Extraction
of Musa acuminate DNA and PCR using
Rapid Primers
By: Nathan Anthony Althaver
LBS 145 Laboratory
Section: W1
Instructors: Brent Atkinson and Rebecca DeGraaf
Abstract
ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ The concept of individual uniqueness has undergone a total transformation in the last 20 years.Ê What was once seen as physical, uniqueness is now being shed in a new light and seen as something that deals with our DNA fingerprint.Ê While outward appearances change over time, DNA fingerprints remains the same for the duration of an individualâs lifetime (Anonymous-1, Unknown).Ê
The purpose of this study was to create a DNA fingerprint for a banana.Ê In doing so, three techniques were used in the process.Ê During DNA extraction, the DNA was extracted from the banana by first breaking open the cells using a salt/soap solution and then extracting it from suspension in an ethanol layer (Anonymous-3, 1996).Ê During RAPD PCR, the DNA was amplified by using free oligonucleotides (Khra et al, 2005).Ê Finally, in the process of gel electrophoresis, the DNA was loaded into a gel and electricity was passed through it.Ê This created the DNA fingerprint (Khra et al, 2005).
In all, four trials were run, and three gels were produced.Ê From these gels, the purpose for this experiment could not be reached due to the inability to produce a DNA fingerprint.Ê Reasons for this result could be seen through a number of problems that occurred during the experiment.Ê Problems such as DNA extraction not being able to extract the DNA, to using too many restriction enzymes in RAPD PCR to making the gel in gel electrophoresis too concentrated or not concentrated enough could have all contributed to this result.
Discussion
ÊÊÊÊÊÊÊÊÊÊÊ The focus of this experiment was to examine Musa acuminate (banana) DNA.Ê In order to study this, three methods (DNA extraction, RAPD PCR and gel elecrophoresis) were devised and carried out.Ê It was hypothesized that in doing these steps, banana DNA would be extracted by first breaking down the cells of the banana in a soap/salt solution and extracting it from an ethanol layer (Anonymous-3, 1996).Ê This DNA would then be amplified using RAPD PCR, which uses free oligonucleotides in a solution to copy the DNA (Khra et al, 2005).Ê Finally, the gel electrophoresis step would create a fingerprint on a gel (Khra et al, 2005).ÊÊ The overall goal of this was to create a DNA fingerprint of the banana.Ê By achieving this, the DNA of bananas could easily be identified and distinguished from other organismâs DNA.
ÊÊÊÊÊÊÊÊÊÊÊ In the first trial, a coconut was used as the original focus of the research.Ê DNA extraction was preformed and a white precipitate was formed.Ê The presence of the white precipitate was an indicator that DNA was present in the precipitate itself (Anonymous-3, 1996).Ê Further testing under a flurometer, however, revealed a low to none concentration of DNA in the white precipitate.
ÊÊÊÊÊÊÊÊÊÊÊ The next three trials were all preformed on bananas.Ê It was believed that the bananas softer fruit could be crushed and mashed easier, making it more conducive to releasing the DNA during DNA extraction (Anonymous-3, 1996).Ê DNA extraction was then preformed, and in all three trails a white precipitate was formed indicating a presence of DNA (Anonymous-3, 1996).Ê This extracted DNA was then run through RAPD PCR in order to amplify the DNA (Khra et al, 2005).Ê Finally, all three trials were run through gel electrophoresis on order to create the DNA fingerprint (Khra et al, 2005).
ÊÊÊÊÊÊÊÊÊÊÊ Overall, the purpose for this experiment was not achieved.Ê As can be seen by the gels, none of the three gels were able to produce a DNA fingerprint for the banana.Ê There are many different explanations for this problem.Ê First, it is possible that the method for DNA extraction was flawed and not releasing the DNA from the fruit.Ê This could be easily investigated by using a working spectrophotometer in order to measure the amount of concentration of DNA in the white precipitate.Ê Since the spectrophotometer was unable to calibrate in the laboratory, this was impossible to perform and the experiment was forced to move onto the next steps without determining the concentration.Ê Second, a problem with the RAPD PCR technique could have occurred.Ê In all three experiments, four samples with two restriction enzymes each were used to undergo PCR.Ê By using fewer restriction enzymes (or none at all) a better result could possibly be obtained.Ê Finally, a problem with the gel used in gel electrophoresis could have also occurred.Ê In all three gels, the concentration of agarose in the gels was the same (0.4grams of agarose for every 50mL of TBE buffer and 1mL of ddH2O).Ê If given more time, different concentrations of agarose in the gels could be tested to determine the right concentration for the DNA to run through (Khra et al, 2005).
ÊÊÊÊÊÊÊÊÊÊÊ In general, DNA fingerprinting is a useful tool for a variety of purposes.Ê It can help to distinguish individuals from members of its own species.Ê It can help us to find similarities and differences between two species.Ê Also, it can aid us in identifying and mapping out inherited diseases (Anonymous-2, Unknown).
ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ
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Figure 1.Ê Gel electrophoresis trials on RAPD PCR
isolated DNA. Trial ran on