Vol1, Issue 1

[Vol1, Issue 1]December 2014

Journal of BiochemistryPolytechnic University of the PhilippinesS.Y. 2014-2015Page 1

Experiment ArticleDNA ISOLATION FROM MUSA ACUMINATA (AA Group) USING RAPID SIMPLE METHOD OF ISOLATIONAyang, Noel Angelo1, Garlan, Paul Trishan2, Jerusalem, Rod Florence3, Sy, Elaine4BS Chemistry 3-2, Department of Physical Sciences, College of Science, Polytechnic University of the Philippines, Sta. Mesa, Manila

ABSTRACT:Just like us, banana plants have genes and DNA in their cells, and just like us, their DNA determines their traits. Thesoft flesh of a banana provides a ready source of DNA. Using a few simple purification steps in a classroom setting, students can yield loads of crudely prepared DNA. The experiment presents the isolation of DNA from banana. First, banana was crushed and detergent /salt solution was added for the cell membrane to be destroyed and for the nuclear membrane to be released. Then, it was reconstituted, precipitated by alcohol and chemically disrupted. Finally it was put to UV-Vis for the DNA to be determined. The result does not meet the accepted A260/A280 ratio for the DNA purity, which is 1.7-2.0, for it was below 1.5. DNA is required for many applications. Proper laboratory practice and care must be taken to ensure reliable and reproducible results.Keywords: DNA, Isolation, Extraction, Nucleic Acid, ReconstituionINTRODUCTION

What do you have in common with a banana? Even though we might not look alike, all living thingsbananas and people includedare made up of the same basic material.Just like houses are made up of smaller units such as bricks, all living things are made up trillions of microscopic building blocks called cells. Within an organism, each cell contains a complete set of "blueprints". These directions determine the organism's characteristics.Deoxyribonucleic acid (DNA) is amoleculethat encodes thegenetic instructions used in the development and functioning of all known livingorganismsand manyviruses.It is one of the most important parts of the cell because it contains the instructions for the synthesis of proteins and RNA inside the cell. With its absence, cell cannot function. Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is calledmitochondrial DNAor mtDNA). A DNA sequence is a specific lineup of chemical base pairs along its strand. The part of DNA that determines what protein to produce and when, is called a gene. Genes control everything from hair color to blood sugar by telling cells which proteins to make, how much, when, and where.DNA isolationis a process of purification of DNA from sample using a combination of physical and chemical methods. Purified DNA has many applications in our modern world. It could be used for Genetic engineering in which they modify DNA sequence for practical application such as in agriculture and in medical research. It could also be used in forensics which forensic scientists identify bodies, cross-contamination of evidence. And it has many other applications.In this paper, we extracted DNA in the fruit of Musa acuminata(AA Group) using a rapid method of isolation by disrupting the cell wall with the use of blender, and separating the DNA from its contaminants by the use of alcohol. We have precipitated the DNA and separated it from the sample through decantation. The separated DNA is then washed with water and tested its purity through the use of UV-Vis spectrophotometer. General Objective: The experiment aims to extract completely the DNA from the banana.Specific Objective: The experiment aims to extract completely the DNA from the banana using a rapid simple method of isolation.Fig 1: Mechanical Disruption of Cell Membrane

METHODOLOGY

The laboratory equipments used in the experiment were test tubes, stirring rod, 250 mL beakers, 500 mL beaker, pipette, aspirator, watch glass and inoculating loop. Additional materials are blender, salt and dishwashingliquid.

Fig 2. Schematic diagram of the procedure

Mechanical Disruption of Cell MembraneUsing your knife, cut your banana into tiny pieces to expose more of the cells. Place your banana pieces in the blender. Add some ice and mix in the blender for some seconds.HomogenizationAdd distilled water. Mix in the blender for 5 to 10 seconds making sure the mixture is not too runny.Chemical Disruption of Cell MembraneAdd a teaspoon of salt. Mix it again. The salt will help the DNA stay together during the mashing process. After mixing, add small drops of dishwashing liquid and gently stir the mixture. You should try not to create bubbles when stirring. The soap helps to break-down cell membranes to release the DNA.

