Monday, October 14, 2019
Extracting DNA From Living Samples
Extracting DNA From Living Samples Karen Stevenson Introduction Collecting DNA samples from animals is often difficult and stressful for the animal, so non-invasive methods of collection are needed. Extracting DNA from animals usually involves one of three methods: Destructive sampling involves the organism having to be killed to get the tissues needed for genetic analysis.Ã Non-destructive or invasive methods require a tissue biopsy or blood sample. These are the most ethically acceptable and humane ways to extract DNA from living organisms as they do not destroy the animal or its habitat and often any DNA from feathers, hair, skin, droppings, etc. can be used, although DNA samples do degrade over time which will subsequently decrease the accuracy of test results. Freeland (2005) discusses a number of processes for DNA preservation including the method we used in the class experiment which is described in this report. High quality DNA shows up in bright contrasting bands on the electrophoresis gel but poor quality DNA displays a blurred or smudged look. Gender will show up as either one or two separate bands. Unlike in mammals where the heterogametic male (XY) will show up as two bands and the homogametic female (XX) will show up on the gel as one band, with birds, this is the opposite and the male is the homogametic and his ZZ genotype shows up as one distinct band while the heterogametic female ZW genotype shows up as two distinct bands on the gel. It is very difficult to determine the gender of very young chicks because there are no visible dimorphisms yet and poultry producers need to determine the sexes well before the animals begin to mature. Modern molecular genetic methods mean we can profile for individual genomes from very small amounts of DNA, whereas historically much larger samples were needed to get accurate results. In this experiment we followed procedures outlined by Hogan, Loke Sherman (2012) in our Prac manual to extract DNA from three tissue types of a domestic chicken to determine the sex of the sample and also to compare the quality and amount of DNA from the three samples. Materials and Methods Tissue Samples. Feathers, muscle tissue and blood samples were supplied by the technicians in the lab. The tissues were taken from a domestic chicken Gallus gallus domesticus. DNA Extraction from Blood, Feather and Muscle Samples We extracted our Our DNA with the Quiagen DNA purification kit DNeasy Blood Tissue Kit (2012). PCR is a faster and more sensitive method of amplifying DNA than cloning, and it produces similar results. We used bird sexing primers to build up the gender-specific loci CHD1W and CHD1Z, which allowed us to determine the gender of the chicken from a method developed by Fridolfsson and Ellegren (1999) using universal avian sexing primers 2250F and 2718R. The class results were collected and graphed so that our individual results could be compared. Negative control, male and female controls were used to conclude whether our hypothesis that Blood and tissue samples would yield a better quality of DNA than feather even though these methods are more invasive than extracting DNA from the blood spot in a feather shaft. In this experiment we extracted DNA from a blood clot in the feather as in the Horvath, Martinez-Cruz, Negro and Goday (2005) procedure, which showed that this was more successful than using material from the tip and this blood clot sample took longer to deteriorate than the tip sample. We did not know how old the feathers were, nor the age of the bird. DNA extraction procedures work by lysing cells, which causes the cell membrane to break free from the cell. Proteinase K can be added to detach the proteins and RNA can be removed with the RNAse. The DNA is then precipitated out using ethanol and further improved using PCR methods and visualized using the electrophoresis procedure. The Section containing the blood spot was cut out using a sharp pair of scissors and cut into tiny pieces and added to 180Ã µL of Buffer ATL before digestion with Proteinase K (180Ã µL pipetted into a sterile 1.5 mL microfuge tube) was then incubated at 56Ãâ¹Ã ¡C for 30 minutes (briefly mixed in the vortex every 10 minutes), after which the cells had been lysed. To precipitate the DNA we added 200Ã µL of 95% ethanol (AR grade) and mixed in the vortex for a further 15 seconds. The lysed DNA was then pipetted into the DNeasy Mini spin column and centrifuged at 8000 rpm (6000 x g) for 1 minute, binding the DNA to the membrane in the spin column, ready for washing. The spin column was placed in a new microfuge collection tube in which 500Ã µL Buffer AW1 was pipetted, centrifuged for 1 minute at 6000 x g (8000rpm) and the flow-through was discarded. Again the DNeasy spin column was placed into a new collection tube, 500Ã µL of Buffer AW2 added and centrifuged for 3 minutes at maxi mum speed (13 Ã¢â¬â 14,000 rpm), removed from the flow-through (which was discarded