DNA Extraction and PCR of Bird DNA for Sex Identification - Lab Report Example

DNA extraction and PCR of bird DNA for sex identification Introduction: DNA is the basic component of genes. It is one of the important molecules of life. The variation in the DNA composition and structure provides variation between the species and within the species. DNA sequence varies among the sexually dimorphic species females and males. The difference in the phenotype is used as the basic identification step. Sometimes the phenotypic identification becomes complex in some species such as birds (they are monomorphic sexually). (Huffman and Wallace 2012). These birds are identified genotypically. The identification of the sex chromosomes will solve the issue. Identification of DNA from the egg shell, feather and blood are the safest way to determine the sex of the birds. (DNA sexing). The development of molecular markers to determine the sex of an individual is an extremely beneficial tool. In mammals , the males are heterogametic and they carry two different sex chromosomes X and Y and the females carry two identical sex chromosomes – X and X. whereas in birds, the males carry the homogametic sex (ZZ) chromosomes and the females carry the heterogametic sex chromosomes( Z and W). (Ellegren 2001). As many birds are not differentiated through phenotype, the use of molecular markers to identify the sex of the birds is practiced now. (Braun 2004). The chromo- helicase – DNA binding gene (CHD) is present in both the W and Z sex chromosomes but they vary in their size. Hence the use of size polymorphism in the CHD gene is the most common way of determining the sex of the birds. (Ellegren 2001). The molecular markers were designed mainly concerning the primer binding regions of the CHD1 gene of the sex chromosome. The genes shared between the non recombining parts of the two types of sex chromosomes are the best means to study the genes in the sex chromosomes. The CHD1Z and CHD1W are found to share a highly conserved nucleotide and amino acid levels among them and they evolve independently. (Fridolfsson and Ellegren 2000). The quality and quantity of DNA obtained from various samples such as blood, feather, eggshell and skin may vary. Blood and organs are very rich sources of DNA and the quantity of DNA obtained from them will be high. (Frankham, Ballou and Briscoe 2004). Whereas the DNA from other sources such as feather, egg shell will have less quantity of DNA and the quality of the DNA will vary because of the presence of the other impurities mainly proteins. (Silvy 2012). Many of the current methods of DNA diversity analysis depends on the PCR which helps in amplification of the specific often small sequences. (Frankham, Ballou and Briscoe 2004). Aim: To extract the DNA from muscle, blood and feather of Gallus gallus and to estimate the concentration of the DNA from the three samples and amplify the CHD1 gene using PCR. After amplification, the DNA is visualized on agarose gel product and the sex of Gallus gallus is determined. Method: DNA extraction and Visualization: DNA extraction from feather: A sharp blade was taken and the feather was cut into very thin slices. It was then added to a micro centrifuge containing 180µl of Buffer ATL. To this mixture, 20 µl proteineaseK was added and vortexed for 15 seconds. The mixture was then incubated at 56ºC for 30 minutes. After incubation, the mixture was again vortexed for 15 seconds and 200 µl of 96-100% ethanol was added and mixed thoroughly by vortexing it for 15 seconds. The sample was then transferred to the DNeasy spin column tube and centrifuged at 8000rpm for 1 minute. The fluid was discarded and the DNeasy mini column was kept in the new collection tube and 500µl of Buffer AW1 was added to the column. The mixture was centrifuged at 8000 rpm for 1 minute and the liquid was discarded. The DNeasy mini column was again kept on a new collection tube and 500µl of Buffer AW2 was added to the column and spinned for 3 minutes at 8000rpm. The supernatant was discarded and the column was again spinned for 1 minute at maximum speed. The DNeasy spin column was removed from the collection tube and kept on the microcentrifuge tube. 100µl of buffer AE was directly added to the DNeasy centre of the membrane and incubated at room temperature for 1 minute and centrifuged at 8000rpm for one minute. The column was discarded and the tube containing the DNA was stored at -20 ºC. DNA extraction from blood: A micro centrifuge tube was taken. To this micro centrifuge tube, 20µl of proteinase K and 166 µl of PBS and 4µl of RNase A was added. One small punched hole card o f preserved blood was added to the tube using the forceps. It was then incubated for 30 minutes at room temperature. 