Volume 4 Number 10 October 1977
Nucleic Acids Research
Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea
James R. Hutton
Department of Biology, Marquette University, Milwaukee, WI 53233, USA Received 18 July 1977
ABSTRACT This paper reports the results of a systematic study of the effects of formamide and urea on the thermal stability and renaturation kinetics of DNA. Increasing concentrations of urea in the range 0 to 8 molar lowers the Tm by 2.250C per molar, and decreases the renaturation rate by approximately 8 percent per molar. Increasing concentrations of formamide in the range from 0 to 50 percent lowers the Tm by 0.600C per percent formamide for sodium chloride concentrations ranging from 0.035M to 0.88M. At higher salt concentrations the dependence of Tm on percent formamide was found to be slightly greater. Increasing formamide concentration decreases the renaturation rate linearly by 1.1% per percent formamide such that the optimal rate in 50% formamide is 0.45 the optimal rate in an identical solution with no formamide. The effects of urea and formamide on the renaturation rates of DNA are explained by consideration of the viscosities of the solutions at the renaturation temperatures.
INTRODUCTION
The complex genomic DNAs of higher organisms require a considerable length of time, sometimes weeks, for a large fraction of their unique sequences to renature when using reasonable DNA concentrations. Since the optimal temperatures for renaturation to occur in aqueous salt solutions are fairly high (65 to 750C, depending on the monovalent cation concentrationl), a considerabile amount of DNA degradation can result from an extended exposure to
those temperatures2'3. In an effort to minimize such thermally induced degradation while still maintaining stringent reaction criteria4, investigators have frequently resorted to studying renaturation rates in aqueous solutions of organic reagents. For excellent reviews see Wetmur5 and Britten, et al.4. These solvent systems allow reasonable renaturation rates and criteria to be achieved at relatively low temperatures. Studier6 has shown that formamide does not chemically degrade nucleic acids at room temperature, and at the lower renaturation temperatures DNA degradation is thought to be much less of
X Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
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Nucleic Acids Research a problem.
Two organic reagents that have been used for this purpose are
formamide and urea7'4. Several investigators have reported limited studies on the effect of formamide on the thermal stability of DNA7-12 and on the renaturation kinetics of DNA7. Several studies of the helix-coil transition of DNA have also been performed in aqueous formamide solutions (e.g. 6, 11-14) in an effort to minimize DNA degradation problems. The reported values for the lowering of Tm per percent formamide in solution range from 0.60 to 0.720C. Although the need for a systematic study of the effect of formamide on renaturation
kinetics has previously been expressed in the literature4, no such studies have been reported for this reagent or for urea. This study endeavors to systematically determine the effects of the variables temperature, monovalent cation concentration, and organic reagent concentration on the thermal stability and the second order renaturation rate of DNA. The results of this study will provide the information that is necessary to critically compare the results of renaturation studies that are performed in aqueous solutions of these organic reagents with the results of similar studies performed in aqueous salt solutions. This should allow conditions to be selected for a given experiment that will provide the desired renaturation criteria with minimal DNA degradation problems.
METHODS
(a)
DNA and Reagents E. coli W3110 was grown in nutrient broth, collected by centrifugation,
washed and resuspended in 0.03 M Tris HC1, 0.01 M EDTA, pH 8.0. The suspension was adjusted to 20 ug/ml with lysozyme (Sigma, Grade I) and incubated
for 20 min. at 240C. An equal volume of 2% sarcosyl, 0.01 M Tris HC1, 0.03 M EDTA, pH 8.0 was added to lyse the cells. The mixture was then adjusted to 200 ug/ml with pronase (Calbiochem, Grade B) and incubated at 370C with gentle shaking for 12 hours. The NaCl concentration was adjusted to 0.15 M and 2 volumes of ice cold ethanol were added to precipitate the DNA. The DNA was spun out of solution on glass rods and resuspended in 0.15 M NaCl, 0.03 M Tris, 0.01 M EDTA, pH 8.0. The solution was adjusted to 20 ug/ml with RNase A (Sigma, type IA) and stirred gently at 240C for 30 min. It was treated with pronase as before and extracted twice with equal volumes of freshly distilled phenol 15 . The aqueous phase was dialyzed exhaustively against 0.50 M NaCl, 10-3 M EDTA, 0.03 M Tris pH 8.2. After dialysis the DNA was fragmented by sonic irradiation with a Biosonik sonicator. The sonicated DNA
