Purification of Genomic DNA from Buccal Cells - Essay Example

? Purification of Genomic DNA from Buccal Cells 0 Purification of Genomic DNA from Buccal Cells 2.0 Introduction 2.1 Extraction of DNA from Buccal Cells and its Purification The process of obtaining DNA samples includes a general procedure regardless of the source of the DNA. First, the investigator has to collect the cells he intends to derive his DNA sample from. This could include all cells of the body but red blood cells (since they contain no nuclear). The next step involves splitting the cells open so as to release their contents. The contents include fats, carbohydrates and proteins. The enzymes that break apart DNA are thereafter destroyed (Bruns 2007, 50). DNA content is then separated from other cell components. The researcher then precipitates the DNA and re-suspends it in a solution suitable for its studies. When extracting DNA from the cheek cells, saline solution used to rinse the mouth helps to prevent the cells extracted from splitting open or lysing too soon. Centrifugation separates the cheek cells from mouth wash used (Johannson 1972, 39). Spinning the mixture in a centrifuge settles the heavier cells to the bottom of the tube to form pellets. Saline solution pours away, leaving the clumped cheek cells at the bottom of the tube. Lysis buffer added to the cell clump splits open the cells to release DNA from inside the nucleus. The buffer contains soap that dissolves and breaks fatty membranes of the cells, buffer that maintains the pH of the solution and ions that increase osmotic pressure outside the cheek cell and aids in ripping open the cell membrane. Incubation in hot water helps denature cytoplasmic enzymes that break up DNA. Concentrated salt solution changes polarity of the solution under study. DNA elements dissolve in ionic solutions. This is as opposed to other components of the solution; proteins, carbohydrates and fats. Further centrifugation separates the dissolved DNA from other junk particles in the mixture. After the un-dissolved particles are filtered out, the remaining solution is mixed with cold ethanol to precipitate DNA out. 2.2 Analyzing DNA Agarose Gel Electrophoresis Gel Electrophoresis is an important analysis tool in understanding the structure of DNA. It is crucial in determining the sequence of nitrogen bases, the presence of a point mutation or an occurrence of deletion or insertion. The process is additionally useful in assessing and distinguishing the variable sizes of alleles. This discerning of allele sizes best takes place with the DNA strands placed at a single locus. Gel Electrophoresis also assesses the quantity and quality of DNA that is present in a sample (Komrakova 2006, 51). This method separates chemical molecules and compounds by charge and size. Substances that are separated are stationed in wells in the agarose gel and an electric field applied. Positively charged molecules and compounds move towards the negative terminal while the negatively charged particles and compounds move towards the positive anode. Larger and longer particles experience difficulty in moving across the mixture to the positive or negative terminal, and are suspended in the gel matrix. Smaller and shorter molecules move easily through the agarose gel matrix and take positions according to their polarity. When strained, the small sized segments form a tight band as they move at relatively the same speed. Type of medium and concentration of the gel determines the gel’s pore size and its ability to segregate same sized fragments. While polyacrylamide gels separate DNA segments differing by a base pair, agarose gels separate fragments of DNA differing by hundreds or more base pairs. Combs forming wells are placed into the gel as it solidifies and cools. The combs are then removed after the gel solidifies. Students can use gel electrophoresis in determining quality and quantity of the DNA matter they extract from their cheek cells. In day-to-day applications, the method is useful in fingerprinting or profiling, DNA sequencing and genetic engineering. Figure 1 below shows the process of Gel Electrophoresis Fig 1 ILRN1 is a member of the interleukin 1 cytokine family. This protein inhibits the activities of interleukin 1, alpha (IL1A) and interleukin 1, beta (IL1B), and modulates a variety of interleukin 1 related immune and inflammatory responses (Lee 1997, 47). This gene and five other closely related cytokine genes from a gene cluster spanning approximately 400kb on chromosome 2. A polymorphism of this gene is reported to be associated with increased risk of osteoporotic fractures and gastric cancer (Coleman 2006, 39). 