In this practical session, an introduction to the principles of DNA extraction and the analysis of DNA molecules using agarose gel electrophoresis was made. You will extract DNA from plant tissue and demonstrate methods for the detection and measurement of the molecular weight of DNA molecules in agarose gels, illustrating the effect of restriction endonucleases and mechanical shearing on DNA molecules (Sambrook, & Green, 2008).
Basically, the following biochemical principles are employed in the course of achieving the aims of this experiment. To start with, all living cells have DNA (genetic code) enclosed in their cell nucleus by the nuclear membrane. Extraction of DNA involves breaking down the cell and nuclear membrane to expose the intracellular components which also comprise the genomic DNA. DNA is basically made up of four bases i.e. AT GC. Different combinations of these bases are what make the complementary strands of DNA. In the cause of extraction, the lysing enzymes and chemical detergents are used to digest the membrane, pH, and heat to denature the proteinous components of the membranes (Sambrook, & Green, 2008).
Naturally, DNA is found in the cell in the protein-DNA complexes called histones. To attain pure DNA, the protein must be removed through the process of protein denaturation. This is achieved by the use of enzymes and detergents. After this, DNA is separated from the denatured protein in the DNA recovery step (Avise, Lansman & Shade, 1979).
Endonucleases are enzymes that are used to cut nucleotide sequences in DNA at specific base-sequence regions referred to as restriction sites. After digesting DNA with endonucleases then their separation in agarose is possible since nucleotide bases migrate with respect to their molecular sizes. The migration is caused by the net –ve charge in DNA which is attracted to +ve charged anode. The shorter DNA fragments are expected to move at a faster rate in comparison with the longer fragments due to ‘fewer collisions with the agarose molecules in the process of migration. After migration, staining is done to ensure visualization of DNA bands in the agarose gel. Staining is done with ethidium bromide (dye). The sequences are identified by the use of a radiolabeled probe and are visualized in an agarose column with the aid of dies (Sambrook, & Green, 2008).
These principles are the building blocks in molecular biology and forensic investigation. The extraction, lysis, and analysis of DNA are used to identify the culprits of rape, robbery, murder, or other social evils in forensic investigation labs. Their small DNA samples are taken from the crime scene, multiplied, and then run in gel alongside those of the suspects. Resemblance (homogeneity) of DNA strands with that of a suspect proves he is the culprit (Winnacker, 1987).
These principles are also applied in disputed paternity cases. Parents who doubt the paternity of their children can be subjected to DNA analysis to know the truth.
1. Where in the cell is DNA found? During DNA extraction, how do we achieve cell lysis?
DNA is found in the nucleolus of the cell nucleus. In the process of DNA extraction, the nuclei membrane and cell membrane are lysed via a combination of SDS (chemical detergent), enzyme, pH, and heat. SDS detergent is used to break the bond between proteins and the membranes i.e. the cell membrane and the nucleus membrane. The enzyme digests the protein components of the membranes, pH, and heat (specific) are for denaturing the protein components of the cells, protein is then denatured and lastly, the DNA is separated from denatured protein (Sambrook, & Green, 2008).
2. A solution of Lambda DNA has a concentration of 0.1 mg/ ml. How much DNA (in g) do we have in a 4 l sample?
Concentration = 0.1mg/ml;
Vol of the sample= 4 l;
If 100 g are in 1000 L what about—-in 4 l;
Assuming the amount of DNA to be y;
(100 g x 4 l)/ 1000 = y;
Therefore y = 400/1000 = 0.4 g.
3. Given that lambda has a genome size of approximately 50 Kb, how many complete lambda genomes are present in the 4 l volume from Question 2? (1 Kb = 1,000 base pairs; 1 Kb = 1.1 x 10-12 g).
Amount of DNA = 0.4 g;
Complete lambda genome = 50 Kb;
Therefore the number of genome = 0.4 g/ (50 x 1.1 x 10-12 g) = 7,272,727,272 genomes are in 0.4 g.
