FACULTY OF APPLIED SCIENCES AND COMPUTING BABS2413 MOLECULAR BIOLOGY PRACTICAL 1 NAME: CHOONG MEL JUNE GROUP: RBS 2 A1 STUDENT ID: 14WAR10521 DATE: 7 OCTOBER 20140 DEMONSTRATER: DR LOKE CHUI FUNG
Title: Spectrophotometry of DNA from Calf Thymus DNA by using Nano-Drops UVSpectrophotometer before and after Reannealing. Objectives: 1. 2. 3. 4.
To determine the purity of calf thymus DNA by using nano-drops UV-spectrophotometer. To measure the absorbance at 260nm to quantify DNA. To produce UV absorbance spectra of DNA as function of DNA sample temperature. To determine the purity of calf thymus DNA b y using nano-drops UV-spectrophotometer after reannealing.
Introduction: A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. When a photon encounters an analyte molecule, then the analyte will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam. Absorption spectrophotometry is a widely used technique in analytical chemistry based on the property of molecules to absorb light at specific wavelengths. The quality or purity of the sample can be determined by comparing the measurements at 260 and at 280 nm (the wavelengths for which DNA and protein absorb). The advantages of spectrophotometry usage are that the process of obtaining result is rapid. The quality of DNA can be assessed to determine the level of degradation. The whole procedure is relatively inexpensive, time saving and the result obtained is very reliable. The machinery is also easy to operate as it is automatable. But, the spectrophotometer is not human DNA specific. Presence of other microorganism DNA will be detected as well. Nucleic acids are characterized and quantified using their absorption spectra. The y can be measured by using spectrophotometry which nucleic acids absorb light in the ultraviolet region of the electromagnetic spectrum. Then, absorption spectrum is produced by measuring the amount of light the nucleic acids absorb at different wavelengths. In this experiment, with the help of nano- drops UV-spectrophotometry, the quality and purity of a DNA can be qualified. Apart from that, it can be used to determine contaminants with the A260/A280 ratios. Purines found in nucleic acids have an absorbance maximum slightly below 260 nm, while pyrimidines have a maximum slightly above 260 nm. Thus, although it is common to hear that the absorbance peak of nucleic acids is 260 nm, in reality, the absorbance maxima of different fragments of DNA vary depending on the base composition. A pure DNA sample with and absorbance 260 = 1 has an approximate concentration of about 50 µg/ml. This value depends on the mole % of G + C in the DNA. The strength of the absorption is also a function of the molecular structure, and has been determined empirically for many compounds; this is known as the “extinction coefficient”. The relationship is described by the Beer-Lambert Law:
A =εcl A ε
c l
absorbance value (no units) extinction coefficient (constant for each substance, units of M cm ) concentration of substance (units of M) light path length (in cm); all specs in common use have l =1 cm
In the case of most compounds, the units involve molarity. But in the case of polymers, where molarity does not depend on length, it is more useful to employ units of mass per volume instead of molarity (moles per volume). For DNA spectrometry, the units of concentration are typically -1 µg/µl and the extinction coefficient has units of (µg/ml) . For DNA, the extinction coefficient is 0.020 per µg double stranded DNA per ml of solution per cm of light-path or 0.020 per µg/mlcm. In DNA sample, proteins may contaminate the DNA preparation, and proteins also absorb in the ultraviolet. By using A260 to calculate the concentration of DNA, it may give deceptively high results. We can reassure ourselves that the contamination by protein is not significant by measuring the absorbance of our DNA preparation at 280 nm, because this is the wavelength at which the aromatic rings on tryptophan and tyrosine absorb. As useful rule, if the absorbance of sample at 260 is more than 1.75 times the absorbance at 280, the DNA should be pure enough to proceed. If the ratio is greater than 2.1, DNA may not probably work or can be said there is salt contamination. If the ratio is less than 1.75, the DNA is badly contaminated with protein and may not behave well in subsequent experiments. As normally isolated in the laboratory DNA exists as an ordered duplex of DNA strands, RNA and contaminating protein. The goal of this experiment is to measure and characterize the purity of a DNA sample isolated from calf thymus DNA and to assess the temperature, Tm, at which DNA transitions from an ordered duplex to a disrupted strands of DNA that have lost some hydrogen bonds, which is called thermal melting. The “melting” temperature can be ascertained from the measurement of absorbance data collected as a function of temperature. A plot of the normalized absorbance versus temperature yields a sigmoidal curve from which the Tm can be measured. The temperature at the mid-point between dsDNA and ssDNA is known as melting temperature (Tm). Each species of DNA has a characteristic Tm value that can be used for identification and characterization purposes. The more G + C bases in DNA, the higher the Tm. The is due to C:G bases have 3 hydrogen bonds, so more energy is required to break the bonds. Then, there will be the increase in absorbance (A260) upon denaturation, which called hyper chromic shift. In this experiment, we also performed renaturation. Renaturation is the process of formation of double stranded DNA from single stranded denatured complementary DNA strands. It involves reannealing or formation of hydrogen bonds between complementary base pairs which effected by cooling. After denaturation, if the temperature is decreased slowly, the strands will reanneal. If the temperature is rapidly decreased, only small a reas of DNA will renature.
