Extraction and Separation of Biological Substances - Coursework Example

PRACTICAL EXERCISE FOUR EXTRACTION AND SEPARATION OF BIOLOGICAL SUBSTANCES Introduction Research in life sciences involves complex structures and molecules that are associated with biological materials like nucleic acids, proteins and other molecules like tissues and cell organelles. Isolation of biological materials is therefore necessary to achieve advances in medical diagnostics and proper treatment through application sin therapeutic monitoring of the environment and industrial processes. Analysis of the isolated biomolecules is crucial to the foundation of molecular biology. The isolated nucleic acids can be used in gene expression studies and in the medical field depending on the efficiency of separation, quality and quantity. Extraction of biomolecules is a tedious process as it involves cell lysis through mechanical or chemical actions to get the crude extracts which must be purified to separate the biological molecule of interest from many other biomolecules through application of techniques like gel electrophoresis, chromatography and dialysis. Proteins are better separated according to their size through SDS-PAGE which ensures that the proteins have similar charges (negative) so that they can migrate towards the positive charge with restricted movements because of their size. The protein that moves the longest distance from the well is the smallest protein in molecular weight while the one that moves the shortest is the heaviest protein in molecular weight. DNA fragments can also be separated through electrophoresis but they first have to be cut using restriction enzymes. The size of DNA can then be estimated depending on the number of base pairs it contains. In this experiment we are going to use SDS-PAGE electrophoresis to separate proteins and use the standard curve to determine the size of the unknown proteins. Further, DNA will be extracted and purified from bacterial cells (E. coli) then separated using gel electrophoresis. DNA analysis will be performed using pure laboratory DNA after restricting the DNA strands. Results Discussion The practical involved use of SDS-PAGE to separate proteins based on their molecular size. In order for this to be, the proteins have first to be denatured such that they no longer have the secondary, tertiary or quaternary structure (Rosenberg, 2005). This is done through heating the protein mixture over steam ensuring that the proteins have the same linear shape for the experiment to be viable. Sodium dodecyl sulfate (SDS) is a detergent that can dissolve the hydrophobic molecules and so if cells are incubated with SDS, its membranes will be dissolved, proteins solubilized and covered in negative charge. This is so that when the proteins are run through the gel, they migrate towards the positive pole because they will be negatively charged (Walker, 2002). Polyacrylamide gel allows protein molecules to move at different rates when they are loaded unto the gel and same charge applied to all of them. The smaller sized molecules are able to move faster through the gel unlike the larger molecules therefore separating the proteins in accordance to their size. Caution has to be taken so that the proteins do not ran out of the gel. After running the proteins, they have to be stained so that they can be visible to enable measurement of distance from the well (Garrett, & Grisham, 2010). As in the case with our experiment, lactalbumin and bovine aprotinin proteins ran out of the gel because they were too small and so they moved very fast through the gel. The heaviest protein was myosin from rabbit muscle which just moved a millimeter from the well. This indication shows that it has more amino acids in its primary structure compared to other proteins. In DNA extraction the first step is obtaining cells which contain DNA. In this case we used calf tissue and bacterial cells. For calf tissue, it had to be grinded to make it easier to get the cells with DNA to get a crude extract that had to be purified (Itoh, Takahashi &Uemura, 2009) The membrane lipids are then removed through solubilizing them in detergent. It is important to remove proteins which act as contaminants through adding proteases or by precipitating the proteins with acetates then DNA can be precipitated with ice-cold absolute ethanol (Carrigg et al, 2007). Chelating agents are added to ensure that the DNAse does not degrade the DNA that is extracted from the cells (Tortajada et al, 2009) The extracted DNA can then be used in different ways like in forensic science or genetic modification. DNA fragment analysis is the procedure of estimating the size of DNA (Suresh & Seong, 2009). It involves using DNA of known size to determine the size of the unknown through construction of a standard curve. The importance of isolation of biomolecules is so that they can be used in further experimentation. Proteins can be separated and used to determine their functions when their amino acid structure is analyzed so that progress can be achieved in the field of medicine. The samples can be used for hereditary studies which determine common ancestry depending on the similarities or differences between the protein and DNA in the organisms (Solomon, Berg & Martin, 2005). Questions Gel electrophoresis of proteins Question 1 Our gel separation of protein based on size was successful