in hazardous waste receptacle), placed back into the collection tube and centrifuged again at maximum speed for a further minute to remove any ethanol. The spin column was then removed from the tube (which was discarded). After placing the spin column into a clean 1.5mL collection tube it was labelled appropriately and 100Ã µL of Buffer AE was pipetted straight onto the centre of the DNeasy membrane and incubated at room temperature for 1 minute, centrifuged for 1 minute at 6000 x g (8000 rpm) to elute it. The DNA was now pelleted in the bottom of the tube, so the spin column was discarded and the pellet stored in its tube in a cold box at -20Ãâ¹Ã ¡C. Electrophoresis Method During electrophoresis, the negatively charged DNA fragments travelled towards the positive cathode causing the smaller protein fragments to move quicker than larger particles. The DNA was visualized as bright bands on the gel, which had been stained with GelRed which is a chemical used to increase mutation rates, multiplies the product and is assumed to be carcinogenic. The agar gel and TAE buffer had been prepared earlier in the microwave and allowing the gel to cool to 50Ã °C. GelRed was carefully added to 150mL of gel for a final concentration of 0.5Ã µL mL-1.The casting tray was carefully put into the gel tank with the black moulding gates at both ends. The comb was inserted after the gel had been poured into the tray inserted, then left for 30 minutes at room temperature to set. 10Ã µL of the DNA chicken feather sample we extracted previously was mixed with the 6x loading dye into a fresh microfuge tube. Wearing rubber gloves, we removed the black casting plates and the comb and then added the TAE buffer until the entire gel was submerged by 5mm. The first and last wells had molecular weight markers Ã »HindIIIand 2-log ladder added and our DNA samples were pipetted into an empty well, noting the position. We applied the cover and connected to the power unit and ran it for 60 minutes at 120V. The DNA proceeded to float from the negative cathode (black cable) to the positive anode (red cable). When finished, we removed the gel tray and transferred it on a plastic container to the Gel Doc System for visualizing the images. PCR method We used the Polymerase Chain Reaction method to expand the DNA so that it could be viewed using electrophoresis. The PCR procedure involved cycles of heating then cooling the DNA which enabled the helix to unwind and bind. We prepared the Mastermix negative and positive controls using 40Ã µL of the PCR Mastermix and 10Ã µL of the DNA sample mixed into a 0.2mL PCR tube. Each group had individually calculated amounts using the chart in the Prac manual. We prepared tubes for male control, female control and one negative control (these were provided by the lab). We then placed the tubes into a thermo-cycler and initiated the program which had been perfected to augment the CHD1W and CHD1Z genes using the primers. When this was done, the DNA was then put on a 1% agar gel comb (that had been microwaved and cooled to 50Ãâ¹Ã ¡C) in a 1 x SB buffer solution for 20 minutes. Wearing gloves, we added 15Ã µL of 3 x GelRed solution to 150mL of agar gel. We prepared the DNA samples by mixing 10Ã µL of PCR with 2Ã µL of 6x loading dye, pipetted it into the gel combined with 5Ã µL of a 100bp molecular weight marker. The sample was pipetted into an empty well in the gel, location documented and after closing and securing the lid, the electrophoresis unit was run at 300V for 20 minutes. When the gel had finished running the power was turned off, gel removed carefully and put into a plastic container and transported to the Gel Doc unit. The bands were then visualised using the Gel Doc System. Results The class groups successfully extracted DNA from all three types of tissue. Due to incorrect or absent labelling of DNA samples, we were unable to use some of the gel images in our report. Figure 1 shows the Gel electrophoresis from a co-operative class Muscle and Blood DNA extraction using Qiagen 2012, DNeasy Blood Tissue Kit, with blood showing up in more distinctive bands, muscle failing to show clear bands and feather samples extracted (on a separate gel image) displayed poorly using electrophoresis. Hogan, Loke Sherman (2012) explain how the DNA concentrations are measured by comparing the brightness of the sample to the 2log Molecular Weight Marker over the amount of DNA pipetted into the well. Figure 1: Blood muscle DNA extraction using (Qiagen 2012, DNeasy Blood Tissue Kit) Figure 2: Feather DNA extraction using (Qiagen 2012, DNeasy Blood Tissue Kit) After extraction and visualization using electrophoresis, our samples were diluted give comparable concentrations. If the band was too faint or not even visible we left it undiluted but most of muscle and blood samples were dilute. Figure 2 shows the Gel electrophoresis from our feather DNA extraction sample with no discernible results. This was expected. Table 1: Mean nucleic acid concentrations