3538
Nucleic Acids Research was dialyzed against 0.40 M NaCl, 10-3 M EDTA, 0.03 M Tris, pH 8.2 and used
in the following studies. Cl. perfringens DNA and an alternative source of E. coli DNA were purchased from Sigma. Bacteriophage lambda DNA was prepared as previously described16. The single-stranded fragment size L of E. coli DNA was determined from the second order rate constant K2 obtained under reaction conditions of known DNA nucleation rate constant KN1,l7. L was calculated from the relation
L1/2=K2NKNj1, where the kinetic complexity N of E. coli is taken as 4.0x106 base pairs 18,19 and KN is 1.7x105 for 0.40 M NaC117. The single-strand fragment sizes of the DNA preparations used in the following studies range from 1,600 nucleotides to 20,000 nucleotides. Unless otherwise indicated, the results reported for the following studies were independent of the DNA fragment size used within this range of L. All chemicals used were reagent grade. Formamide was Fisher F-95 and Urea was obtained from Sigma. (b) Melting Temperatures The methods used to measure the DNA melting temperatures were essentially those previously describedl6. Sonicated E. coli DNA was adjusted to 0.015 M Tris, pH 8.2, 5x10-4 M EDTA and the desired concentrations of NaCl and organic reagent. The samples were placed in cuvettes and overlaid with mineral oil to prevent evaporation. The absorbance at 275 nm was measured in a recording Gilford model 2400 spectrophotometer equipped with a reference compensator and a temperature programmer to monitor the absorbance of four cells and the temperature of the cell compartment. A recirculating Haake bath was equipped with a motor to rotate the thermostat control of the contact thermometer. The rate of temperature increase was 0.50C per minute. Absorbance at 275 nm was used to monitor the denaturation and renaturation reactions in order to minimize the contribution of formamide to the absorbance of the solution. E. coli, Cl. perfringens and bacteriophage/N DNAs each exhibited a hyperchromicity of 40% at 275 nm for each of the solutions studied. (c)
Renaturation Kinetics Solutions with DNA concentrations ranging from 30 to 150 ug/ml were placed in cuvettes and overlaid with mineral oil. The absorbance at 275 nm was determined for a native DNA solution at the desired renaturation temperature using a Gilford 2400 spectrophotometer. This A275 was taken as A . The cell chamber of the Gilford was then switched to a 1000C Haake bath and the chamber temperature raised to Tm+100C or greater. The absorbance of the
3539
Nucleic Acids Research fully denatured DNA at Tm+100C was taken as AO. The chamber was then switched back to the Haake bath regulated at the temperature desired for renaturation. Absorbance at 275 nm and temperature were recorded as a function of time. After the renaturation reaction had been monitored, the chamber was switched back to the 1000C Haake bath to recheck AO. The rate data were treated according to the procedure of Wetmur and Davidsonl. Only the first
half of a reaction, during which simple second order kinetics are followed, was used to determine the rate for a given sample. DNA solutions that demonstrated Tms greater than 800C were placed in a water-jacketed fused silica spectrophotometer cell that was connected directly to a recirculating Haake bath. The melting profiles and renaturation curves were then determined as previously described16.
All renaturation rates reported in this study are expressed relative to the optimal rate for the same DNA in an aqueous 0.20 M sodium chloride solution. That is, each reported rate is obtained by dividing the rate determined for a given set of conditions by the rate determined for the same DNA
preparation at the same concentration in 0.20 M NaCl at 650C. RESULTS AND DISCUSSION
(a)
Thermal Stability of DNA in aqueous solutions of urea Solutions of sonicated E. coli DNA were adjusted to 0.20 M NaCl, 0.015 M Tris HC1, 5x10-4 M EDTA and the desired urea concentration with a final pH of 7.7. The DNAs were melted as described in the methods section. The temperature at which half the cooperative absorbance change had occurred (Tm) is plotted in figure 1 as a function of the molarity of urea in the solution. As can be seen from figure 1, the melting temperature of the DNA has a linear dependence on the concentration of urea. Increasing concentrations of urea
in the range studied lowers the Tm by 2.250C per molar added urea.
100__
oc
Figure 1. 3540
0 2.0 4.0 60 8.0 MOLES/LITER Melting temperature versus urea concentration.
