DNA Extraction and Purification - Lab Report Example

INTRODUCTION All life forms bear genetic material, chains of deoxyribonucleic acid (DNA) that for their characteristics, including biological sex. Each characteristic has two or more traits, and for organisms that undergo sexual fertilization, having one over the other depends on several factors. During fertilization, the combination of genes from the ovum and sperm cell (heterogametes) should be the first determinant of trait. For humans, the heterogametic XX and XY, each letter corresponding to the gene coming from a parent, is the definitive determinant of biological sex, and is the code for female and male trait, respectively. The effects of other processes, such as genetic imprinting, are important in determining the traits for other characteristics of the organism. Sequencing the genetic material of a species, and determining all coding sequences and their corresponding proteins are vital projects in the field of molecular biology. The human genome project had been successful in sequencing the DNA of humans (Venter et al., 2001). The studies of (Kyrylkova et al., 2012; Yu et al., 2012) determine the characteristics corresponding to a particular genetic sequence by preventing the transcription of the latter and observing the changes afterward. A sequence identified to determine a particular characteristic can also be used as a molecular marker to determine the presence or absence of trait, even without actually seeing it in the organism. For example, markers for sex can help distinguish between males and females in monomorphic species or their young. The objective of this experiment was to demonstrate the use of DNA samples to determine the characteristics of the organism, particularly its sex. Specifically, the activity aimed to isolate DNA from different sources, blood, muscle and feather. After purification, the DNA isolates underwent polymerase chain reaction (PCR) using sexing primers 2250F and 2718R to replicate the sequence for sex determination. Agarose gel electrophoresis of the PCR products were compared with that of known male and female samples to identify the sex of the chicken from which the samples were obtained. METHODS DNA Extraction and Purification Qiagen DNeasy Blood and Tissue Extraction Kit was used for extraction. Briefly, the tissues sample was lysed by incubating it in a solution containing 20 µl proteinase K, 4 µl RNAse A and 166 µl phosphate buffer solution (PBS) (blood) or 180 µl Buffer ATL (muscle or feather) for 30 min. After mixing with 200 µl Buffer AL for 15 sec, the DNA was precipitated by addition of 200 µl 95% ethanol. The resulting solution was then loaded onto the spin column, and centrifuged. Contaminants were removed further through centrifugation with 500 µl buffer AW1 and AW2, separately. The DNA in the membrane was incubated with 100 µl Buffer AE for 1 min, and the solution was centrifuged to elute the DNA. All centrifugation were done at 8000 rpm for 1 min. The eluent containing the DNA was stored at -20°C. In the visualization of isolated DNA through agarose gel electrophoresis (AGE), 150 ml 1% agarose gel with 0.5 µg/ml ethidium bromide was prepared, and was poured onto the casting tray inside the gel tank, the comb was placed to produce loading wells on the gel. Meanwhile, 10 µl DNA sample was mixed with 4 µl 6x loading dye. 5 µl molecular weight markers λ HindIII and 2-log ladder (1.0 µg) were loaded into the first and last wells, and the same amount of DNA samples were loaded in between. Electrophoresis was run at 120V for 60 min, after which the gel was visualized using the Gel Doc apparatus. PCR and Visualization In the PCR, 10 µl DNA solution and 40 µl PCR master mix were mixed in 0.2 ml PCR tube. The primers used were 2550F and 2718R to amplify CHD1W or CHD1Z gene variants. The tubes underwent PCR in these conditions: 94°C for 5 min (1 cycle), 94°C, 55°C and 72°C for 30 sec each (35 cycles), and 72°C for 5 min (1 cycle). PCR products underwent electrophoresis and visualization. RESULTS Visualization of DNA Extracts As seen from figure 1, 4-6 ng/µl genomic DNA can be extracted from blood or muscles. In contrast, minimal, if any, were obtained from feathers. The size of this extracted DNA is approximately 23 kbp. Visualization of PCR Products Amplification of CHD1W or CHD1Z gene variants was shown in figure 2. Negative (-VE) controls in wells 7 and 14 for 1st gel and 7 and 15 for 2nd gel did not show any bands. The male and female controls are characterized by having 1 and 2 bands having 0.9 to 1.2 bands, respectively. Based on these controls, the sexes from which the samples extracted were derived. For the first gel, samples in wells 2, 5, 8-10 and 13 were obtained from male chickens, while samples in wells 3, 4 and 11 are from females. On the other hand, wells 2, 4, 8, 11 and 17 for the 2nd gel are from males, and those in 3, 5, 9, 10, and 12 are from females. Despite the difficulty in detecting DNA from isolates themselves, PCR of DNA extracts from all tissue types produced detectable bands (figure 2). DISCUSSION There are many reasons that no bands were seen from some DNA extracts, particularly those from feathers. First, the improper storage of DNA may result to its degradation from nucleases that contaminate the solution. However, greater amounts of DNA can be obtained from blood and muscles than from feathers. This is because the former two have a higher cellular content than the latter. In fact, most structures of the feather are acellular, except its superior tip. Even then, it is still a poor source of DNA. In comparing muscles and blood, the latter is a better DNA source because the mass of muscle is based on cell enlargement, and not cell division (Rawlence et al., 2009). However, obtaining feathers is the least invasive way of extracting DNA from chicken. Getting blood or muscles needs intravenous or dissection of the bird. For female chicken-derived extracts, the two bands correspond to ZW chromosomes, while those from male have one band, corresponding to two Z chromosomes. The band with a bigger weight is Z, which is about twice as large as W. Such distinction between males and females becomes an effective measure of determining the sex of a monomorphic organism (Mendonca, Carvalho and Clarindo, 2010). Sexing is important in industries such as poultry, agriculture and cockfighting. It helps in the determination of which eggs to cultivate for hatching. The primers used in this experiment are universal, in that it can be found in sex-determining gene sequences of almost heterosexual organisms. However, it should be noted that using 2550 as a primer is not optimally efficient in amplifying sex-specific introns, and thus may result to inconclusive results, especially in samples that do not contain a lot of DNA material (Liu, Zhao and Li, 2010). REFERENCES Kyrylkova, K., et al. 2012. Determination of gene expression patterns by in situ hybridization in sections. Methods Mol. Biol., 887, pp. 23-31. Liu, W., Zhao, C. and Li, J. 2010. A Non-Invasive and Inexpensive PCR-Based Procedure for Rapid Sex Diagnosis of Chinese Gamecock Chicks and Embryos. Journal of Animal and Veterinary Advances, 9(5), pp. 962-970. Mendonca, M. A. C., Carvalho, C. R., and Clarindo, W. R. 2010. DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry. J. Histochem. Cytochem.,58(3), pp. 229-235. Rawlence, N. J., et al. 2009. DNA content and distribution in ancient feathers and potential to reconstruct the plumage of extinct avian taxa. Proc. R. Soc. B., [online] Available at: [Accessed 01 October 2009]. Venter, J. C., et al. 2001. The Sequence of The Human Genome. Science, 291(5507), pp. 1304-1351. Yu, M., et al. 2012. Deficiency in nucleotide excision repair family gene activity, especially ERCC3, is associated with non-pigmented hair fiber growth. PLoS One, 7(5), pp. e34185 Read More