Bird's Sex Identification Using DNA Markers - Lab Report Example

LAB REPORT WRITTEN ASSIGNMENT: DNA EXTRACTION AND PCR OF BIRD DNA FOR SEX IDENTIFICATION Your name Subject Date Introduction: In this study, DNA was isolated from three types of samples these are: blood, feathers and muscle tissue. The collection of blood as a sample for DNA extraction could be from a live bird or a dead specimen. The sample should be fresh to maintain high quality and quantity of DNA and to prevent contamination. Fresh blood samples can be stored in 95% ethanol at room temperature or frozen. Feather samples could be obtained by plucking the bird and these provide a less painful collection method than drawing blood. The feathers should be stored in a sterile sample bag immediately they are plucked from the bird to avoid contamination. Muscle tissues are extracted from dead birds and immediately frozen for preservation of quality. When freezing of tissues is not possible, one can place minced tissues in a vial of buffer (Seutin at el. 1991) or place small pieces into 95% ethanol for dehydration. Ultra-cold freezing represents the closest thing to a museum-quality tissue preservation method. Generally determination of the sex of birds is quite difficult before puberty but in monomorphic species it is difficult even after puberty. Chickens are difficult to sex morphologically hence the use of PCR for sex identification. The Z and W sex chromosomes evolved differently in birds from the mammalian X and Y chromosomes. In Chickens females are heterogametic and carry a copy of Z and W sex chromosomes but males are homogametic and carry 2 copies of the Z sex chromosome. The most critical question in sex identification of chickens is how the 2 types of sex chromosomes play a role. It is not clear yet if female characteristics are developed by female specific W chromosomes or male characteristics are defined by the dose of chromosome Z. Even though it is female specific, the W chromosome has a similar structure to the mammalian male Y chromosome, except for its poorer gene structure, smaller size, and richness in heterochromatin and repeat sequences. There are 2 genes defined on the W chromosome. These are chromo helicase DNA binding protein (CHD1W) and ATP synthesis α-sub unit (ATP5A1W). Both genes are located in the non-recombined part of the W chromosome and their similar homologues (CHD1Z and ATP5A1Z) exist in the Z chromosome. This study aims to help breeder identify the sex of chickens without having to wait for the physical characteristics to manifest as they grow. The breeders normally have large numbers of birds and it would be tedious to physically inspect each bird for its sex. Method: Purification of genomic DNA from blood, muscle tissue and feather sample. Protocol 1a The purification of genomic DNA from preserved blood was as follows: after pipetting 20µl of proteinase K into a sterile 1.5 ml microcentrifuge tube,166µl of PBS and 4µl of RNase A was added into the tube. A sample of preserved chicken blood was transferred using sterile forceps. This was incubated for 30 minutes at room temperature before adding 200µl of buffer AL and then it was mixed thoroughly using vortexing for 15 seconds.A lid lock was placed on the tube and it was further incubated for 25-45 minutes. 200µl of 95% ethanol (AR grade) was added to the sample and thoroughly mixed by vortexing for 15 seconds. The DNA precipitated and was ready to be bound to the membrane. Protocol 1b The purification of genomic DNA from muscle tissue was as follows: a sterile razorblade was used to macerate the muscle tissue of approximately 20mg (the size of a match stick head) in a sterile petri-dish. The tissue was then transferred into a sterile microcentrifuge tube.180 µl of buffer ATL was added to the tissue and was vortexed for 15 seconds. 20 µl of Proteinase K was added next a mixed thoroughly through vortexing for 15 seconds and was incubated at 56º C for 30 minutes. It was vortexed every 10 minutes during the incubation. After removal from the incubator the sample was vortexed again for 15 seconds. 200µL Buffer AL was added to the sample and mixed thoroughly by vortexing for 15 seconds. Added 200 µl ethanol (96-100%) was added and thoroughly mixed by vortexing for 15 seconds. The DNA precipitated and was ready to be bound to the membrane. Protocol 1c A small section of the feather shaft was cut using sharp razor blade. The pieces of feather were put in a sterile 1.5 ml microfuge tube containing 180 µl of Buffer ATL. 20µl of proteinase K was added and mixed thoroughly through vortexing for 15 seconds and was incubated at 56ºC for 30 minutes. The sample was vortexed briefly every 10 minutes during incubation to disperse the sample.After incubation it was vortexed fro a further 15 second before adding 200 µl Buffer AL to the sample. It was vortexed for 15 seconds before adding 200 µl ethanol (96-100%) and again mixed