Alcohol PrecipitationTransfer an aliquot of the mixture using pipette into test tubes, about of its size. Carefully pour very cold 95% ethanol down the side of the glass stopping near the top. Let it sit undisturbed for about four to five minutes or so in a 500mL beaker with ice. Do not shake. The white material coming out of solution as a precipitate is DNA. ReconstitutionDip the glass rod into the tube, slowly rotating it to spool out the bananas DNA. Using the inoculating loop, scoop out the DNA precipitate. Put the precipitate in a watch glass and allow the remaining alcohol to evaporate. After a minute, reconstitute it on water to remove excess alcohol that is not completely eliminated through evaporation.

Fig 3: Sample mixture in test tubesInstrumentationAfter reconstitution, we now test the DNAs purity through the use of UV-Vis Spectrometer. Make a 3mL sample mixture of DNA and water into a new test tube. Setting the wavelengths to 260nm and 280nm, we now read and record its absorbance.

Fig 4: Adding of dishwashing liquid

Result and Discussion

Nucleic Acid absorbs ultraviolet light in a specific pattern. The collected DNA samples undergo a spectrophotometric analysis. Table 1 shows the Absorbance of the samples @ 260 nm and 280 nm.

SampleAbsorbance @ 260 nmAbsorbance @ 280 nm

10.4220.378

20.2660.221

30.0080.006

Table 1. Absorbance of collected DNA samples from Banana @ 260 and 280 nm.DNA concentration estimation by Spectrophotometric Absorption

Quantitation of Nucleic Acid were obtained using the formula:

Concentration (g/mL) = Abs@260 50 g/mL dilution factor

At a 1-cm path length, the Absorbance (optical density) at 260 nm (OD260) equals 1.0 for a 50 g/mL solution of double stranded DNA. Table 2 shows the calculated concentration of the isolated pure DNA samples from Banana.

SampleConcentration (ppm)

142.2 ppm

237.8 ppm

30.8 ppm

Table 2.Concentration of collected DNA samples from Banana @ 260 nm.

Nucleic acids and proteins have absorbance maxima at 260 and 280 nm, respectively. The ratio of absorbance at these wavelengths has been used as a measure of purity in both nucleic acid and protein extractions. A ratio of ~1.8 is generally accepted as pure for DNA; a ratio of ~2.0 is generally accepted as pure for RNA. Table 3 shows the calculated ratio of absorbance at 260/280 nm from the isolated DNA samples.

SampleAbsorbance @ 260 nmAbsorbance @ 280 nmRatio (Abs 260/280)

10.4220.3781.1

20.2660.2211.2

30.0080.0061.3

Table 3.The ratio of absorbance obtained at wavelength 260 and 280 nm.

Discussion

Extraction of DNA from Banana

Banana was use as source of DNA since bananas are soft and dense, without a lot of stringy or gritty material which might be present in some fruits(a pear, for instance). Their softness makes it easy to release their DNA without a lot of work. Crushing of bananas by the use of blender helps break down the tough walls of the cells to release the cell contents. Addition of detergent to the slurry helps to break down the cell and nuclear membrane. Detergent is made up of Sodium Laurel Sulfate, which dissolve the fats and lipid bilayer of membranes and release the cellular contents, including DNA. Once the DNA is released from the cells, the addition of salt to the solution enables the DNA strands to come together, or aggregate. Since DNA is not soluble in alcohol, addition of cold Ethanol dehydrates the DNA by removing the water. This dehydrated molecule then forms the DNA precipitate, while the other remaining materials remain in the solution.

Concentration of isolated DNA samples

The calculated concentration of DNA samples from Banana accounts only for the concentration of the double stranded DNAs present in the sample. Table 1.2 shows the calculated concentration of isolated DNA sample using the formula:

Concentration (g/mL) = Abs@260 50 g/mL dilution factor

Assessment of Nucleic Acid purity

The ratio of absorbance at wavelength260 and 280 has been used as a measure of purity in both nucleic acid and protein extractions. It is important to note that the A260/A280 ratio is only an indication of purity rather than a precise answer. A ratio of ~1.8 is generally accepted as pure for DNA; a ratio of ~2.0 is generally accepted as pure for RNA.

