Forensics: Isolation of DNA from whole blood.
Abstract
In this lab extraction of DNA FlexiGene DNA kit was used. The extraction process included effective disruption of cells, denaturation of nucleoprotein complexes, inactivation of nucleases and other enzymes, removal of biological and chemical contaminants, and then DNA precipitation which resulted in successful extraction of DNA pallet which was the dissolved again for storage purposes. The DNA was run on 2% Agarose gel which showed a great amount of resolution leading to the conclusion that the DNA that was extracted was enough to use for PCR and had a reasonably good quality. The quality of the DNA was determined by the clarity and how resolved the bands on the gel were. The few bands whose strands were smeared were due to poor loading techniques.
Introduction
DNA (Deoxyribonucleic Acid) is composed of two polynucleotide strands (the polymers of nucleotides). The Nitrogenous Bases in DNA store the instructions for making polypeptide chains, essentially coding for every feature of the entire organism. It is a polymer (long chain molecule) made of nucleotides. The nucleotides polymerise by forming bonds between the carbon of the sugar and an oxygen atom of the phosphate. The bases do not take part in the polymerization. DNA is unique to each person that is to say no two people have the identical DNA except for identical twins. This implies that it can be used as a tag to identify many individuals. If it is an issue with identification of an individual, it is concerned with 13 (or more) loci on the DNA molecule that are obtained from the whole DNA after it’s extraction. The 13 loci are then used to construct a DNA profile. The extraction process is very important to obtain the dna molecule (Adnan, 2016).
Extraction of DNA from whole blood is process that requires caution when handling the samples as the blood may contain a number of diseases and bloodborne pathogens. DNA can be extracted from all the nucleated cells in the body such as hair, tissue and blood among others. Erythrocytes do not have DNA because they do not have a nucleus. The best source of DNA are the leukocytes. DNA extraction method used in this lab work follows some common procedures intended to achieve effective disruption of cells, denaturation of nucleoprotein complexes, inactivation of nucleases and other enzymes, removal of biological and chemical contaminants, and then DNA precipitation. Purified DNA is required for many applications such as studying DNA structure and chemistry, examining DNA-protein interactions, carrying out DNA hybridizations, sequencing or PCR, performing various genetic studies or gene cloning. In this case the DNA to be obtained is to be used for PCR. DNA extraction generally involves five steps which are: step 1. Breaking cells open to release the DNA: Step 2. Separating DNA from proteins and other cellular debris: Step 3. Precipitating the DNA with an alcohol: Step 4. Cleaning the DNA: Step 5. Confirming the presence and quality of the DNA. Lysis: in DNA extraction refers to the breaking of the cellular membranes (most importantly, the plasma and nuclear membranes. Precipitation is a series of steps where DNA is separated from the rest of the cellular components (Griffiths and Chacon. 2014).
After extraction Agarose Gel Electrophoresis for DNA quantification and quality analysis is necessary to come up with valid conclusions and deductions. This method of quantification is based on the ethidium bromide fluorescent staining of DNA. Ethidium bromide is a fluorescent dye, which intercalates between the stacked bases. The fluorescent yield of the dye:DNA complex is much greater than the unbound dye. During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a positive and negative terminal. The DNA to be analysed is forced through the pores of the gel by the electrical current. Under the influence of an electrical field, DNA will move to the positive terminal and away from the negative terminal. Several factors influence how fast the DNA moves these include; the strength of the electrical field, the concentration of agarose in the gel and most importantly, the size of the DNA molecules. Smaller DNA molecules move through the agarose faster than larger molecules. DNA itself is not visible within an agarose gel. The DNA will be visualized by the use of Ethidium bromide that binds to DNA (Barbas, et al. 2007).
Materials and Methods
Refer to the practical schedule: Forensic Genetics 2 (BSFS 204) 2016, Practical schedule: Protocol; Isolation of DNA from 100-500µl Whole Blood: Practical 1.
Aims and Objectives
Extract DNA from human blood sample.
To quantify the amount of DNA extracted from the sample available.
Run the extracted DNA on 2% Agarose gel.