Nucleic Acids Research (b)
Renaturation of DNA in aqueous solutions of urea
The renaturation rate of sonicated E. coli DNA in 0.20 M NaCl was studied as a function of temperature and of urea concentration. Figure 2 presents the rate-temperature profiles obtained for solutions containing 0, 4.0, 6.0 and 8.0 M urea. Figure 3 depicts the dependence of the renaturation rate
at the optimum renaturation temperature (i.e. the temperature that allows the fastest renaturation rate) on the concentration of urea in the solution. As indicated by the slope of the linear curve, the renaturation rate decreases by approximately 8 percent for each added mole/liter of urea. I
I
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1.0 .8 .6 < .4
.2 _0
I
I
I
I
40
50
60
70
%
I
80
oc Figure 2. Relative renaturation rate versus renaturation temperature. All M 0.015 M Tris pH 7.7)5 x 10-4 M EDTA and 140 ug 0.20 were solutions NaCl, DNA/ml. 03: no urea; 0: 4.0 M urea; *: 6.0 M urea; A: 8.0 M urea. I
I
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1.0i_ .8
.4
.2 N0
I
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2.0
40
I
60
MOLES/LITER
I
8.0
Optimal renaturation rate versus urea molarity. The maximum renaturation rates obtained from rate-temperature profiles such as those presented in figure 2 are plotted as a function of urea concentration. All solutions contained 0.20 M NaCl, 0.015 M Tris pH 7.7 and 5 x 10-4 M EDTA.
Figure 3.
3541
Nucleic Acids Research The effect of urea on the renaturation rate of DNA can be explained by considering the viscosities of the renaturation solutions. The nucleation rate for DNA renaturation has been shown to be inversely proportional to the microscopic viscosity of the reaction solutionl20. The linear curve in figure 3 can be derived from the measured viscosities of the urea solutions at
Tm-250C (e.g. the viscosities of 0 M urea at 660C, 4.0 M urea at 520C and 6.0 M urea at 520C relative to H20 at 250C are respectively 0.499, 0.76 and 0.88) by simply graphing the ratio of the viscosity of the 0 M urea solution at 660C to the viscosity of the renaturation solution as a function of urea concentration. From the above studies it is obvious that urea has a rather severe effect on the renaturation rate of DNA relative to its effect on the Tm. Therefore, this reagent was considered to be of little probable value in minimizing thermally induced DNA degradation and was not studied further. (c) Thermal stability of DNA in aqueous solutions of formamide The thermal stability of sonicated E. coli DNA was studied as a function of formamide concentration and of sodium chloride concentration. Figure 4 illustrates the dependence of the Tm of E. coli DNA on the percent formamide contained in the solution for several different sodium chlodride concentrations. From consideration of the curves in figure 4 and similar curves (not shown) obtained for other sodium chloride concentrations, it can be concluded that for DNA solutions containing NaCl concentrations ranging from 0.035 to 0.88 M increasing the formamide concentration in the range studied lowers the
100s _
1
U
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_
0
80
60
0
20 40 PERCENT FORMAMIDE
60
Figure 4. Melting temperature versus percent formamide. DNA solutions contained 0.015 M Tris pH 8.2, 5 x 10-4 M EDTA, the indicated percent (volume/ volume) of formamide and sodium chloride to give 1.12 M,V ; 0.20 M, A ; 0.10 M, O ; and 0.035 M,O . DNA single strand molecular weight was 7 x 105 daltons. 3542
Nucleic Acids Research Tm by 0.60+0.010C per percent formamide added. At higher NaCl concentrations the dependence of the Tm on percent formamide was found to be slightly greater (e.g. dTm/d percent formamide equals -0.66 for 1.12 M NaCl) with the exact linear slope depending on the NaCl concentration. The reason for the greater dependence at higher salt concentrations is the shift of the Tm plateau region to lower NaCl concentrations with increasing formamide concentration as indicated in figure 5. Figure 5 presents the results obtained for the dependence of the Tm of E. coli DNA on the NaCl concentration for several different formamide concentrations and for Cl.perfringens DNA at zero percent formamide. It has
long been known that at high salt concentrations the salt dependence of the Tm of DNA will level off and even reverse signl,2l-24. The data in figure 5 indicates that this is also true for DNA in aqueous-formamide solutions. As is evident from figure 5, the NaCl concentration at which the salt dependence of Tm levels off decreases with increasing formamide. For each of the formamide concentrations studied the data for NaCl concentrations less than 2.0 M are fit quite well by two intersecting straight lines. The intersection point (i.e. the minimum NaCl concentration above which the Tm of E. coli DNA will no longer increase with additonal NaCl) can be estimated from figure 5
for each of the formamide concentrations studied. If these estimated minimum NaCl concentrations listed in Table 1 are graphed as a function of percent
oc~~~~~~~~~~~~~
60
F |l
.04
.10
MO LAR ITY
1.0
5.0
Melting temperatures of E. coli (curves B-G) and Cl. perfringens (curve A) DNA versus sodium chloride concentration for several different concentrations of formamide. DNA single strand molecular weight was 7 x 105 daltons. Formamide concentrations are - curve A, 0%; B, 20%, C, 30%; D, 40%; E, 50%; F, 60%; and G, 70%.
