using a stick, the DNA clumps were removed on the alcohol layer. The steps above were repeated 3 times to obtain 3 samples of DNA clumps. Results Blank Data Sample Table Concentration of DNA4.75mL ( 10% ) Trial 1 Trial 2 Trial 3 Data Collection and Data Processing Raw Data Collection Concentration of DNA in 10 mL of saliva compared to 47.5 mL of the mixture solution: Concentration of DNA4.75mL ( 10% ) Trial 1 19 mL (40% of mixture solution) Trial 2 23.75 mL (50% of mixture solution) Trial 3 23.75 23.75 mL (50% of mixture solution) Processed Data: Concentration of DNA in 10 mL of saliva compared to 47.5 mL of the mixture solution: Concentration of DNA 10% Trial 1 19 mL (40% of mixture solution) Trial 2 23.75 mL (50% of mixture solution) Trial 3 23.75 mL (50% of mixture solution) Total 66.5 mL (140%) Average 22.17 (46.67%) Standard deviation 2.74 Calculations: Conversion of DNA Concentration into volume: Graph 1: DNA concentration in 3 trials Graph 2: DNA volume in 3 trials Discussion The salt ions interfere with the hydrogen bonds formed between the DNA molecules and water. The DNA molecules are then precipitated in a subsequent sequence with the addition of alcohol. In the presence of salt ions, alcohol induces a change in the structure of the DNA molecules (Rudin & Inman, 2001). This causes the molecules to aggregate and form precipitations from the solution. The salt is used as an extraction buffer to break the protein chains around the molecules of the nucleic acids and the detergent dissolves the fats in the cheek cells and cell wall (Mozayani & Noziglia, 2007). The buffer provides the much needed access to DNA from where they are located in the cells. The quantity of cheek cells to be removed from the mouth decreases as the collection of the cells continues and regenerates after some time (Rice, 2012). The cells can only be collected by spitting saliva, which should be done several times with a possibility of getting varied results (Bailey, 2010). From both graph 1&2, it is shown that the concentration and volume of DNA varies, indicating the effect of other parameters. The parameters that may affect the results of DNA extraction include temperature, the mixing power and the quantity of cheek cells in a given sample of saliva (Mozayani & Noziglia, 2007). The number of cells collected in each spit differ, thus, causing different amounts of DNA molecules in each quantity of saliva used in the experiment. The difference in the DNA concentration data clearly illustrates this difference (Tan & Yiap, 2009). Sources of Errors The experiment had numerous sources of errors and limitations. The first source of error was failure to maintain a constant temperature throughout the experiment. The temperature affects the rate at which protein histones and enzyme protease react. It also affects the precipitation as well as the condensation process when alcohol is added to the mixture (Nedel, André, Oliveira, Tarquinio, & Demarco, 2009). Another probable error could result from approximation of the DNA concentration as it is difficult to count the exact number of DNA clumps that are visible after the extraction process. Other errors could also result from inaccurate measurements of materials used in the experiment. The major limitation for this experiment was insufficient saliva as the difficulty in collection increases as more trials are needed. Different people have different quantity of cheek cells in their saliva (Zdanowicz, 2010). However, one has to spit several times to get sufficient amount of saliva for all the trials. If the mixing power is insufficient, there may be no a homogenous concentration in the mixture, which may cause difference in material concentration (Roe, Crabtree, & Khan, 1996). Too high mixing power on the other hand may break the enzyme protease reducing the clumping ability of the DNA strands. Improvements Further improvements in future experiments can help reduce these errors and limitations. First, the time for collection of sufficient quantity of saliva should be increased. Second, the experiment should be carried out at a constant temperature to reduce the effect of temperature variation on the results obtained. The counting and weighing errors could be minimized by using more accurate measurement scales. Conclusion There are probabilities that the results obtained in DNA extraction differ, even though the same materials and method is followed in every test. This can be explained by the variation in temperature during the experiment as it affects the rate at which materials used in DNA extraction react. Other factors that can affect the results of DNA extraction include the quantity of cheek cells in each sample of saliva and the mixing power. In most cases, the effects by these factors may be insignificant and therefore, ignored. References Read More