200µl of Buffer AL was added to the tube and vortexed for 15 seconds. The sample was again incubated for 25 – 45 minutes at 56ºC. Finally 200µl of 95% ethanol was added to the sample and mixed thoroughly and vortexed for 15 seconds. The sample was then transferred to the DNeasy spin column tube and the procedure for extraction was the same for feather DNA extraction using the DNeasy kit. DNA extraction from tissue: 20 mg of tissue was macerated in a sterile petriplate and 180µl of Buffer ATL was added and vortexed for 15 seconds. To the mixture 20 µl of proteinase K was added and vortexed for 15 seconds. The sample was then incubated at 56ºC for 30 minutes. After incubation 4 µl of RNase was added and the sample was vortexed for 15 seconds and incubated at room temperature for 2 minutes. The sample was then vortexed for 15 seconds and 200 µl of buffer AL was added to the sample. The sample was again vortexed for 15 seconds and 200 µl of ethanol was added and again vortexed for 15 seonds. The Dneasy spin column was taken and the sample was added to it and the extraction procedure is similar to that of the feather DNA extraction using the DNeasy kit. DNA visualization using Electrophoresis: 1% agarose gel was prepared using 1 X TAE buffer by boiling the agarose until it gets dissolved and was allowed to gel cool to 50 ºC and 15 µl of 5mg mL-1 ethidium bromide was added to the agarose gel. The gel was then poured into the casting tray and allowed to solidify. 10µl of the DNA was mixed with 4 µl of 6X loading dye and kept ready for electrophoresing. 1X TAE buffer was added to the tank and the molecular weight markers λ HindIII and 2- log ladder were added to the first and last wells. The DNA sample was then pipette into the empty well and position was marked. The electrophoresis was run at 120 volts for 60 minutes and the gel was removed from the tank and visualized under the gel documentation system. PCR and Visualization: Master Mix : Final concentration required in 50 µl reaction Number of reactions per batch 4 reactions + 3 controls + 1 pippeting error= 8 reactions. 1X PCR buffer 40 µl 200 µM dNTP’s 40 µl 1U Taq DNA polymerase in 1X PCR buffer 8 µl 2pmol 2550F forward primer 8 µl 0.5mM MgCl2 8 µl Sterile milliQ water 212 µl Sample DNA or milliQ water 10 µl Of the three controls, 2 were positive controls. The first was male positive control and the second was female positive control and the third one was the negative control containing no DNA sample. PCR was then run with the above master mix and the DNA was amplified. The amplified DNA was run in the electrophoresis for band visualization. 1% agarose gel was prepared using 1X SB electrophoresis buffer and 15 µl of 5 mg mL-1 ethidium bromide was added to the gel and allowed to cool in the casting tray for 30 minutes. 10 µl of the PCR product was mixed with 4 µl of 6X gel loading dye and kept ready for electrophoresis. 1X SB buffer was added to the electrophoresis tank and the gel was kept in the tank. 5 µl of the 100bp molecular weight marker was added to the first well and 10 µl of the PCR mix was loaded in the empty well with proper marking. The unit was run at 300V for 20 minutes. The gel was then removed from the tank and viewed under Gel Documentation system. The positions of the PCR bands were then compared with the standard bands in the marker. The sizes of the PCR products were determined. Results: Figure 1: DNA extraction and visualization: The figure shows the gel containing the 2-log molecular weight marker in the first lane and lambda Hind III marker in the last lane. In between these lanes are the DNA extractions from muscle, blood and feather of Gallus gallus. It is found clearly that the DNA bands were visible for the muscle samples than the other samples. A clear thick band was observed in the muscle samples. The DNA bands are seen close to the first band of the lambda HindIII marker which is of 23,120 bp. This refers to a concentration of 120 ng from the 2-log ladder. The concentration of DNA in the given sample is 120 ng DNA in molecular weight reference band / 10µl of sample DNA = 10 ng/ µl. Figure 2: PCR gel : The figure shows the PCR amplification for the DNA samples. From the results, it was found that the DNA isolated from the feather F12.16 belongs to a male Gallus gallus. The blood DNA and the muscle DNA from 12.16 confirm that it is male. Similarly 12.17 is female Gallus gallus and 12.15 is also a female Gallus gallus. 12.18 is male, 12.19 is female, 12.21 is female, 12.22 is male, 12.23 is female, 12.24 is male, 12.11 is female and 12.12 is male. So out of the 11 samples, 5 samples were male and 6 samples belong to female. There was difference in the amplification of the samples. Some samples were amplified very well and some were not. The occurrence of thick distinct bands for the muscle samples compared to the other samples details the variation in the amplification. One band was seen for the male samples having an average molecular weight of 550 base pairs and the female samples had two distinct bands of molecular weight around 550 base pairs and 400-500 base pairs. The amplification of the DNA samples varied between the tissue types. The muscles DNA were amplified very well than the blood and feather DNA samples. The negative controls were not amplified. Thus the results from the PCR correlate with that of the actual sex of the samples. Discussion: The DNA was extracted from the blood, muscles and feather of the Gallus gallus. The extracted DNA was then amplified using the molecular markers designed for targeting the highly conserved primer flanking regions within the chromo-helicase-DNA