Experiment 11 DNA Composition by HPLC 1,2 Introduction This experiment illustrates quantitative analysis by high performance liquid chromatography and biochemical methods of sample preparation. The sample is calf thymus DNA (Clostridium perfringens). A suspension of the sample is denatured to give singlestranded DNA by incubation at 100 C for 10 min. The heat-denatured DNA is then digested with Nuclease S 1, (50 C for 1 hr) to give 5'-mononucleotides (abbreviated C, T, A, and G): In double-stranded DNA, C is hydrogen bonded to G and A is hydrogen bonded to T. Therefore the concentrations of C and G are equal and the concentrations of A and T are equal. DNA from different organisms has different relative amounts of (C + G) and (A + T). When DNA is hydrolyzed by the enzyme nuclease S 1, it is cleanly broken into the four nucleotides. These nucleotides are separated and quantified by use of a reverse phase HPLC column. Standard solutions of the four nucleotides are used to calibrate the elution times and absorbance signals (260 nm). The peak areas of A, T, G, and C are determined for the standards and for the DNA sample. You will determine the relative concentrations of [A]/[G], [C]/[G], and [T]/[G] and find the fraction of [C + G] nucleotides in this DNA. (1) Wietstock, S. M. J. Chem. Ed. 1995, 72, 950-952. (2) Harris, P.C. "Quantitative Chemical Analysis" 6 th Ed. 2003. w\vw.whfreeman.com/gca. 1
Background Information 1 Over the years, the most common method utilized for the determination of the combined mole fraction of G + C (X gc ) is the melting temperature (T m ) of the DNA as determined by a spectrophotometric method (1-3). This involves observing the 260 nm hyperchromic shift of the DNA as it is transformed from double stranded DNA to single stranded DNA (denaturing the DNA greatly increases the absorbance at this wavelength in comparison to the double stranded DNA). The DNA in this assay is suspended at a given concentration in a buffer system of known ionic strength, typically a 0.05-0.15 M sodium chloride-sodium citrate buffer solution. This sample is placed in a spectrophometer with a water-jacketed cell holder, and a microtemperature probe, connected to a recording system, is placed in the sample. The DNA suspension is heated slowly while the absorbance at 260 nm (A 260 ) is recorded. A plot of the A 260 versus incubation temperature is prepared and the T m of the DNA is read at the midpoint of the hyperchromic shift. If the salt concentration and the T m of the DNA solution are known, then the X gc can be determined utilizing the equation: T m = 16.6(log C s ) + 41 X gc 81.5 where C s is the total salt concentration of the sample (1). Determining X gc by this method is not a trivial task when dealing with X gc greater than 0.6 because the T m occurs near 95 C. A new method for the determination of X gc has been developed that requires only a sample of heat denatured DNA, a buffered nuclease S 1 solution, and a reverse phase isocratic high performance liquid chromatography (HPLC) system. Results are obtained in two hours time, and the need for an extensive temperature-regulated spectroscopy system is eliminated. The HPLC procedure gives good results for X gc when compared to values obtained from the literature. This is in contrast to those values for X gc obtained by the thermal denaturation method where experimental values obtained by students deviated from theoretical values up to 10%. Instrumentation 1. Liquid chromatograph A commercially available liquid chromatograph (PM-80 pump and CC-5 Injector, Bioanalytical Systems, West Lafayette, IN) is used in this experiment where separation is performed on a reversed phase column (#MF 8954). Chromatograph and column settings are as follows: Flow rate: 0.2 ml/min Minimum pressure setting: 250 PSI Maximum pressure setting: 4000 PSI Column dimensions: 3(i.d.) x 100 mm Column has silica packing (3µm particle size) and is functionalized with ODS (C 18 ). ph range is 2-7. 2. Detector Detection is by absorption spectroscopy with the UV lamp set at 260 nm. Detector settings are as follows: Rise time: 0.2 s Wavelength: 260 nm Range: 0.50 absorbance units (full scale). 3. Data acquisition Data will be recorded on line using a voltmeter and a computer, using the Timed Data Acquisition software. The following settings should be used: Time per point: 0.1 s Time per chromatogram: 15 min initially 2
4. Saving data Each group should bring at least two flash drives to save data on. Care and Maintenance of the HPLC Columns These reversed phase columns are often used with mobile phases that contain mixtures of acetonitrile and water (as in Exp. 9.) In the present experiment you will use a 100% aqueous phase with 10 mm phosphate buffer at ph 7.2. This eluent works very well for the present separation, but prolonged exposure of the column to these conditions will ruin the column. Therefore, the columns have been stored in 20% acetonitrile and 80% water. The acetonitrile will interfere with the desired separation. Hence, the columns must be equilibrated with the aqueous phosphate eluent by flushing it for 20-30 min prior to starting separations (this will have already been done before you come to lab). After all the separations are completed, the column should be flushed with 20% acetonitrile / 80% water (the TA will do this for you when you have completed the lab). Procedure Pre-made solution: Enzyme Buffer: 0.6 mm ZnCl 2, 0.050 M Acetate Buffer @ ph 5.3. The TA will weigh out and add enzyme (0.3 mg/100 ml) on the day of the lab. Standard Solution: Only one standard is needed for this experiment. All groups will work together to prepare one standard solution for the entire class. Four groups should weigh out 0.20 mmoles of the