DNA Extraction and Polymerase Chain Reaction of Bird DNA for Sex Identification - Lab Report Example

Name Tutor Date Introduction The methods of collection, storage and tissue types may affect the quality of DNA. The quality of the starting samples affects the yield and quality of the DNA isolated. The best results can be obtained from a fresh material. If the material is not used immediately, it can be frozen immediately in a mixture of ethanol and dry ice or in liquid nitrogen and stored at low temperature of -20°C or -70°C. Repeated freezing and thawing of stored samples leads to precipitation of the DNA and reduced fragment size. Also, the yields of genomic DNA decrease if the material samples are stored at -20°C without earlier treatment. Heterogametic sex is the sex of a species whose sex chromosomes are not similar. In birds’ species, females are heterogametic. They carry a copy of both Z and W sex chromosomes, but males are homogametic and carry 2 similar copies of the Z sex chromosome (Baquero et al., 2009). Most chicks and species of birds are sexually monomorphic and cannot be easily be distinguished as females and males from their phenotype (Takagi et al., 1972; Ogawa et al., 1998; Jensen et al., 2003). Universal markers can be used to genetically determine the sexes of all species of birds or their young ones. The molecular markers targets the highly conserved primer flanking section in the chromo - helicase - DNA binding gene 1 - CHD1, in the birds’ sex chromosome. The sex identification can be determined by detecting the difference in size between the CHD1 on the Z sex chromosome (CDH1Z) and CHD1 on the W sex chromosome (CHD1W) (Doosti et al., 2009). This can be done through extraction of genomic DNA from a small tissue sample like feathers, blood or any other tissue of a species and then the CHD1 genes is amplified using PCR. The results of PCR product is visualized using gel electrophoresis. Tissues from male individual will produce one band, while those from female individuals produce two bands on the gel (Caetano and Ramos, 2008). The use of PCR in the genetic sexes identification of birds is ideal since it requires just a small sample, like a blood drop or a feather for DNA extraction, minimizing individual bird’s trauma (Itoh et al., 2001). The technique is cheap, high sensitive and smart for sex identification of monomorphic birds. On the other hand, other techniques such as avian laparoscopy and laparotomy, biochemical surgical examination and others are inconvenient such as costly, time consuming, low sensibility or cause harm to the organism (Doosti et al., 2009). The aim of this study was to determine the sex of individual species using different samples DNA from blood, muscle and feather of Gallus gallus. The concentration of the extracted DNA was estimated and CHD1 gene was amplified using polymerase chain reaction (PCR). The PCR product was then visualized on an agarose gel to determine the sex of the sample. This method of sex identification is a valuable when sexing the genetic samples from different species. It is applicable to other closely related species given the conserved nature of this DNA region. Sex identification is important when rearing birds especially in many commercial units, due the need for different goals (Thear, 1998). Method In this experiment, DNA from different tissue types of blood, muscle and feather was extracted, in order to determine the sex of a domestic chicken (Gallus gallus). In the first part of the experiment, the DNA samples were purified using a commercial DNA purification kit called Qiagen DNeasy Blood & Tissue Kit. Each sample type was first lysed using proteinase K and then buffering conditions are adjusted to give appropriate optimal DNA binding conditions. The lysate mixture was loaded onto the DNeasy Mini spin column and Centrifuged for 1 minute a speed of 6000 x g (8000 rpm). The flow-through liquid was discarded into the hazardous waste container. The collection tube into the tip was also discarded. DNA was selectively bound to the DNeasy membrane as contaminants pass through during centrifugation. Remaining contaminants and enzyme inhibitors were removed in two efficient wash steps. The column was incubated at a room temperature for 1 minute, and then centrifuged for 1 minute at a speed of 6000 x g (8000 rpm) to elute DNA. DNA was eluted in buffer or water, ready to be used in the next practical. Finally, the tube containing the DNA was placed in the esky at the front of the lab and all samples are stored at a temperature of -20°C until the next practical. 