Abstract

The focus of the experiment was to determine quantitatively the value of the ultraviolet measurement of the -phage DNA via spectrophotometry and to extract the bacterial chromosomal DNA from E. coli. The experiment was done in two consecutive weeks, starting with ultraviolet measurement and denaturation of isolated DNA in the first week and DNA extraction in the second week. The methods were modified from the manual due to restricted resources. The results of the experiment were recorded and further discussed, with provision of suggestions and recommendations to improve the procedures and outcomes. The DNA extracted from the bacteria was kept for future experiment. It was concluded that the higher the ratio of A260/A280 of the DNA sample, the purer the sample is.

Introduction

Nucleic acid is a macromolecule composed of high molecular weight biopolymers chains of monomeric nucleotides. This molecules carry genetic information of generate cell structures. Deoxyribonucleic acid (DNA) and ribonucleic acid are the most common nucleic acids. DNA is the hereditary material in almost all living organisms. Most DNA is located in the chromosomes of cell nucleus. There are four types of bases exist in DNA, which are adenine (A), guanine (G), cytosine (C) and thymine (T). The original DNA double helix exists as separated single strands after denaturation.

Denaturation of DNA happens when the complementary strands of the double helix are separated when a solution of DNA is heated above physiological temperature (about 100°C) or under a high pH condition. The complete denaturation of DNA is reversible. Single strands often meet their complementary strands and reform regular double helices when heated solution of denatured DNA is slowly cooled. In this experiment, DNA denaturation was monitored by measuring the absorbance of ultraviolet light passed through a solution of DNA. DNA maximally absorbs ultraviolet light at a wavelength of about 260nm. It is the nucleotide bases that are fundamentally responsible for this absorption.

The easiest means of determining DNA concentration is through spectrophotometric analysis. Since nitrogenous bases absorb UV light, the more concentrated the DNA solution, the more UV light it will absorb. The concentration of pure double-stranded DNA with an A260 of 1.0 is 50 mg/ml. Thus, one can use the following formula to determine the DNA concentration of a solution:

Unknown (mg/ml)/ Measured A260 = 50 (mg/ml)/ 1.0 A260

Since there is a linear relationship between absorbance and DNA concentration, some simple algebra can be used and reformulated as follows:

Unknown mg/ml = 50 mg/ml x Measured A260 x dilution factor

The assessment of the purity of a nucleic acid sample is often performed by a procedure known as the A260/A280 ratio. It is commonly used to assess purity of nucleic acid samples. The standard A260/A280 ratio falls in the range of 1.8 to 2.0. If the ratio is too low, this implies that the proteins in the DNA is very high and so that PCI solution and proteinase K need to be added into the DNA solution to digest the proteins.

DNA extraction is a process in which the DNA from the cells or viruses is removed. It is normally used to detect bacteria and viruses as well as diagnosing disease and genetic disorders. The cells or virus containing the DNA of interest are broken into small fragment during DNA extraction. The basic procedures of DNA extraction are briefly explained below:

1. Break open (lyse) the cells or virus containing the DNA of interest - This is often done by sonicating or bead beating the sample. Vortexing with phenol (sometimes heated) is often effective for breaking down proteinaceous cellular walls or viral capsids. The addition of a detergent such as SDS is often necessary to remove lipid membranes.

2. DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease. Precipitation of the protein is aided by the addition of a salt such as ammonium or sodium acetate. When the sample is vortexed with phenol-chloroform and centrifuged the proteins will remain in the organic phase and can be drawn off carefully. The DNA will be found at the interface between the two phases.

3. DNA is the precipitated by mixing with cold ethanol or isopropanol and then centrifuging. The DNA is insoluble in the alcohol and will come out of solution, and the alcohol serves as a wash to remove the salt previously added.

4. Wash the resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the pellet.

5. After pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a buffer such as Tris or TE.

6. Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light.