Materials and Apparatus: Calf thymus DNA (0.001g), TE buffer(ml), autoclaved microcentrifuge tube (1.5ml), water bath (90°C), ice bath, nano-drops spectrophotometer, gloves, electronic digital balance, P1000 micropipette, autoclaved micropipette tips, stopwatch. Methodology: A 0.001g of calf thymus DNA was measured by using an electronic digital balance and placed in to 1.5ml of autoclaved microcentrifuge tube. The microcentrifuge tube was labelled as master tube. A 1000 ul of TE buffer was inserted into the master tube by using P1000 micropipette. Then, 10 ul of mixture from master tube was transferred to a new 1.5 ml of autoclaved microcentrifuge tube and labelled with sample tube 10 ug/ml, followed by 990 ul of TE buffer was added in the tube. After that, 50 ul of mixture from sample tube 10 ug/ml was transferred to a new 1.5 ml of autoclaved microcentrifuge tube and labelled with sample tube 50 ug/ml, followed by 950 ul of TE buffer was added in the tube. Following, 100 ul of mixture from sample tube 50 ug/ml was transferred to a new 1.5 ml of autoclaved microcentrifuge tube and labelled with sample tube 100 ug/ml, followed by 900 ul of TE buffer was added in the tube. Next, 500 ul of mixture from sample tube 100 ug/ml was transferred to a new 1.5 ml of autoclaved microcentrifuge tube and labelled with sample tube 500 ug/ml, followed by 500 ul of TE buffer was added in the tube. All the sample tubes prepared were then measured by using nano-drops UV-spectrophotometer to determine the concentration and purity of DNA samples and recorded in data sheet 1.1. Sample tube 100 ug/ml was chosen to further undergo the renaturation process. A 300 ul of mixture from sample tube 100 ug/ml was transferred to three of new 1.5 ml of autoclaved microcentrifuge tube and labelled fast cooling, slow cooling and room temperature respectively. Sample tube labelled room temperature was maintained at room temperature with left it in a blank beaker. The other two sample tube (slow cooling and fast cooling) were placed in a 90°C water bath for 15 minutes. After the incubation period, the sample tubes were removed. Sample tube labelled fast cooling was quickly cooled in an ice bath and sample tube labelled with slow cooling was slowly cooled to room temperature over a period of about 30 minutes. After 30 minutes, all the three sample tubes were measured by using nanodrops UV-spectrophotometer and the absorbance at wavelength from 220 to 350 (nm) were recorded in data sheet 1.2. Calculation:
Results:
Discussion: In this experiment, after DNA was done prepared, then we proceed to measure the purity of DNA through the concentration of yielded DNA by using nano-drops UV-spectrophotometer. We were using 260/280 ratio for the measurement. From the results we obtained, all of the samples were mostly have half yielded of concentration of DNA detected by nano-drops UV- spectrophotometer as shown as below: Prepared sample volume (ug/ml) 10 50 100 500
Detected sample volume (ug/ml) 5.3 25.2 60.1 288.4
However, the ratio of 260 / 280 measurement for samples 50, 100 and 500 ug/ml was in the correct range. Only sample 10 ug/ml was out of the range. The ratio of 260/280 measurement of each sample were shown as below: Sample (ug/ml) 10 50 100 500
Ration of 260/280 1.69 1.84 1.83 1.86
A ratio of 1.8 is generally accepted as pure DNA; a ratio of 2.0 is generally accepted as pure RNA. Abnormal 260/280 ratios usually indicate that the sample is either contaminated by protein or a reagent such as phenol or that there was an issue with the measurement. A low A260/A280 ratio may be caused by: Residual phenol or other reagent associated with the extraction protocol. Residual proteins associated with the extraction protocol. A very low concentration (>10 ng/ul).of nucleic acid.
