Extracting DNA From Living Samples

Extracting desoxyribonucleic acid From Living SamplesKaren StevensonIntroductionCollecting desoxyribonucleic acid attempts from animals is often difficult and trying for the animal, so non-invasive modes of collection are needed. Extracting deoxyribonucleic acid from animals usu bothy involves one of three methods Destructive sample distribution involves the existence having to be killed to get the create from raw material papers needed for transmissible analysis.Non-destructive or invasive methods require a tissue biopsy or blood sample. These are the most ethically acceptable and humane ways to extract desoxyribonucleic acid from livelihood organisms as they do not destroy the animal or its habitat and often whatsoever desoxyribonucleic acid from plumages, hair, skin, droppings, etc. can be exampled, although desoxyribonucleic acid samples do degrade over cartridge clip which will subsequently decrease the accuracy of test results.Freeland (2005) discusses a number of processes for desoxyribonucleic acid preservation including the method we used in the figure experiment which is described in this report. High quality DNA shows up in glinting contrasting bands on the cataphoresis mousseatine but poor quality DNA displays a addled or smudged look. Gender will show up as either one or both start out bands. Unlike in mammals where the heterogametic male (XY) will show up as two bands and the homogametic feminine (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 genetic constitution 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 thither are no visible dimorphisms yet and poultry producers need to determine the stires well before the animals arrive 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 MethodsTissue 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 stemma, Feather and Muscle SamplesWe 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 attain 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) apply universal avian sexing primers 2250F and 2718R. The course results were collected and graphed so that our individual results could be compared. cast out figure, male and female controls were used to conclude whether our hypothesis that Blood and tissue samples would yield a better quality of DNA than plumage 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 curdle in the feather as in the Horvath, Martinez-Cruz, Negro and Goday (2005) procedure, which showed that this was more successful than victimization material from the tip and this blood clabber 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 origination 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 outside with the RNAse. The DNA is past precipitated out apply ethyl alcohol and further improved using PCR methods and depictd using the electrophoresis procedure.The Section containing the blood spot was calamity out using a sharp pair of scissors and cut into tiny pieces and added to 180L of Buffer ATL before digestion with Proteinase K (180L pipetted into a sterile 1.5 mL microfuge tube) was then incubated at 56C for 30 minutes (briefly mixed in the vortex every 10 minutes), by and by which the cells had been lysed. To precipitate the DNA we added 200L 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 fit(p) in a modern microfuge collection tube in which 500L Buffer AW1 was pipetted, centrifuged for 1 minute at 6000 x g (8000rpm) an d the flow-through was discarded. Again the DNeasy spin column was placed into a new collection tube, 500L of Buffer AW2 added and centrifuged for 3 minutes at maximum speed (13 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 designate appropriately and 100L 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 bum of the tube, so the spin column was discarded and the pellet stored in its tube in a stone-cold box at -20C.Electrophoresis MethodDuring electrophoresis, the negatively charged DNA fragments travelled towa rds the positive cathode create the smaller protein fragments to move quicker than larger particles. The DNA was visualized as bright bands on the gel, which had been varnished 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 50C. GelRed was carefully added to 150mL of gel for a final submergence of 0.5L mL-1.The plaster bandage tray was carefully adjust into the gel tank with the drab 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.10L 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 well had molecular weight markers HindIIIand 2-log ladder added and our DNA samples were pipetted into an vacate well, noting the position. We applied the cover and connected to the power whole 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 methodWe 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 alter the DNA which enabled the helix to unwind and bind.We prepared the Mastermix negative and positive controls using 40L of the PCR Mastermix and 10L 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 dumbfound on a 1% agar gel comb (that had been microwaved and cooled to 50C) in a 1 x SB buffer solution for 20 minutes. Wearing gloves, we added 15L of 3 x GelRed solution to 150mL of agar gel. We prepared the DNA samples by mixing 10L of PCR with 2L of 6x loading dye, pipetted it into the gel combined with 5L 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.ResultsThe class groups su ccessfully 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 illumination of the sample to the 2log molecular(a) 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 earn comparable concentrations. If the band was too fai nt or not even visible we left it concentrated 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 techniqueFrom table 1, results show us the average DNA concentration of the three tissue types and softens 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 put away produced the highest class of extracted DNA in comparison to the blood samples. The feath er sample still showed the poorest DNA quality, which related with our expected outcomes.LadderMale controlFemale controlNegative controlJacks sample DNASample DNAKaren Feather DNA SampleNegative controlFemale controlMale controlLadderFigure 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 Jacks 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).DiscussionThe 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 imperil species are involved. Invasive blood sampling provided a high quality of DNA in scathe 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 feather 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 violent death the animal for muscle tissue (Mundy et al. 1997) and usually can be collected from nests or off the make 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 -20C to preserve the samples in between both practical sessions. While do the practical sessions, our DNA was generally kept at room temperature which could possibly have caused some declivity 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 practicals 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.ReferencesFreeland, J (2005).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, 116 121.Hogan, F., Loke, S., and Sherman, C. (2012)SLE254 Genetics mulish 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, 126-1 29.Qiagen. (2012).Sample Assay Technologies DNeasy Blood Tissue Kit.Retrieved September, 11th2012Taberlet, P. Waits, L. and Luikart, G. (1999). Noninvasive genetic sampling look before you leap. Trends in Ecology and Evolution. 14, 323 327.