binding gene 1 ( CHD1). The difference in the size between the CHD1 on the W chromosome of the female and the CHD1 variant in the Z chromosome of the male was determined. These DNA were amplified using the PCR and the final product was electrophoresed with suitable markers. It was observed that two distinct bands were formed for the female samples of muscle, blood and feather and one distinct band was obtained for the male samples of muscle, blood and feather. The amplification rate of the muscle, feather and blood DNA samples varied. The quantity of DNA obtained among the samples also varied. The muscle tissues were found to be the best source for DNA extraction and the amplification rate of muscle cells in all the samples were very good. The blood samples produced less amount of DNA than the muscle cells. The rate of amplification was comparatively good as that of the muscle samples. The feather samples had the less amount of DNA and the quality of the DNA obtained was also very less. Many resources have found that DNA can be extracted from blood, muscle, feather, egg shell, hair, scal, teeth, bone and buccal swab from the birds and the quantity and quantity of DNA obtained from them will vary. (Herrmann 1994). The studies also indicate that the quality of DNA obtained from blood, muscle, egg shell and feather will be good than the hair, scal, teeth, bone and buccal swab. The quantiy of DNA extracted from blood and muscle will be high when compared to feather and the quantity will be very poor in the other samples. (Silvy 2012). The results obtained from these samples were consistent with the expectations. In the feather samples, some bands were not visible. The reason may be low concentration of DNA in the sample or the extraction procedure may not be perfect. In the feather sample, the concentration of the DNA was very less. When compared to muscle and blood samples, feather contains less amount of DNA. (Silvy 2012). Similarly sample collection and preservation are very important for improving the quality and quantity of DNA. Blood samples should be preserved in EDTA, muscles in buffer solution, feathers must be stored as dry as possible, so do the egg shell, hair, teeth and bone. If the preservation method is not proper, then the chance for the degradation of the samples is very high. These degraded samples will not give correct bands. (Silvy 2012). Depending on the species of interest, destructive sampling, non-destructive sampling is done. Destructive sampling includes sampling the wild animals by killing and collecting their muscle, liver, heart and embryo tissue. Non-destructive sampling refers to collection of genetic sample without sacrificing the animal such as feather, blood, shell membrane, skin, hair, feces and urine. The samples thus obtained are stored in ice, dessicated, refridgerated and stored in a buffer. (Silvy 2012). CHD1 gene is shared by the sex chromosomes in the non recombining part. The coding sequence of the CHD1 gene is also shared by the Z and W sex chromosomes CHD1Z and CHD1W. (Ellegren, 2001).These two genes work independently but have highly conserved nucleotide sequence. The percentage of identity was 82.9% in the CHD-W gene of the Gallus gallus male and female. (Griffiths and Korn 1994). ( Ellegren 1996). CHD1 gene is used as the universal marker for the determination of sex among the Gallus gallus. Forward and reverse primers for the CHD1 gene are used for the analysis of the gene. These universal markers simplify the task of sex determination in the birds. These molecular markers help us to identify the rare monomorphic birds and increase their population. (Hu et al. 2003). For nestling of the correct sex, universal markers are very useful. Thus Universal markers are used for the identification of the sex in Gallus gallus. References: Braun, CE., 2004. Techniques for Wildlife Investigations and Management, Wildlife society. Ellegren, H., 1996. First Gene on the avian W chromosome (CHD) provides a tag for universal sexing of non-ratite birds, Proceeding/Biological Sciences, Royal Society, Vol. 263, No.1377, pp. 1635 – 41. Ellegren, H., 2001. Hens, cocks and avian sex determination: A Quest for Genes on Z or W?, EMBO reports, Vol.2, No.3, pp.192 – 196. Frankham, R., Ballou, JD and Briscoe, DA., 2004. A Primer of Conservation Genetics. Cambridge University press. Fridolfsson, AK and Ellegren, H., 2000. Molecular Evolution of the Avian CHD1 genes on the Z and W Sex chromosomes, Genetics, Vol.155, No.4, pp.1903-12. Griffiths, R and Korn, RM., 1994. A CHD1 Gene is Z chromosome linked in the Chicken Gallus Domesticus, Gene, Vol.197, No.1-2, pp. 225 - 229. Herrmann, B., 1994, Ancient DNA: Recovery and Analysis of Genetic Material from Paleontological, Archaeological, Museum, Medical, and Forensic Specimens, Springer. Huffman, JE and Wallace, JR., 2012. Wildlife Forensics: Methods and Applications, John Wiley and sons. Hu, RY., Geng, X., Ma, J., Chen, YS., Li, ZK and Ding, XY., 2003. A Simple and Universal method for Molecular Sexing of Birds, Shi Yan Sheng Wu Xue Bao, Vol.36, No. 5, pp. 401- 4. Silvy, NJ., 2012. The Wildlife Techniques Manual: Volume 1: Research. Volume 2: Management, JHU press. Read More