• • •
High 260/280 purity ratios are not indicative of an issue. Although purity ratios and spectral profiles are important indicators of sample quality, the best indicator of DNA or RNA quality is functionality in the downstream application of interest. If the purity ratio is significantly higher than expected, it is best to review the spectral profile as a primary means of troubleshooting. Normally, when 260/280 ratio is higher than 2.0, we conclude it as RNA contamination. Some researchers encounter a consistent 260/280 ratio change by using nano-drop spectrophotometer. The three main explanations for this o bservation are listed below: Change In Sample Acidity Small changes in the pH of the solution will cause the 260/280 to vary. (William W. Wilfinger, March 1997 , pp. 22:474-481) Acidic solutions will under-represent the 260/280 ratio by 0.2-0.3,
while a basic solution will over-represent the ratio by 0.2-0.3. It is important to ensure that the pH of an undiluted sample measured on the instruments is at the same pH and ionic strength. 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.
Nucleotide Mix In The Sample The five nucleotides that comprise DNA and RNA exhibit widely varying 260/280 ratios. (Leninger, 1975 ) The following represent the 260/280 ratios estimated for each nucleotide if measured independently:
Guanine: 1.15 Adenine: 4.50 Cytosine: 1.51 Uracil: 4.00 Thymine: 1.47 The resultant 260:280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260/280 ratios for the four nucleotides present. It is important to note that the generally accepted ratios of 1.8 and 2.0 for DNA and RNA respectively, are "rules of thumb". The actual ratio will depend on the composition of the nucleic acid. From the graph of data sheet 1.2, the ratio of 260/280 was in the correct range for DNA, which is above 1.8 but below 2.0. With these, we could see that the reannealing was taking in process after denaturation. The renaturation of regions of complementary between different nucleic acid strands is known as hybridization. The highest ratio of 260/280 was the cooling at room temperature for 45 minutes, followed by fast cooling and then slow cooling. Theoretically, the sample tube cooling at room temperature should ranked at the top, followed by slow cooling and then fast cooling. However, there is a different in our results. There may be some errors occurred when doing the experiment. Several factors contribute to renaturation efficiency. The most 3 important are temperature, DNA concentration and renaturation time. In this experiment, we applied the factor of renaturation time as manipulated. The best temperature for renaturation of a DNA is about 25°C below its melting temperature. This temperature is low enough that it does not promote denaturation, but high enough to allow rapid diffusion of DNA molecules and to weaken the transient bonding between mismatched sequences and short intra-strand base-paired regions. With this statement, we able to support the results that the highest ratio of 260/280 was the cooling at room temperature. This is due to when we conducting the experiment, the airconditioner had decreased the room temperature to around 25°C. And, obviously, from data sheet 1.2, the longer time allowed annealing, the more renaturation occurred. However, there is a shift between fast cooling and slow cooling. Supposing, fast cooling allows only the formation of local regions of dsDNA and formed by the base pairing or annealing of short regions of complementary within or between DNA strands. This will leads to decrease in A260. On the
other hand, slow cooling allows time for the wholly complementary DNA strands to find each other and the sample could become fully double-stranded with the same absorbance as the original native sample. Hence, this will leads to increase in A260. With the errors occurred between fast cooling and slow cooling, it may due to there is contamination in sample tube slow cooling or the DNA concentration of sample tube slow cooling is not in correct amount.
Renaturation of DNA by fast cooling and slow cooling Conclusion: The ratios of 260/280 for calf thymus DNA were 1.69, 1.84, 1.83 and 1.86 for 10 ug/ml, 50 ug/ml, 100 ug/ml and 500 ug/ml respectively. The ratios of 260/280 for calf thymus DNA of 100 ug/ml were 1.83, 1.85 and 1.84 for slow cooling, room temperature and fast cooling respectively. References: 1. Campbell, N. A., Reece, J.B., Meyers, N., Urry, L. A., Cain, M.L., Wasserman, S.A., Minorsky, P.V., Jackson, R.B. (2009), Biology (8th, Australian version ed.), Sydney: Pearson Education Australia. 2. William W. Wilfinger, Karol Mackey, and Piotr Chomczynski, Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity: BioTechniques 22:474-481, March 1997. 3. Leninger, A. L. Biochemistry, 2nd ed., Worth P ublishers, New York, 1975. rd 4. Philip C. Turner, Molecular Biology, 3 ed. Instant Notes, 2005.