4. Figure 1 shows typical results from this practical session. Explain the results describing why the different treatments results in the different banding patterns seen in each lane.
I.) II.) III.) IV.)
Figure 1: results obtained after electrophoresis experiment control. (2) Results of physical shearing. (3) Results of HinfI restriction digestion. (4) Molecular weight markers.
The results of sample 1 (untreated control) show one DNA band after visualization at 23.13 kB. This might be a result of breakage of genomic DNA from physical stress in the cause of extraction owing in mind DNA breaks easily.
From sample 2, (physical shearing), no bands were visualized at all. This might be due to the fact that the mechanical shearing never happened or either it happened to a very small extent resulting in no DNA fragments for migration (Winnacker, 1987).
In sample 3, (Results of HinfI restriction) several bands can be visualized. The bands are 4.36 kB downwards in size. This shows that the restriction endonuclease HinfI cut the DNA at several restriction sites resulting in several fragments that can be visualized as opposed to the case in Sample 1 & Sample 2 which don’t employ endonuclease (Winnacker, 1987).
5. What is a restriction endonuclease, describe how these can be used to investigate two types of polymorphism found in DNA?
Restriction endonucleases are enzymes that cut DNA at particular points referred to as restriction sites. These restriction sites are distinguished by certain nucleotide sequences. Restriction endonucleases are classified into three classes on the basis of whether the cleavage/restriction sites are separate from one another or if they clip the DNA at their specific cleavage types (Pingoud, Alves, & Geiger, 1993).
Restriction enzymes can detect particular sequences in a nucleotide in which they establish a cut on the DNA. Most of these restriction sites have the same base sequence and are the same when read from backward and also from forward. Mirror-like palindromes read the same from backward and forwards, for instance, GTAATG (on the same strand) while mirror-like palindromes read the same from backward and forward but the forward and backward sequences exist in opposite strands of double-stranded DNA (Avise, Lansman & Shade, 1979).
Restriction endonucleases are used to identify polymorphism in DNA by working on the DNA being tested. After digestion, the fragments of DNA are separated via electrophoresis in accordance with their length. The DNA fragment that is homologous to a nucleon is then identified by the use of a probe. The probe is usually similar to the nucleon being investigated but it is labeled with a radioisotope. From this, any fragment that is identical to the nucleon wholly or partially is identified. Here, nucleomorphs with one or more restriction sites can be identified (from the nucleon region).
DNA is the genetic code found in the nucleus of the cells of living organisms. DNA is extracted broken down and analyzed for various purposes. DNA is extracted from cells by the use of enzymes and detergents to disrupt the membranes. Endonucleases are enzymes that clip DNA at particular sites called restriction sites. After breaking down, DNA is run in agarose gel where it separates in accordance with its respective length. The bands can be visualized after treating the gel with a dye (Avise, Lansman & Shade, 1979). In medicine, this technology can be used in controlling certain disease-causing microorganisms and drug making. In day-to-day life, it can be used to prove the parenthood of a child in case of a dispute and also identify criminal culprits in forensic investigations.
- Sambrook, R. Green, J. (2008) Molecular Cloning. (7th ed. ed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory.
- Roberts J. (1976). Restriction endonucleases: CRC Crit. Rev. Biochem. 4 (2): 123–64.
- Pingoud A, Alves J, Geiger R (1993). Restriction Enzymes: Enzymes of Molecular Biology. Totowa, NJ: Human Press. pp. 106–201.
- Micklos A., Bloom V. & Freyer A. (1996). Laboratory DNA science: an introduction to recombinant DNA techniques and methods of genome analysis. Menlo Park, Cummings Pub. Co.
- Winnacker L. (1987). Isolation, Identification, and Characterisation of DNA fragments: From Genes to Clones. VCH.
- Avise, J. Lansman R. & Shade, O. (1979) The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations: Population structure and evolution in the genus Peromyscus. Genetics 92: 278-294.
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