standards (one group weighs out A, one weighs out C, etc.) and another group will weigh out 1.6 mmoles of sodium hydroxide. NOTE All of these weighed out standards are then dissolved together in a single 1L volumetric flask using the furnished super pure water. DNA Solution: Calf thymus DNA will be weighed out by a TA. The solution will be 10 ml with a concentration of 0.1 mg/1 ml. Each group will pipet 1 ml (with a Pasteur pipet) of this solution into a centrifuge vial. Close the cap and label your vial on the top of the cap. Then put the vial in the large oven set at 100 C for 15 minutes to allow the DNA to denature. After this time take the vial out and put it in ice for about 30 seconds. Transfer 0.1 ml (use the micropipette with two 50 µl portions) of the DNA solution to another vial containing 0.1 ml (again, two 50 µl portions) of the enzyme solution. Put the vial in the heating block for an hour at 50 C. Filtering the DNA digest with a syringe filter: Remove the blunt tip needle from the injection port and attach it to a disposable syringe. Draw your digested DNA up into the syringe. Pull some additional air into the syringe to ensure that all of your sample is out of the needle. Remove the needle and replace it with a syringe filter. Filter the sample into a clean centrifuge vial. Rinse the blunt tip needle with DI water and set aside for reuse. Data Acquisition: You will use the timed data acquisition program on the computer desktop to obtain data. The run time needs to be set to 15 min in the software. 3
The pump flow rate is preset to 0.2 ml/min, and the UV lamp is preset to 260 nm. Press zero on the detector in order to tell the instrument that the eluent is your blank. Rinse a syringe with several volumes of DI water and fill the syringe with the solution to be separated. With the INJECTOR handle in the LOAD position, inject 25 µl of solution through the septum. DO NOT REMOVE THE SYRINGE. Move the INJECTOR handle to the INJECT position to inject the sample onto the column and Click Start on the computer screen immediately. At least three runs are needed for the standard solution. More may be needed if the peaks are not separated. This can be done while the DNA solution is in the heating block for 1 hour. Then do 3 runs of the DNA solution. NOTE Following clean up to be completed by the Teaching Assistants ONLY: Turn off the pump, and after the pressure reaches zero change the flow rate on the pump to 0.5 ml/min. Switch the inlet from the mobile phase to the flush solution. Be sure to rinse the inlet line with DI water prior to putting it on the flush solution (20% acetonitrile). Allow 10 minutes to flush the column and line. Empty your waste beaker into the waste bottle. Data Analysis: Data should initially be opened in Excel. Make a plot of your standard runs, and print it out. Note the times when a peak starts and stops. In order to use Origin to integrate the peaks, you have to calculate them one at a time. Copy the data corresponding to one of your peaks from Excel into Origin. Make a line plot of the data, then go to Analysis Calculus Integrate. The text box that appears gives you the peak area. After you have all the areas of the first peak, find the average of those areas. Do this for all subsequent peaks. Make a table of these averages and label them (elution time: C<T<G<A). You will now treat peak G similar to an internal standard; divide the average area of G by each of the other peaks' average areas. Measure the response factor (f) of each component to G and add them to your table. An example calculation is provided below. (Average Area X)/[X] = (f x )*(Average Area G)/[G] Now repeat the integration process for the DNA runs, being careful to identify the correct peaks based on the retention times from the standard runs. Record the peak areas in a new table. Using your individual DNA peak areas and your previously calculated response factors, determine the relative concentrations [A]/[G], [C]/[G], and [T]/[G]. Find the fraction of [C + G] nucleotides with the following equation. Calculate this value for each DNA run. The accepted literature value for calf thymus is 0.42. Graphs and Tables to include in your lab report: - Graph of each standard ran and each DNA ran (6 graphs in total). - Table of standard peak areas, average standard peak areas, and response factors. - Table of DNA peak areas, relative concentrations, and experimental [C + G] fractions. 4
Operation of Finnpipette Pipetman This pipet can deliver a range of 5 µl to 50 µl. The volume can be adjusted by turning the knob on the top of the pipet. The volume to be delivered is read on the side of the pipet. - Start by adjusting the pipet to the appropriate volume to be delivered. - Attach a disposable tip to the pipet. Tap the pipet several times while still in the box to ensure a secure fit. - Depress the plunger to the first stop. Immerse the tip into the solution to be pipeted. Be sure to keep the pipet vertical or improper measurement may result. - Release the plunger slowly to draw the sample up into the tip. The tip contains a built in filter to help prevent liquid from entering the pipet mechanism. DO NOT lay the pipet down on its side while there is solution in the tip. This will allow liquid to enter the pipet mechanism and will mess up its calibration and operation. - Place the tip of the pipet within the container where the solution is to be delivered. Depress the plunger to the first stop. Wait a second or two for any remaining liquid to settle and then depress the plunger to the second stop to expel all of the liquid. - Remove the used tip by depressing the ejector button and dispose of it properly. - Always use a new tip for a new solution. - Return the pipetman to its box. 5