Materials and Apparatus

Escherichia coli cell cultureLysis bufferPhenol-chloroform-isoamyl alcoholPotassium acetateEthanolSodium dodecyl sulphate (SDS)TE bufferStandard DNAIceMicrofuge tubesWater bathQuartz cuvetteSpectrophotometer for UV measurementsTable-top centrifugeMicropipettes and disposable tips

Methodology

Ultraviolet Measurement and Denaturation of Isolated DNA

1. Standard DNA was diluted with 0.9ml of the TE buffer.2. 200µl of the TE solution was transferred to a quartz cuvette and A260 was determined

and recorded after the reading was first blanked with TE buffer before DNA solution was measured. Similar procedure was done for the measurement of A280. The A260 and A280 of the DNA sample was determined and recorded.

3. 2ml of the DNA solution was prepared in the TE buffer at a DNA concentration of 20µl/ml. The A260 and A280 of this solution were measured and recorded.

4. The DNA solution was pipetted into four microfuge tubes, each with a volume of 0.5ml. One tube was maintained at room temperature and the other three were placed in a 90C water bath for 15 minutes.

5. The tubes were removed when the incubation period was up. One heated tube was quick-cooled in an ice bath, the other heated tube was cooled slowly to room temperature over a period of 1 hour and the third tube was immediately poured into the quartz cuvette for spectrophotometry measurement.

6. Final A260 readings of each of the four tubes were measured and recorded.7. The A260/A280 ratio was calculated.8. The A260(T)/A260(25C) was also calculated for each of the four tubes.

DNA Extraction

1. 1 ml of an overnight E. coli culture was added to a 1.5ml microfuge tube.2. The tube and its content were centrifuged at 12000 rpm for 2 minutes.3. The supernatant was removed.4. Steps [1] until [3] were repeated three times.5. The cell pellet was re-suspended in 600µl of Lysis Solution before being incubated at

80C for 5 minutes for cells lyses.6. The tube contents were cooled to room temperature.7. To the cell lysate, 600µl of phenol-chloroform-isoamyl was added.8. The tube was vortexed for 20 seconds to mix.9. The tube was centrifuged at 12000 rpm for 3 minutes.10. The supernatant-contained DNA was transferred to a clean 1.5ml microfuge tube with

the content of 600µl of room-temperature absolute ethanol and 300µl of potassium acetate.

11. The content of the tube was gently mixed by inversion.12. The tube was placed inside a refrigerator for 15 minutes.13. The tube was taken out to let the content melt at room temperature.14. The tube was centrifuged at 12000 rpm for2 minutes.15. The supernatant was carefully poured out and the tube was drained on clean absorbent

paper.16. 600µl of room-temperature 70% ethanol was added to the tube and the tube was

gently inverted several times to wash the DNA pellet.17. The tube was again centrifuged at 12000 rpm for 2 minutes.18. The ethanol was carefully poured out and the tube was drained on clean absorbent

paper.19. The pellet was allowed to air-dry for 10 to 15 minutes.

20. 50 µl of TE buffer was added to the tube and was put in the 37C water bath for 10 minutes before the DNA was rehydrated by incubating at 65C for 1 hour.

21. The tube was kept inside the refrigerator for future use.

Results

Table 1: The data for ultraviolet measurement and denaturation of isolated DNA experiment

Sample Absorbance A260

Absorbance A280

Ratio of A260/A280

Concentration( µg/ml)

Ratio of A260(T)/A260(25̊+C)

Pure 0.079 0.043 1.8 3.95 1.000Control 0.186 - - - 0.235

Quick-cool 0.374 - - - 0.473Slow-cool 0.370 - - - 0.468Immediate 0.428 - - - 0.542

Calculation

Concentration of A260 = 0.079 x 50 x 1(path-length) x1(dilution factor)= 3.95µg/mL

Concentration of control group sample = 3.95 µg/ml x dilute factor = 3.95 µg/ml x 4 = 15.8 µg/ml

Percentage error of concentration after denaturation of isolated DNA

= ( 20.0 µg /ml−15.8 µg /ml20 .0 µg /ml )×100 % , [the initial concentration of DNA sample is 20.0

µg/ml]= 21%

Discussion

Table 1 has shown that the absorbance of 260 nm and 280 nm wavelengths are measured. The ratio of A260 and A280 are calculated as well. For the denaturation of isolated DNA, there are four sample groups that comprise of the control group, quick-cool, slow-cool and immediate of DNA samples. In this part, the standard λ DNA of the absorbance of control group A260 is 0.079 while the absorbance of A280 is 0.043. The ratio of A260/A280 is 1.8. The result of the standard λ DNA is within the range of the ratio, indicating that the sample is quantified as very pure. The standard λ DNA is procured from a supplier with state-of-the-art laboratory where the preparation of the sample provides less contamination in the DNA.