muscle, blood and feather DNA extraction using nanodrop technique From table 1, results show us the average DNA concentration of the three tissue types and reveals that compared to feather, muscle samples provided the best quality of extracted DNA, followed closely by the blood samples. Our test yielded 5 muscle samples, 6 feather samples and 8 blood samples as well as the 2 unspecified class samples. Because 1 feather sample and 1 blood sample failed to clearly show any visible DNA (see figures 1 2), they influence the averages. In the face of this, however, the resulting average sample DNA concentrations reveal that muscle still produced the highest class of extracted DNA in comparison to the blood samples. The feather sample still showed the poorest DNA quality, which related with our expected outcomes. Ladder Male control Female control Negative control JackÃ¢â¬â¢s sample DNA Sample DNA Karen Feather DNA Sample Negative control Female control Male control Ladder Figure 3 shows the Gel electrophoresis from our feather DNA extraction sample with the male, female and negative controls. DNA had been amplified from the extraction and visualized using electrophoresis to determine the sex of the bird that our sample was taken from. Results successfully indicate that sexes were able to be determined. Our PCR result matched the expected result and we determined our sample to be ZW female and JackÃ¢â¬â¢s sample to be ZZ male. This experiment matched the Fridolfsson and Ellegren (1999) procedure except that we used a 1% agar gel to visualize the DNA fragments via electrophoresis and Fridolfsson and Ellegren used a 3% gel as well as our use of a commercial kit (Quiagen 2012). Discussion The quality of DNA extracted varied between our different tissue samples although all we were able to amplify all of them using the non-invasive technique PCR. Extracting DNA from a blood clot of a feather is an option when alternative methods (blood or muscle) are not suitable. The destructive muscle samples provided a better class and measure of DNA in comparison to the feather samples, however destructive methods of DNA extraction necessitate the slaughter of the organism and is not typically ethically acceptable particularly when endangered species are involved. Invasive blood sampling provided a high quality of DNA in terms of results and should be used in preference to destructive methods if non-invasive methods are not possible. The disadvantage of blood sampling is that if the procedure is done in the field, it necessitates the capture of the organism to extract the blood sample as well as the storage while out in the field as DNA deteriorates over time. Although DNA from fea ther samples gives a lower quality than the other two methods discussed, they are usually easier to obtain in the field because capture, plucking and release are far less invasive that taking blood or killing the animal for muscle tissue (Mundy et al. 1997) and usually can be collected from nests or off the ground without having to involve capturing the animal at all. This experiment was conducted over a number of weeks. DNA deteriorates over time and storage is therefore very important. Freeland (2005) discusses the importance of preserving DNA to circumvent DNA molecules from re-arranging and so affect the results when amplified by the PCR technique. We froze the DNA at -20Ã °C to preserve the samples in between both practical sessions. While performing the practical sessions, our DNA was generally kept at room temperature which could possibly have caused some deterioration but this is not very likely to cause large variations of DNA quality as all our samples were exposed to the same conditions. Cold-boxes were used to store the DNA samples but all products including the DNA were kept at room temperature for the duration of both practicalÃ¢â¬â¢s and this could easily have been avoided by asking the students to me mindful of the importance of preserving the DNA in order to get better quality DNA for extraction. References Freeland, J (2005).Ã Molecular Ecology. Wiley. Chichester. Fridolfsson, A and Ellegren, H. (1999). A simple and universal method for molecular sexing of non-ratite birds. Journal of Avian Biology. 30, 116 Ã¢â¬â 121. Hogan, F., Loke, S., and Sherman, C. (2012)Ã SLE254 Genetics: Practical Manual 2012~ Sex Determination of the Domestic Chicken (Gallus Gallus).Deakin University. Burwood. 1-46. Horvath, M. Martinez-Cruz, B. Negro, J. Kalmar, L and Goday, J. (2005). An overlooked DNA source for non-invasive genetic analysis in birds. Journal of Avian Biology. 36, 84-88. Mundy, N. Unitt, P., and Woodruff, D. (1997). Skin from feet of museum specimens as a non-destructive source of DNA for avian genotyping. Auk 114, 126-129. Qiagen. (2012).Ã Sample Assay Technologies: DNeasy Blood Tissue Kit.Retrieved September, 11th2012 Taberlet, P. Waits, L. and Luikart, G. (1999). Noninvasive genetic sampling: look before you leap. Trends in Ecology and Evolution. 14, 323 Ã¢â¬â 327.