by vortexing for 15 seconds. The DNA had now precipitated and was ready to be bound to the membrane. PCR The liquid in protocol 1a, 1b and 1c was then transferred separately into 3 DNeasy spin columns without touching the white membrane at the bottom of the tube. It was then spun for I minute at 6000x g(8000 rpm) and it was then ready for washing. After discarding the flow-through liquid and collection tube, the DNeasy mini spin column was placed in a new 2ml collection tube and 500µl Buffer AW1 was added. This was centrifuged for 1 minute at 6000 x g(8000 rpm). The flow through container was discarded in the hazard waste container and the collection tube into the tip discard. The DNeasy mini spin column was placed in a new 2ml collection tube and 500 µl Buffer AW2 was added. It was centrifuged for 3 minutes at a maximum speed of 13-14000 rpm to dry the DNeasy membrane. The DNeasy mini spin column was then removed carefully so as not to touch the flow through liquid. The collection tube was emptied into a hazard waste container and the spin column was placed back into the collection tube and centrifuged for 1 minute at a maximum speed of 13,000- 14,000 rpm. The DNeasy mini spin column was placed in a clean labeled 1.5ml microcentrifuge tube. 100 µl of Buffer AE was added directly onto the DNeasy center of membrane without touching it with the tip of the pipette. The column was incubated for 1 minute at room temperature and the centrifuged for a further 1 minute at 6000 x g8000 rpm to elute. The column was then discarded. For the PCR of the DNA a tube of master mix for control was prepared for the whole group. Each person in the group added 40µl of the PCR master mix into a 0.2 ml PCR tube, 10µl of diluted DNA/ neat DNA or Water was added into the tube. One male control and one female control were added for the group. The tubes were all labeled correctly. The PCR tubes were then placed into the thermocycler and run in a program optimized to amplify the CHD1W and CHD1Z gene variants with the 2550F- 2718R primer set. To prepare the gel for electrophoresis, the casting tray was inserted into the gel tank and the black gate was placed at each end of the casting tray. The gel was poured and the comb was inserted and left to set for about 30 minutes at room temperature. 10µl of PCR product was mixed with 2µl of 6x loading dye and the tube was labeled. The black casting plates and the gel comb were carefully removed. 1x SB electrophoresis buffer was slowly added until the buffer just covered the whole gel by about 5mm. 5µl of the 100bp molecular weight marker was put into the first well. The DNA sample was put into an empty well and the well position of the sample on gel was recorded as a loading guide. The cover was applied to the electrophoresis unit and connected to the power unit. 300v was run for 20min. The power was turned off and the gel tray was carefully removed from the tank and the gel was transferred to a plastic container. The gel was visualized using Gel Doc system Result from electrophoresis. Figure 1: Concentration of DNA Calculations showing how you estimated the concentration of DNA extracted from each tissue type ng DNA in MWM reference band _________________________ = ____ ng/μL μL of sample DNA Protocol 1a 120 ng= 24 ng/µl 5µl Protocol 1b 61 ng= 12.2 ng/µl 5µl Protocol 1c 32 ng= 6.4ng/µl 5µl we get, ng DNA in MWM reference band = 0.045  μg/μl (0.000045 ηg/μl) μl marker = 10 μl 0.045 0.000045 = 10 μl Sample Concentrated ng/ μl 1 Blood tissue 0.000042922 2 Organ tissue 0.000007154 3 Feather tissue 0.000000107 Blood tissue gave the greatest yield of the DNA (0.000042922). The quality of feather tissue was not of good quality than the blood tissue. In figure 1the brightness in the samples varies with the concentration of the DNA. Protocol 1a sample was very bright and bold and hence has a higher concentration of DNA which according to the calculation is 24ng/µl. Protocal 1c is not bright and has the least concentration of DNA which according to the calculation is 6.4ng/µl; this is four time less that the concentration in protocol 1a. Protocol 1a is blood sample and has the highest concentration of DNA material, the muscle tissue follows in DNA concentration then the feather had the lowest DNA concentration. M M M F M M F F M F M M F Figure 2 The results from the PCR in figure 2 show the sex of the birds, there were differences in amplification due to the quality of the DNA. The very bright bands are from the purified blood sample which had very high quality DNA. Discussion In this study the DNA was isolated from chicken blood, feathers and muscle tissue. The concentration of DNA varied in the experiment. Blood had the highest concentration of DNA followed by muscle tissue then the feather. The observation from these samples however showed that the identification of sex of the chickens could be determined from all the sample types accurately. The well