The other three sample groups are the quick-cool, slow-cool and immediate. The quick-cool absorbance is 0.374 while the slow-cool absorbance is 0.370. The difference in the absorbance of between these two groups is just a fraction although it is anticipated to be of greater difference, with the slow-cool having a lower absorbance when compared to that of the quick-cool. This difference is contributed by the phenomenon of hyperchromicity and hypochromicity of DNA. The increase of the absorbance DNA is called hyperchromicity while the decrease of the absorbance DNA is called hypochromicity. For the quick-cool method, the ice is used to rapidly cool down the temperature of DNA. Therefore, the DNAs

do not have enough time to anneal the DNA during the renaturation process. The sequences of the DNA are not uniformed and the overhangs of DNA occur. The single stranded DNAs are still can found after quick-cool method. DNA bases absorb UV light due to aromatic rings. Hence, the quick-cool method causes the exposure of aromatic rings to affect the absorbance of DNA. The absorbance of single stranded DNA has higher absorbance compared to that of the double stranded DNA. In the contrary, the slow-cool method allows the renaturation of the DNA after denaturation of isolated DNA. The DNA samples are able to renature to double stranded DNA. Slow-cool method has a lower absorbance because the stacking of aromatic rings stabilized by hydrophobic force and induced dipole. The aromatic rings of DNA do not expose to UV light, so the lower absorbance we obtain. For the immediate group, the sample is immediately measured once it is taken out from the water bath. With the absorbance of 0.428, the largest when compared to the rest of the sample groups, it indicates that the nucleotide bases are in loose forms as the DNA are in denatured state.

The phenol-chloroform-isoamyl alcohol is used to isolate the DNA. It is corrosive because it contains phenol. Hence, glove and lab coat should be worn while conducting the experiment. TE buffer is used to dissolve and stabilize DNA. The 70% ethanol is used to wash the DNA pellet. In this experiment, the proteins are degraded by proteinase K. Vortex the lysate cell with phenol-chloroform-isoamyl alcohol (PCI) solution which is used to isolate the DNA. The addition of SDS reagent is often necessary to remove the lipid membrane of DNA. After the sample had been vortexed, the sample can be found at the interface between the two phases and it can be drawn carefully. TE buffer can stabilize and protect the DNA from degradation. DNA is insoluble in ethanol and it acts as a wash to remove the salts (potassium acetate) previously added.

The human-induced errors like pipetting the sample DNA contributes to inaccuracy when the sample DNA is extracted from the tube. The pipetting duration must be as short as possible. This is because the longer period the sample is being pipetted, the more exposure on the DNA. The longer exposure time of the DNA will affect the purity of the DNA sample. The absorbance values of the extracted DNA have been measured by the spectrophotometer. There are some errors when we used the spectrophotometer. The incorrect position of cuvette causing the UV light cannot pass through the DNA sample. The negative values of absorbance have been obtained from the spectrophotometer. The cuvette must be clean enough to get the accurate results. When holding the cuvette, the surface of the cuvette should not be touched to avoid the fingerprints smudge on the cuvette. The absorbance values of the DNA sample will become inaccurate due to the smudge as it hinders the absorbance rate. The edges of the cuvette are the place to hold and gloves should be worn to avoid the errors.

Conclusion

1. The absorbance value increases as DNA denatures.2. The higher the A260/A280 ratio, the purer the DNA sample.

Reference

Seidman, L.A., & Moore, C. J. (2000). Basic Laboratory Methods for Biotechnology Textbook and Laboratory Reference. New Jersey: Prentice Hall.

Garrett, R. H., & Grisham, C. M. (2005). Biochemistry Third Edition. California: Thomson Brooks/Cole.Weaver, R. F. (2008). Molecular Biology. New York: McGraw-Hill