2.3 PCR Amplification DNA sample obtained from the cheek cells should be amplified so as to improve the probability of obtaining viable results from them. One of the methods used to amplify the DNA fragments is the Polymerase Chain Reaction (PCR). The technique is so far one of the fastest means of amplifying DNA samples. It is also specific and dependable. The method, however, require that the sample be cloned and previously sequenced using other available traditional methods. It call for two oligonucleotide primers, template DNA, buffer, thermostable enzyme Taq polymerase and dNTPS. Oligonucleotide primers are responsible for determining the extent of the sample to be amplified and initiate the amplification reaction (Dingman 1998, 29). Polymerase Chain Reaction is an in vitro enzymatic procedure conducted on a programmable thermocycler. The PCR machine reaction is specified by the primers used. The sequence of a primer matches that of flanking regions of targeted DNA fragments. In a few hours, PCR can amplify a given region of DNA by up to 106. Segments of DNA targeted for amplification must always be flanked by a preset DNA sequence. The sequences are used to design two oligonucleotide primers chemically synthesized to complement opposite threads of DNA duplex situated at either ends of the DNA targeted. These primers form nuclear hybrids with complementary single strand template DNA strands resulting into short double-strand regions that extend by a DNA polymerase in 5’ to 3’ direction. This results in an exponential buildup of the definite target section, approximately 2n where n is the amount of cycles of magnification performed. Figure 2 below shows a pictorial expression of PCR. Fig 2 3.0 Aim and Hypothesis The aim of this project was to investigate purification of genomic DNA from buccal cells by use of Agarose Gel Electrophoresis. Practical 1: Purification of Genomic DNA from Buccal Cells 4.1 Methods and Materials 4.1.1 Materials 10 ml of 0.9% saline, sterile 50 ml Falcon Tube, centrifuge, stop watch, pipette, Chelex beads, heater (Bunsen burner) and a freezer. 4.1.2 Method 10 ml of 0.9% saline was agitated in the mouth for about 20 seconds and the specimen/suspension collected in sterile 50 ml Falcon Tube. The tube was labeled with a permanent marker pen indicating name and date. The specimen was then placed on a bench-top centrifuge and set on rotation for 10 minutes at 2000 rotations per minute. A pipette was then used to remove and discard supernatant liquid into the bleach spots. The pellets were saved at the bottom of the tube. The pellets were further agitated by flicking the base of the tube with fingers to assist re-suspend them. An end off a blue tip was cut and 500 ul of chelex beads was pipette into the tube containing the pellets. Using the same tip, the cells were re-suspended in the tube by pipetting up and down repeatedly. Further, 500 ul of chellex/cell solution was pipette into 1.5 ml ependorf. The tube was labeled with the experimenter’s initials. The solution was boiled in water bath for 10 minutes. It was then placed in a freezer for 5 minutes to cool. The tube was span in a centrifuge at 13,000 rotations per minute for 3 minutes. The supernatant that contained denatured DNA was transferred into a clean eppendorf tube The tube was labeled with the initials of the experimenter and placed in ice bucket at -20oC. 5.1 Results The end result of the above procedure was isolated, denatured sample of DNA from cheek cells. Resulting suspension from agitated mouth wash contained cells that were successfully ruptured to release DNA fragments, 6.1 Discussion Saline solution used to rinse the mouth helps to prevent the cells extracted from splitting open too soon. Centrifugation separates the cheek cells from mouth wash used. Spinning the mixture in a centrifuge settles the heavier cells to the bottom of the tube to form pellets. Saline solution pours away, leaving the clumped cheek cells at the bottom of the tube. Lysis buffer added to the cell clump splits open the cells to release DNA from inside the nucleus. The buffer contains soap that dissolves and breaks fatty membranes of the cells, buffer that maintains the pH of the solution and ions that increase osmotic pressure outside the cheek cell and aids in ripping open the cell membrane. Incubation in hot water helps denature cytoplasmic enzymes that break up DNA. Concentrated salt solution changes polarity of the solution under study. DNA elements dissolve in ionic solutions. This is as opposed to other components of the solution; proteins, carbohydrates and fats. Further centrifugation separates the dissolved DNA from other junk particles in the mixture. After the un-dissolved particles are filtered out, the remaining solution is mixed with cold ethanol to precipitate DNA out. The experiment worked out properly and DNA samples were collected. Practical 2 Part A: DNA Quantization and Estimation of Purity 4.2A Methods and Materials 4.2A.1 Materials 10 ml of 0.9% saline, sterile 50 ml Falcon Tube, centrifuge, stop watch, pipette, Chelex beads, heater (Bunsen burner), a freezer, water, UV Cuvette, water and eppendorf tube. 4.2A.2 Method Previously prepared DNA solution was diluted in the ratio 1:10 with water. 