at which no band appeared was the well which a sample prepared with just water was put it therefore had no DNA materials or chromosomes. Samples intended for genetic analysis should be stored correctly to maintain the quality and quantity of the DNA and to prevent contamination. Blood and organ tissue samples should be frozen for storage, this keeps them fresh and there isn’t any degradation of the DNA. Blood and muscle tissue can also be stored in 95% ethanol at room temperature for a long time. Feathers should be plucked from the bird and stored in a sterile sample bag; shed feathers should be selected picking the freshest and cleanest samples also stored in a sterile sample bag. This prevents contamination from the environment and during handling. There was no evidence of DNA degradation in any of the tissue type used in the experiment. The tissues were kept in low temperatures throughout the experiment. The different types of tissues used for genetic studies have they’re advantages and disadvantages. Blood has high concentration of DNA but the collection of the sample is invasive as it involves puncturing with a needle. In wild animals this exercise would be challenging and would involve sedating the wild animal so as to acquire a blood sample. The concentration of DNA in muscle tissue is also relatively high but to acquire a sample a sample to muscle tissue one would have to kill the animal. Feathers have a low concentration of DNA but are easy to collect using noninvasive techniques. Feathers can be plucked from the bird or collected where they have been shed off by the bird. The shed feathers should however be freshly shed. Universal markers can identify the sex of bird species, the markers target the chromo-helicase-DNA binding gene 1(CHD1) this is carried in the sex chromosome of the bird. The female is identified by CHD1W and the male by CHD1Z. By using PCR we amplify the genes and then use electrophoresis to visualize them in agarose gel using UV light. If one band is observed it is male (ZZ) if two bands are observed it is female (WZ) The experiment gave results of 6 females with double bands and 6 males with single bands. The samples proved adequate in determining the sex of the chickens. Using the universal sexing molecular markers makes it easy to identify the sex of monomorphic bird species especially in large numbers. In the case of chicken, early sex identification help to sell the right type of chick for its intended purpose. Females can be sold as layer birds and the males can be sold as broilers. The right feeding is then applied so as to get optimum result from these birds. The ability to rapidly and reliably determine the sex of birds is therefore very important for successful captive-bird breeding programs, as well as for field research. Visual inspection of adult birds is sufficient for sexually dimorphic species, but nestlings and monomorphic species are difficult, if not impossible, to sex by sight only. The study has determined that can be successfully extracted and amplified from a variety of noninvasive and moderately invasive sample type. The study has also determined which sample types yield high quality and quantity of DNA for use in bird sexing DNA. Blood samples yield high quality and quantity of DNA whilst feathers yield a lower quality of DNA but still equally effective in determining the sex of the bird and is non invasive in sample collection. Conclusion The use of non invasive techniques of sample collection in determining the sex of monomorphic birds is more effective than the use on invasive methods of sex determination like laproscopy where the bird has to undergo incisions to allow the laproscope into the body. The after effect of this type of surgery could result in swelling and blood clots which pose a further danger to the bird especially to small chicks. The invasive methods are also slow and would not work well in the commercial breeding industry or for endangered species of birds. The isolation of DNA from feathers yielded PCR products of similar quality to those derived from blood samples. The storage of bird feathers in sealed sample bags for a short period of time does not affect the ability to correctly sex the birds usind the DNA derived from the feather. List of references Baquero A, Puerta A, Gutierrez G (2009). Magnitude Effects of Sexual Reinforcement in Japanese quail (Coturnix japonica). Int. J. Comp. Psychol. 22: 113-126 Cerit H, Avanus K (2007). Sex determination by CHDW and CHDZ genes of avian sex chromosomes in Nymphicus hollandicus. Turk. J. Vet. Anim. Sci. 31(6): 371-374. 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. Freeland, J (2005) Molecular Ecology. Wiley. Chichester Griffiths R. 2002. Sex identification using DNA markers. In: Molecular methods in Ecology (BACKER A. J., Ed.), pp. 295–321, Blackwell Science, London. 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. Read More