10 μL of diluted DNA, water or neat DNA was added into the tube. One male (♂) control, one female (♀) control and one negative control are included. The prepared PCR tubes was then placed into the thermocycler and a program optimized run to amplify the CHD1Z and CHD1W gene variants using the a set of universal avian sexing primers 2250F and 2718R developed by Fridolfsson and Ellegren (1999). The technique used to separate DNA molecules on a gel matrix is called Gel electrophoresis. A comb inserted into one end and then molten gel was poured into a casting tray. When the comb was removed it left behind small wells. The gel was placed into an electrophoresis tank and then the reservoir was filled with a buffer; electrophoresis buffer. The DNA was mixed with the loading dye then the samples are loaded into the wells. Since the loading dye solution is a dense, DNA sinks into the well that contains coloured indicator dyes. The process of electrophoresis was then monitored as the dyes travel at a predictable rate through the gel. To visualize the DNA, a small amount (0.5µg mL-1) of the chemical ethidium bromide was added into the gel. The bands produce are then visualized. The brightness of the sample on the gel was compared to the various bands in the molecular weight marker, in one of the wells. The concentration of the sample was obtained by marching the brightness of the of the DNA band to the brightness of the band in the standard. Also the size of the sample was estimated by comparing the band of the sample with the size in the standard. Results Section 1 Figure 1. Figure 1 above shows the concentration of DNA in the three tissues types. A DNA concentration standard (molecular weight marker) was included in one of the wells. The concentration and size of DNA fragment was estimated by comparing sample band with the size marker. Results are recorded in table 1. In figure 1 Wells 3, 6 and 14 does not show any band. DNA concentration determination The concentration of DNA in the sample use the following calculation ng DNA in MWM reference band = ng/µL µL of sample DNA Table 1. The concentration of DNA compared to the reference band 2 3 4 5 6 7 8 9 10 11 M12.29 6.56ng B12.23 F12.21 2.03ng B12.22 2.32 F12.20 B12.24 0.56ng M12.30 6.56ng F12.18 4.36ng M12.25 23.13ng B12.20 4.36ng 12 13 14 15 16 M12.269.42ng F12.19 2.03ng B12.21 M12.28 6.56ng M12.27 2.32ng Concentration of DNA in blood sample 2.413 ng = 0.2413 ng/µL 10 µL Concentration of DNA in muscle sample 9.09 ng = 0.909 ng/µL 10 µL Concentration DNA in feather sample 2.87 ng = 0.287 ng/µL 10 µL Table 2. Combined results Blood sample Muscle sample Feather sample 0.2413 ng/µL 0.909 ng/µL 0.287 ng/µL There is difference in the concentration of DNA extracted from the three types of tissues as shown in table 2. The muscle sample has the highest concentration of DNA of approximately 0.909 ng/µL followed by the feather that has DNA concentration of 0.287ng/µL. The blood sample has the least DNA concentration of 0.2413ng/µL. Section 2 The figure below shows the gel showing the PCR product for the blood, muscle and feathers tissues, positive controls for male and female, negative control and size marker. Figure 2 The figure 2 above shows an image for sex identification based on intronic length polymorphism on agarose gel. The PCR was amplified with sexing primers 2250F and 2718R developed by Fridolfsson and Ellegren to determine the sex of the domestic chicken form the three samples. The known-sex sample is indicated MALE and FEMALE. The male sample has one band while the female sample has two bands. Column 1 contains size markers. Columns 2 - 11 show samples from different single DNA extractions. The sex of the samples shown on the Gel: Table 1 2 3 4 5 6 7 8 9 10 11 B12.22 Male M12.30 Male F12.20 Male B12.24 Male Control Control Male Control Female M112.29- Female F12.21 Female B12.23 Female B12.21 Female M1228 Male F12.19 Female M12.27 Female Control Male Control Control Female M12.25 Female F12.18 Male B12.20 Male Polymorphism is only shown in the 2550F/2718R region. The approximate base pair size of the W and Z genes is 1000bp. The number of samples for male was 9 while number of females was 11. Amplification of the feather samples was found to be inferior to those obtained from blood and muscle. Sample F12.21 at column 9 in figure 2 was not amplified. Also the negative controls were not amplified. The results obtained from the PCR does not seemed to correlate with the actual sex of the individual, for example column 10 with sample F12.18 indicated in the table that it is a male but it has more than one band in the gel in figure 2. Also control sample in column 6 has two bands which show that it a female, but the sample type is indicted as a male. Discussion There was an obvious difference in the concentration of DNA extracted from the three types of tissues. The muscle sample has the highest concentration. The feather sample has more concentration than the blood sample. The result is not consistent with the expectation. The blood is expected to have more concentration of DNA than muscle sample and feather sample. The feather sample is expected to have the least concentration. The absence of visible band on the gel in figure 1 Wells 3, 6 and 14 in the practical does not necessarily mean that the extraction was unsuccessful. It depends on a number of reasons than affect the quality of the starting materials like storage. If the sample is not stored properly at a temperature of below 20oC the material is degraded. It also depends on the size of sample and the efficiency of extraction. The DNA might be present, but not enough to stain well. Genomic DNA extraction may yield very large pieces of DNA or pieces of random sizes. The large stuff produce a visible band, and the smaller size stuff will just produce a smear, which may not have a distinct band, especially if the DNA sample is degraded (Itoh et al., 2001). It is important to store samples that are to be used for genetic analysis correctly since it affects the quality of DNA. Incorrect storage may result in DNA