Roger Steinerl and Bruce Hudson Harvard University Cambridge, Massachusetts 02138
I I
The Helix Coil Transition of DNA Observed with an inexpensive uv photometer
The helix-to-coil transition of DNA is a potentially useful experiment for an advanced freshman class because it introduces several concepts of general physical interest such as weak forces, cooperative phenomena and spectral changes due to interactions between neighboring units. Changes in the median temperature and temperature range over which helix-to-coil transition takes place may he used to demonstrate photochemical changes or interactions with organic dyes. An attractive feature of this experiment is that it utilizes contemporary student interest in molecular biology and illustrates an application of thermodynamics, statistical mechanics, and spectroscopy to a problem of some biological interest. The most easily measured change which occurs when DNA is converted from the double helix to its random coil form is the approximately 40% increase in intensity of the principle absorption maximum a t about 2600 A. This increase in intensity usually occurs in the temperature range of 60 to 100°C over a transition range of several degrees. The equipment necessary for this experiment therefore consists of an ultra-violet photometer operating near 2600 A and equipped with a variable temperature cell. Commercial versions of such instruments are sufficiently expensive that their availability for individual use by large classes is unusual. The device to be described reduces the cost of this experiment considerably. More importantly, however, it is constructed by the student who thereby learns the basic principles of spectroscopy on a first-hand basis. Experimental The Photometer The light source for the photometer is a low pressure germicidal mercury lamp which emits about 99% of its power in the 2537 A mercury line (1). This Lamp Radiation is Very Harmful to the Eyes. T h e Student Must be Warned Not to T u r n The Lamp on Except When i t is Entirely Covered. This Warning Must Be Enforced. The light is filtered by a NiSO&oSO4 solution (2) and a 9863 Corning filter. The other components of the photometer are a quartz sample cell mounted in an aluminum block wound with a heating element, and an inexpensive photosensitive resistor coated with salicylic acid which fluoresces with a high efficiency when excited by 2537 A irradiation. The arrangement of the components is shown in Figure 1. The aluminum hlock and mercury lamp are supported by standard laboratory clamps. The other elements are mounted and masked with hlack photographers' masking tape. After addition of the sample cell the unit is made light tight by liberal use of hlack tape. The resistance of the photoresistor was measured with a vacuum tube voltmeter. A less expensive volt-ohmmeter would be adequate, and greater accuracy could he obtained by use of a Wheatstone bridge. Observation of the midpoint and shape of the helix-tocoil transition does not require a knowledge of the absolute absorbance of the sample but only a measure of some
Mercury lamp
\
Photoresistor
I$
~ S a / i c y / i c&id coaling
Figure 1. Schematic representation of the photometer. Black photographer's masking tape covers the entire apparatus when in use. The quartz sample cell fits inside the heating block. A thermometer is seated in a hole in the block. The black is wrapped with Nichrome wire tor heating.
quantity which is proportional to the absorbance, since the final data are then normalized to the total chanee ohserved. The only calibration necessary is a measurement of the resistance of the ohotosensitive element for a dilution series which is in {he range of approximately 0.1 to 2.0 OD. The hest test solution to use, naturally, is the DNA solution to he used for the melting experiment. The student should understand that this photometer, like all others, will fail at some sufficiently high value of the optical density. In this case the principal cause of failure will be the inability of t h e filters to completely hlock the mercury lines at 3650 A and in the visible range which are not absorbed by DNA. These will, of course, mask the transmitted 2537 A line a t high OD. The necessary check, for expected stray light is shown in the table.
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The Heating Block The aluminum hlock which holds the sample cell is covered with glass tape, wrapped with Nichrome wire, and then wrapped with more insulating tape. The ends of the wire are connected to a line cord. The unit is then powered with a variable nutn-transformer. The temperature is measured hy placing a thermometer in a hole in the block containing mineral oil and, for calibration, another thermometer in the sample cell which is iilled with Volume 50, Number 2, February 1973 / 129
water. The voltage is raised and the temperatures measured at several values after thermal equilibrium is reached. In actual aperation the sample cell is capped with a greased stopper to prevent evaporation, being careful tbat none of the UV absorbing grease gets into the sample. During a run the temperature is determined from the thermometer in the block and corrected for any small differences between sample and black.