A260/280 ratio present at Table 3 do not met the generally accepted ratio for a pure DNA. The values of the ratio that are obtained are relatively smaller than of the standard ratio for a pure DNA. Fig 5: Extracted DNA

Several factors influence the accuracy of our A260/A280 ratio.A low A260/A280 ratio may be caused by residual phenol or other reagent associated with the extraction, and a very low concentration (> 10 ng/ul) of nucleic acid. In the case of our experiment, the extraction of DNA from banana was properly done but in some cases, it may be contaminated by other reagents or substances. During the spooling of DNA precipitates from the test tube, some of the bananas cell components were also spool out of the test tube together with the DNA precipitates which causes the inaccuracy of our A260/280 ratio. Moreover, the DNA concentration of the samples may also influence the inaccuracy of our result. Due to a very small amount of DNA samples that were collected, it reflected to the very low concentration of DNA present in our samples.

Wavelength Accuracy of the Spectrophotometers

Although the absorbance of a nucleic acid at 260 nm is generally on a plateau, the absorbance curve at 280 nm is quite steeply sloped. A slight shift in wavelength accuracy will have a large effect on 260/280 ratios. It is possible to see as much as a 0.4 difference in the 260/280 ratio when measuring the same nucleic acid sample on two spectrophotometers that are both within a 1 nm wavelength accuracy specification. Two different types of Spectrophotometers may measure two different readings.

Nucleotide Mix in the samples

The type(s) of protein present will also have an effect. Absorbance in the UV range by proteins is primarily the result of aromatic ring structures. Proteins are composed of 22 different amino acids of which only three contain aromatic side chains. Thus the amino acid sequence of proteins would be expected to influence the ability of a protein to absorb light at 280 nm.For example bovine serum albumin (BSA) has an extinction coefficient value of 0.7 for a 1 mg/ml solution at 280nm, while streptavidin has an extinction coefficient of 3.4, absorbing almost five times as much light at 280nm at the same concentration.

Due to the different absorption spectra, the nucleotide composition of the bases present in DNA will have different A260/A280 ratios.

NucleotideA260/A280 ratio

Adenine4.50

Cytosine1.51

Guanine1.15

Thymine1.47

Table 4.A260/A280 ratios for nucleotides.

Therefore the ratio will be approximately equal to the weighted average of the A260/A280 ratios estimated for each nucleotide if measured independently, which explains why the accepted ratio of 1.8 for pure DNA is an approximation.The actual ratio will depend on the composition of the nucleic acid.

Conclusion

Fig 6: Alcohol precipitationIsolation/extraction of high quality, intact pure DNA is required for many applications. Proper laboratory practice and care must be taken to ensure reliable and reproducible results.A good quality DNA sample should have a A260/A280ratio of 1.7-2.0 and an A260/A230ratio of greater than 1.5, but since the sensitivity of different techniques to these contaminants varies, these values should only be taken as a guide to the purity of your sample. Our result do not met the general accepted ratio for a pure DNA. The reasons for this inaccuracy may cause by an error in the preparation and extraction of the DNA precipitates and the presence ofother reagents or contaminants thatcause a low A260/280 ratio of our DNA samples.To improve the accuracy of DNA concentration determination, allowance should be made for any impurities in the solution. This can be estimated by adjusting the A260 measurement for turbidity which is measured at an absorbance of A320. A reading at 320nm will indicate if there is turbidity in the solution, another indication of possible contamination. Therefore, taking a spectrum of readings from 230nm to 320nm is one of the suggested and informative ways to accurately determine the purity and concentration of DNA.

REFERENCES

1. http://www.sciencedaily.com/articles/d/dna.htm

2. http://ghr.nlm.nih.gov/handbook/basics/dna

3. Dahm, R (January 2008). "Discovering DNA: Friedrich Miescher and the early years of nucleic acid research.".Human Genetics122(6): 56581.doi:10.1007/s00439-007-0433-0.PMID17901982.