because all bands could be identified. This is an indication that the samples used were pure. However, some proteins like the lactalbumin and bovine aprotinin ran out of the gel, an indication of their small size. Question 2 Sample A had one band indicating that the plasma protein present had similar molecular weight as B-Galactosidase (116000) based on the distance traveled by sample A. The results show that Johnny does not have enough normal fibrinogen and will need FFP Sample B Sample B had only one band, which is of trypsinogen obtained from bovine This indicates that the sample extracted from the cow’s pancreas was pure. One can tell the purity of the trypsinogen if the sample has only one band indicating that no other protein is present in the extract. Sample C Sample C had one band of myosin from rabbit muscle. Its presence indicates muscle wasting which indicates cachexia. Myosin Sample D The results indicate that there is no presence of GDH meaning that Suzy’s sample is not usable. Mutations in GLUD1 can be identified by direct sequencing which is the most sensitive technique for identifying mutations. Exons of GLUD1 gene which encode the allosteric and catalytic domains of GDH are amplified using PCR and sequenced using BigDye Terminator v3.1. The sequencing reactions can then be analyzed on ABI 3730 and compared with published sequences like M37154 using Mutation Surveyor software. DNA extraction (Question 3) The DNA extraction techniques were successful because we were able to obtain the DNA from E. coli and calf tissue. (Question 4) There are different techniques for extracting DNA from bacterial cells and from eukaryotic cells. This is because in eukaryotic cells there is a nuclear membrane that has to be lysed to access the DNA through grinding. The prokaryotic DNA is suspended in the nucleoid region making extraction easier in prokaryotes than eukaryotes. (Question 5) DNA extraction can be used in genetic engineering of plants and animals through gene splicing, mutation detection, DNA sequencing and gene localization (Aygan, 2006). It can also be used to gather evidence in crime scenes for forensic DNA matching and DNA fingerprinting in addition to being used to diagnose medical conditions like cystic fibrosis, hemophilia A, Down’s syndrome, Tay-Sachs disease, sickle-cell anemia, Huntington’s disease and fragile X syndrome. (Question 6) Though DNA is useful in forensic studies the ethics of who should have the access to genetic information. Psychological impact and stigmatization resulting from the genetic differences in individuals is not considered. When being used in reproductive studies, it is unknown if the parents know of the risks and limitations of genetic technology. Analysis of DNA fragments (Question 15) The method that is more precise in determining the fragment size of DNA is the one of measurements on the semi-log graph (Laird, 2010). This is because the values are extracted from the graph through constructing perpendicular lines to the curve then reading the figure on size of base pair coinciding with the distance travelled by the sample. Directly reading the distance from the gel is tasking because at times bands may overlap making it hard to get precise figures. (Question 16) Errors that may affect the results of the experiment include the DNA extract not being pure due to contamination resulting from the equipment used. The quantity of DNA might be too small to be restricted effectively. When constructing the standard curve, some points may not be indicated accurately which means that the standard curve is misleading. The experimental errors can however be minimized by ensuring that the material used are sterile and that accuracy is maintained when constructing the standard curve. References Aygan, A. 2006. Nucleic Acid Extraction from Clinical Specimens for PCR Applications.Turkish Journal of Biology, Vol. 30 Issue 2, p107-120. Carrigg, C., Rice, O., Kavanagh, S.,Collins, G. and O'Flaherty, V. 2007. DNA extraction method affects microbial community profiles from soils and sediment. Applied Microbiology & Biotechnology, , Vol. 77 Issue 4, p955-964, DOI: 10.1007/s00253-007-1219-y Garrett, R and Grisham C. M. 2010. Biochemistry. 4th Edition. Mason: Cengage Learning. Itoh, Y., Takahashi, K. and Uemura, Y. 2009 Solvent Extraction of DNA with a Hydrolysable Double-chain Surfactant. Separation Science & Technology, Vol. 44 Issue 2, p466-475, DOI: 10.1080/01496390802437222 Laird, P. W. 2010. Principles and challenges of genome-wide DNA methylation analysis. Nature Reviews Genetics, Vol. 11 Issue 3, p191-203, DOI: 10.1038/nrg2732 Rosenberg I. M. 2005. Protein analysis and purification: benchtop techniques. Springer Solomon, E.P., Berg, L.R. and Martin, D.W. 2005. Biology. Thomson Learning, Inc.: Belmont, CA. Suresh, K. K. and Seong, H. K. 2009. Microchip-Based Capillary Electrophoresis for DNA Analysis in Modern Biotechnology: A Review Separation & Purification Reviews, Vol. 38 Issue 3, p.p.242-288, DOI: 10.1080/15422110903095136 Tortajada, M., Martínez-Culebras, P., Navarro, V., Monzó, H. and Ramón, D. 2009 Evaluation of DNA extraction methods for PCR detection of fungal and bacterial contamination in cocoa extracts. European Food Research & Technology, Vol. 230 Issue 1, p79-87, DOI: 10.1007/s00217-009-1139-4 Walker J. M. ed. 2002. The protein protocols handbook. 2nd Edition. Totowa, NJ: Humana Press. Read More