150? ?l of DNA sample was transferred into eppendorf tube and 1.35 ml of water added into it. It was then pipette up and down for a 2 minutes. The 1.5 ml diluted mixture was transferred into a UV Cuvette. Spectrophotometer was zeroed with water blank. Using the Spectrophotometer, absorbance readings were taken at both 280 nm [protein/phenol] and 260 nm [DNA] for sample against water blank. 5.2A Results Absorbance was observed and recorded as in the tables below At 280nm (DNA) At 260nm (protein/phenol) Fig 3 5.2A.1 Result Analysis DNA concentration (Tg/ml) = OD260 x 50 x L x RDF Where OD260 is you absorbance reading at 260nm Where L is the length of the light path (1cm) Where RDF is reciprocal of the dilution factor used (so a 1:10 dilution has a RDF of 10) DNA sample B 0.085 x50 x1 x10 = 42.5 ng/ul or 42.5/1000 = 0.425 ug/ul DNA sample C 0.101 x50 x1 x10 = 50.5 ng/ul or 50.5/1000 = 0.505 ug/ul Purity of sample B = 0.439/0.085 = 5.164 Purity of sample C = 0.451/0.101 = 2.634 DNA purity = OD260 OD280 = 5.164/3.065 = 1.932 6.2A Discussion From the results of the above analysis, a DNA purity of 1.932 shows that the sample obtained was clean. This implies the tubes used in the experiment were sterilized and the sample was carefully handled to avoid contamination. The experiment was a success. Practical 2 Part B: PCR Amplification 4.1B Methods and Materials 4.2B.1. Materials 10 ml of 0.9% saline, sterile 50 ml Falcon Tube, centrifuge, stop watch, pipette, Chelex beads, heater (Bunsen burner), a freezer, 10x PCR buffer (NEB) B9014S 25 ?l, 10 uM Forward primer 25 ?l, 10 um Reverse primers 25 ?l, 1 mM dNTP’s 50 ?l, Water 75 ?l, Forward & reverse primers, IL1RNGR: TCCTGGTCTGCAGGTAA, IL1RNCR: TCCTGGTCTGCAGCTAA, IL1RNF: CTCAGCAACACTCCTAT 4.2B.2 Method 200 ?l of PCR mixture was pipette into an eppendorf tube and placed on ice. 50 ?l of Taq polymerase was added into the mixture and the tube kept in ice. Each pair labeled 4 PCR tubes carefully with a permanent marker pen indicating numbers 1-4. Aliquot 45 ?l of PCR mixture to each PCR tube 5 ?l of sterile water was then added to tube labeled 1, 5 ?l of DNA into sample A (tube labeled 2), 5 ?l into sample B (tube 3) and 5 ?l positive control DNA to tube 4. The tubes were stored on ice, and reactions in the program below cycling 40 times: Denaturation 94?C 30secs Annealing 60?C 30secs Extension 70?C 30secs Completing any unfinished extensions 72?C 5 min Fig 4 After preparing the agarose gel, combs and metal dividers were removed from the gel former 1x TBE buffer was poured into the tank until the level was about 1mm above the gel. 3 ?l of loading dye and 10 ?l of each PCR reaction was mixed in eppendorf tube. 12 ?l of each sample was pipette into the wells size markers were loaded onto the well on both sides of the sample. The lid of the tank was closed cautiously. Connection from power pack to the tank was then done. Voltage of 1-10V was applied per cm of gel. The electrophoresis was stopped before the bromophenol blue die ran off to the end of the gel. Once the separation was complete, the gels were viewed on UV transluminator and a photographic image was captured. The results were then evaluated as directed in the prerequisite video using DNA markers. 5.2B Results The photographic image on the UV transluminator was as shown in figure 5 below Fig 5 6.2B Discussion Substances that are separated are stationed in wells in the agarose gel and an electric field applied. Positively charged molecules and compounds move towards the negative terminal while the negatively charged particles and compounds move towards the positive anode. Larger and longer particles experience difficulty in moving across the mixture to the positive or negative terminal, and are suspended in the gel matrix. Smaller and shorter molecules move easily through the agarose gel matrix and take positions according to their polarity. When strained, the small sized segments form a tight band as they move at relatively the same speed. From figure 5, the bands observed were discrete and evenly spaced. This implies that the DNA fragments collected from the check cells were of similar length. In addition, the bands are dark, insinuating that concentrations of DNA in the investigated samples are densely populated. 7.0 Conclusion Results of the practices 1, 2A and 2B reveal that the experiment was successfully conducted. The aim of the experiment to investigate purification of genomic DNA from buccal cells was reached. The check cells were successfully extracted, DNA released and isolated from them. In addition, quantization and estimation of DNA purity was successfully carried out and results revealed that DNA samples were pure. DNA purity measure of 1.932 fell in between the acceptable limits of purity; between 1.8 and 2.0. 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