Obtain a DNA pallet that will be used in another lab practical.
Results
A tabulated representation of results as obtained after a step in which physical change was observed.
Table of results only available in the downloadable document. Search for it here
Illustrative images also included in the file.
Discussion
The first steps in DNA isolation are based on getting the DNA out of the cell. Thus, the cell has to be broken and the cytoplasmic contents released. After which, the nucleus has to be broken to release the DNA. A lysis buffer is a buffer solution used for the purpose of breaking open cells. In this lab protocol the buffer choice was already suggested by the extraction kit that was used, Buffer FGI. Most lysis buffers for extraction of proteins, membranes, and organelles contain detergents. The choice of detergent is determined empirically and depends on the proteins and the tissue source. After addition of this buffer a pellet is observed (Griffiths and Chacon. 2014).
Proteinase K is an enzyme that cleaves the peptide bond in proteins next to the carboxyl group of hydrophobic amino acid residues. High temperatures, detergents, and chaotropic salts help to denature proteins, exposing many more hydrophobic amino acid residues that would normally be hidden in the hydrophobic core of the protein. Proteinase K is a protein that is resistant to denaturation by heat, detergents, and chaotropic salts and will continue to function as long as the temperature and concentration are kept in a reasonable range. It is stable and functional at higher temperatures and also functions in temperatures at room temperature. It is used at higher temperatures because most nucleases that would degrade DNA are inactivated at these temperatures. After the addition of the Buffer FG2/QIANGEN Protease the solution is incubated in 65 degrees Celsius to allow protein digestion to take place. This was indicated by the colour change from red to olive green colour (Lalwani, et al. 2014).
The precipitation of DNA was done by isopropanol (100%). DNA is insoluble in isopropanol, the addition of alcohol, caused the DNA proteins to come out of the solution. When DNA concentration in the sample is heavy, the addition of isopropanol caused a white precipitate to form immediately. These appeared as threads in the tube. More DNA was further precipitated after centrifugation of the resultant solution. The visibility of the white thread like DNA as precipitate indicates that the leukocytes count of the sample was sufficiently high. The pellet was then re-suspended in hydration buffer in this case the buffer was Buffer FG3 and it dissolved. However, the time for vortexing was increased do as to allow the pellet to completely dissolved (Lalwani, et al. 2014).
After running gel electrophoresis, the DNA was clearly visible as separates band on the gel. This indicated that the extracted DNA was of a larger amount and the yield for DNA in this practical lab was high and is sufficient to conduct PCR with. However, band – and --- had some smears, this could have been due to practical technique handling error were the gel may have been pincered in the well upon loading (Yeates, et al. 1998).
Conclusion
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References
Adnan, A. (2016). Role of DNA in Forensics. Biotecharticles.com. Retrieved 4 November 2016, from http://www.biotecharticles.com/Genetics-Article/Role-of-DNA-in-Forensics-110.html
Barbas, C., Burton, D., Scott, J., & Silverman, G. (2007). Quantitation of DNA and RNA. Cold Spring Harbor Protocols, 2007(11), pdb.ip47. doi:10.1101/pdb.ip47
Griffiths, L. & Chacon-Cortes, D. (2014). Methods for extracting genomic DNA from whole blood samples: current perspectives. Journal Of Biorepository Science For Applied Medicine, Volume 2, 1-9. Retrieved from https://www.dovepress.com/methods-for-extracting-genomic-dna-from-whole-blood-samples-current-pe-peer-reviewed-fulltext-article-BSAM
Lalwani, S., Millo, T., Pooniya, S., Raina, A., & Dogra, T. (2014). Quality and quantity of extracted deoxyribonucleic acid (DNA) from preserved soft tissues of putrefied unidentifiable human corpse. Journal Of Laboratory Physicians, 6(1), 31. doi:10.4103/0974-2727.129088
Yeates, C., Gillings, M., Davison, A., Altavilla, N., & Veal, D. (1998). Methods for microbial DNA extraction from soil for PCR amplification. Biological Procedures Online, 1(1), 40-47. doi:10.1251/bpo6
