ADVANCED BIOMEDICAL LAB REPORT 1
Advanced Biomedical Lab Report
Advanced Biomedical Lab Report
The aim of this report was to:
Extract DNA samples from a liver tissue from a mouse
To establish the genomic composition of the mouse DNA using the polymerase chain reaction (PCR) method.
The experiment involved performing a DNA sequencing of the gene region of a sample from a mouse in a polymerase chain reaction (PCR). The experiment was performed using a DNA extracted from the liver of a mouse as the starting template. During the sequencing reactions, a mixture of deoxynucleotide triphosphate (dNTPs, dGTP, and dTTP) was used in the sequencing reactions. It also involved the use of specific concentrations such as the ddNTP that will be regarded as the usual dNTP at different nucleotide positions in the resulting DNA chain.
The sequence in which the reactions occurred was cleaned up involved the addition of 25µL of 100% ethanol to 1 µL of 125mM dEDTA and 1 µL 3M Sodium Acetate and the PH was maintained at 1.5 mL in a micro centrifuge tube. The tube was labeled with a letter that represents the sample number. 10 µL was pipetted into the mix and left at room temperature for 20 minutes to enable precipitation of a number of length extension products. The tubes were centrifuged for 15 minutes at 14000 rpm and hinged outside to know the orientation of the pellet. The experiment was performed as illustrated in the figure below.
Figure. 1. The sequencing procedure during the analysis of the DNA
A pipette was used to remove the supernatant and care was taken to prevent disturbance to the pellet.
The forward primer reagents used were 2.0 µL at 50 ng/ µL. The PCR reaction involved making dilutions of 50 ng/ µL. A working dilution of 20 ng/ µL was required during sequencing reactions. It was kept in ice at all times during the experiment. This enabled amplification of a section of unknown gene and the acquisition of a number of copies of a particular region. The PCR set-up involved: 1 µL of BDT (v 3.1), 1.5 µL Buffer (5x), 1 µL Primer (20 ng/ µL), 2 µL PCR (10-20ng), and 4.5 µL dH20.
The annealing temperature was calculated using the formula: 2(A + T) + (4(G + C) – 6 = Annealing temperature. The objective of annealing was to bind the primers to the template DNA.
The sequencing reactions included a process that occurred in three stages: the first stage involved denaturing of the double stranded DNA to single strands, the annealing of the primers to enable their binding to the template DNA, the extending process where dNTPs lined up in the right location to form a synthesized strand.
The resulting gel image was as illustrated in the figure below.
Figure. 2 Gel Image from the electrophoresis process
From the gel image obtained during the electrophoresis, it is observed that the movement of the DNA molecules occurred from the negative electrode towards the positive electrode. It was also observed that the DNA samples with smaller base pairs moved further away from the negative electrode towards the positive electrode while those with larger base pairs remained closer to the negative electrode.
Figure 3. The chromatogram results of the study
During the visualization of the DNA samples when the electric current was passed, it was observed that there were bars of different loop sizes according to the sizes of the DNA samples. The resulting visualized image was as illustrated in Figure 3 above.
Discussion and Conclusion
The study involved performing an electrophoresis using agarose gel in which DNA samples of tissues from a mouse liver were used. During the experiment, the tissues were placed in the gel container after which an electric current was passed. This was followed by making observation of the manner in which the DNA samples move across the container towards the positive electrode. The movement of the DNA samples towards the positive electrodes is based on the fact that they are negatively charged and when an electric current is passed, they tend to move towards the positive electrode. Another observation that was made is that DNA molecules with smaller base pairs moved closer to the positive electrode faster compared with those with larger values of base pairs. This is because, the smaller the sample, the higher its ability to move when an electric current is passed across the gel (Hudnall et al. 2008). This resulted into the generation of a unique, distinguishable band patterns as represented in figure 3. Due to the manner in which the DNA samples moved across the gel, it was possible to classify them based on sizes.
The experiment also involved visualizing the movement of the DNA samples across the gel during electrophoresis. The visualization of the DNA samples was achieved by the use of fluorescent dyes that were used to stain them.
In conclusion, the experiment involved designing of a rapid robust PCR-based method that enabled identification of genes of a mouse from a liver tissue on the basis of size when they move across the gel during electrophoresis. The use of a mouse tissue constituted an important component for studies that enable the identification of the sex of a particular organism. The experiment was also important in understanding the sizes of DNA samples and enabling separation of the samples based on size.
Hudnall, S. D., Chen, T., Allison, P., Tyring, S. K., & Heath, A. (2008). Herpesvirus prevalence and viral load in healthy blood donors by quantitative real‐time polymerase chain reaction. Transfusion, 48(6), 1180-1187.