The Major Emission Lines of Mercury, Their Relative Intensities, the Transmission of the Filter Assembly and the Final Transmitted Intensity
Intensity Relative to 2537 A-
AtAl
Transmission
Transmitted Relative Intensity
DNA Solutions
Commercial calf thymus DNA (highly polymerized) is suitable for this experiment. This material suffers from the disadvantage that the transition is relatively broad due to base composition heterogeneity from molecule to molecule. This can be avoided by purchasing bacterial DNA's hut these are much more expensive. Solutions are made up in 0.05 M pH 7 phosphate buffer which is available in prepackaged form as a pH standard. Solutions take several days to a week to prepare with constant stirring carried out in a cold room or refrigerator. A few drops of chloroform should be added to the solution to kill bacteria, and the solutions should be stored in the cold. A 1 OD solution contains about 4&50 micrograms of DNA per milliliter.
Figure 3. The measured melting curve
Figure 2. The photometer calibration
curve
Calibration Figure 2 shows the photometer calibration curve. Resistance is plotted against optical density (OD), normalized by the values for a pure buffer sample. Thus the values plotted are (OD of the DNA sample - OD of the pure buffer) versus (resistance of the DNA sample - resistance of the pure buffer). By employing this calibration curve on subsequent trials, the pure-buffer normalized resistance of the photoresistor may be immediately converted to OD. A sample cell of pure buffer was heated in the apparatus in e i aetly the same manner as a sample of DNA. The optical density was found to vary with temperature in a systematic manner; this is a combination of reversible heat effects on the buffer and on the photoresistor. A value is found for change in OD a t a given temperature, and this "alue is then subtracted from the OD found at tbat temperature during trials with DNA samples. The temperature effects mentioned were not found to interfere with the usefulness of the photometer, but they might be reduced by passing a stream of coal compressed air in front of the phatoresistor. Adequate protection must, of course, be taken against the entrance of stray light. This cooling procedure is a refinement found to be unnecessary with our photometer; after the two relatively simple steps of calibration and control, through which the operator gains familiarity and experience with the apparatus, the photometer is ready for use. DNA Hyperchromism
Having completed these calibrations, t h e basic experimental procedure consists of measuring resistance of t h e photoresistor a s a function of temperature. After suhtracting t h e constant resistance value of a pure buffer sample
130 /Journal of Chemical Education
of calf thymus DNA
from t h e measured resistance, Figure 2 provides t h e corresponding normalized O D value. Correcting for a n y heat effects, as described above, t h e desired d a t a i n t h e form of optical density a s a function of temperature are derived. A plot of (relative absorbance) / (total relative absorbance change) versus temperature is then produced (Fig. 3) which shows a break a t t h e so-called melting temperature, T,. T h e time required for a trial is about two hours. Figure 3 shows a hyperchromic shift of 60%. T h e midpoint of t h e curve, T,, is 86.Z°C; the value for calf thymus D N A reported by Marmur (3) is 86°C. T h e rather large span of temperature over which t h e transition takes place is due t o t h e heterogeneity of calf thymus D N A with respect t o base composition a n d molecular weight. An experiment of this type was also performed on a sample of D N A which h a d been irradiated with t h e unfiltered uv l a m p for 15 miu. Obvious changes d u e t o t h e photochemical action of t h e irradiation were observed including smaller hyperchromism and broader transition. T h e genetic a n d physical effects of uv irradiation in uivo and in uitro are well reviewed i n t h e literature (4-6). Other possible modifications of this experiment include t h e effect of t h e interaction of cations with D N A (7) a n d t h e changes i n T , with base composition of t h e DNA (8). Literature Cited 111 "American lnsfifufe of Physics Handhoak." McCraw-Hill. N e w York, 1957, p. 7799
121 Kssha. M.. J.Ool. Soc Am.. 38.9291194SI. ~J.. J. M~~ I B~OI., . ~ 5.208(19611. ~ . (a M (4) Setlaw. R.B..%OF. Nvcl. Acid. Res, 8,257 1196Rl. (SI Setlow. R.B..Science. 155.379 11966). 16) Hsrhers, E.."lntroduetion!otheNucleieAeid~,"binhoid,NelvYork. 1968. (7) Gilrin. Y,R., andDoty, P..B i o c h ~ mBmphya. . Aelo., 61.458(19621. (81 Mahler. H. R.. and Corder. E. H.. "Bioiogieal Chemirfry." Harpor and Row York. 1 9 7 1 , ~238. .