degradation. The starting materials should be of high quality as it affects the yield of DNA. The best results are obtained when fresh materials are used or when the materials are immediately frozen at a temperature -20oC or -70oC in frozen liquid nitrogen or a mixture of ethanol and ice. Repeated frozen and thawed samples may reduce the fragment size and precipitation of the DNA, and may also reduce yield of DNA. DNA is also subjected to acid hydrolysis if they are stored in water. It should be stored at a slightly alkaline pH like in Buffer AE from QIAGEN or TE buffer. Previous treatment is important because poor quality starting materials may reduce the yield and length of DNA (Itoh et al., 2001). Poor storage may also lead to chemical modification of DNA which results in decrease in the quality of DNA and shorter fragments upon purification. This may also explain the absence of the visible band on the gel for a feather sample obtained in this experiment (Baquero, 2009). DNA was observed after the addition of a small amount of 0.5µg mL-1 of ethidium bromide chemical to the agerose gel. This chemical intercalates with DNA molecules and fluoresces under UV light, which enabled DNA to be visualised as fluorescent bands in each lanes. When the electric current is applied to the gel, the negatively charged DNA molecules move through the gel matrix toward the positively charged anode, and away from the negative charge of the cathode. Smaller DNA molecules move through the gel at a faster rate than larger DNA molecules. The universal primers, 2550F/2718R used in this experiment were used to amplify the two band pattern that was used to identify the sex of an individual. It amplified the regions of the sex-linked CHDZ and CHDW genes enabling the identification of monomorphisms and polymorphisms in the region of the CHDZ. Females sample showed two bands in agarose gel analysis and were different from the male sample which showed one band (Itoh et al., 2001). Invasive sampling method involves capturing of specific individuals. The method can be beneficial to endangered population and in behavioral studies. It enables sampling of a large number of animals even in secretive species, making it possible to estimate important population parameters and obtain more information. The use of feces can provide important information on pathogens, reproductive status and diet. On the other hand, the method is stressful to the population and the quantity of DNA obtained through this method is generally not large enough and is of low quality and quantity. Destructive method destroys the individual (Horvath et al., 2005). There are a number of advantages of using universal sexing molecular markers. It is used in sex identification, genetic and infectious disease identification, parentage testing and marker assisted selection. The use of feathers sample instead of blood sample as a genomic DNA source simplifies sampling and reduces the stress on the bird, specifically when analysing large bird species like ostrich. In some cases, the bird’s size is small like parrots and juvenile bird; therefore, the blood vessels are small in size making the extraction process to be difficult. In addition, since such DNA-based sex identification can be performed on plucked feathers, and involves no anesthesia or surgery, it means that it can be performed without the help of a veterinarian (Griffiths et al., 1996). DNA analysis can be performed in all species of birds and on any age of bird, and this enables the sex identification of young birds, which can be coupled to an early diagnosis characteristics linked to gender. The information of the sex of bird obtained from this analysis has a high accuracy of over 99% and the information enhances the value of the bird. Sex determining of animals in natural environment is important for a number of reasons such as understanding population structure and dynamics. DNA examination used as a tool for sex identification may be applied to biological samples of unknown sex, like preserved non-invasively collected specimens (Hauge, 1997). References Baquero A., Puerta A., Gutierrez G., (2009). Magnitude Effects of Sexual Reinforcement in Japanese quail (Coturnix japonica). Int. J. Comp. Psychol. pp, 113-126. Caetano L. C., Ramos ES (2008). MHM assay: Molecular sexing based on the sex-specific methylation pattern of the MHM region in chicken. Conserv. Genet. pp, 985-987 Doosti A,, Fathpour H,, Moshkelani S., (2009). Sex identification in the Canary using DNA typing methods. BJVM, pp, 207-211. Freeland, 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 Griffiths R., Daan S., Dijkstra C., (1996). Sex identification in birds using two CHD genes. Proc R Soc Lond B pp, 1251-1256. Hauge J. G., (1997). From molecular genetics to diagnosis and gene therapy. Adv Vet Med pp, 1–49 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. Jensen T., Pernasetti F. M., Durrant B., (2003). Conditions for rapid sex determination in 47 avian species by PCR of genomic DNA from blood, shell membrane blood vessels and feathers. Zool. Biol. 22: 561-567. Itoh Y., Suzuki M., Ogawa A., Munechika I., Murata K., Mizuno S., (2001). Identification of the sex of a wide range of carinatae birds by PCR using primer sets selected from chicken EE0.6 and its related sequences. Am. Genet. Assoc. 92: 315-321. Read More