Figure 5.
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Table 1. NaCl concentrations above which the thermal stability of DNA in formamide solution no longer increases with additional NaCl. The values for zero percent formamide are for Clostridium perfringens DNA (26.5% GC) and all other values are for E. coli DNA (50% GC).
20
Maximum Tm (OC) 91.0 85.0
30 40 50 60 70
78.0 72.0 66.0 58.5 52.0
% Formamide 0
NaCl (moles/liter) 1.06 .85
.75 .70 .62
.46 .40
formamide, a reasonable fit is obtained to a curve that can be defined by the linear equation NaCl molarity = -0.0096 x percent formamide + 1.06. Within the range of formamide concentrations studied, this equation can be used to obtain an estimate of the minimum NaCl concentration for a given formamide concentration above which the Tm no longer increases with additional NaCl (i.e. the minimum NaCl concentration that will yield the maximum DNA Tm for the formamide solution). If the maximum Tms for E. coli DNA listed in Table 1 are graphed as a function of percent formamide, a linear curve is obtained that is defined by the equation
Tm(maximum) = -0.66 x percent formamide + 98.50C. These equations suggest that E. coli DNA should have a maximum Tm of
98.50C in aqueous NaCl solutions at pH 8.2, and that this maximum should occur at a minimum NaCl concentration of 1.06 M. As seen from curve A of figure 5, the minimum NaCl concentration at which Cl. perfringens DNA demonstrates a maximum Tm of 910C is approximately 1.06 M.
If the value of 33
is taken as dTm/dXGC for DNA in 1 M NaC124 where XGC is the mole fraction of GC composing the DNA, the Tm of 910C for Cl. perfringens DNA with XGC=0.265 can be converted to the equivalent Tm of 98.80C for E. coli DNA with XGc= 0.50 in excellent agreement with the equations. The slopes of the low salt regions of the curves in figure 5 are contrary to previously reported results for formamide solutions. Record1O has reported a dTm/d log Na+ value of 20.20C for calf thymus DNA in 50% formamide. Even if the counter ion contributions of the buffer and formamide degradation products are taken into consideration, the maximum value of dTm/d 3544
Nucleic Acids Research log Na+ that can be obtained from the data of figure 5 for E. coli DNA in 50% formamide is 9.50C.
Several experiments were performed in an attempt to re-
solve this difference. The NaCl dependence of the Tm of E. coli DNA in 50% formamide was deter-
mined using three different sources of formamide: Matheson Coleman and Bell formamide, and Fisher Scientific Co. F95 and F82 formamides. The conductivities determined for 50% solutions of formamide in distilled H20 were 830, 1200 and 1380 micromhos for MC/B, F95 and F82, respectively. This is equivalent to the conductivity of 0.005 to 0.01 M NaCl. The same value of dTm!d log Na+ was obtained for each of the three formamides, so the formamide does
not appear to be the likely source of the difference. Since record25 has shown that the value of dTm/d log Na+ for DNA in aqueous solutions is a function of pH, this was considered a likely source for the discrepancy. The values of dTm/d log Na+ for 50% formamide were deter-
mined at several different solution pHs. Figure 6 depicts the dependence of the solution pH as measured with a glass combination electrode on the percent
formamide for several buffer systems. Figure 7 presents the graphs of the Tm versus sodium ion concentration for 50% formamide solutions of E. coli DNA at four different pHs and Cl. perfringens DNA at one pH. The results of figure 7 are summarized in Table 2 along with the values obtained for 40% DMSO
solutions.
I
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I A
10
8
C
pH
D _
6
Ewt -
4~~~~~~~'
X -~~~
0
PERCENT
SO
Figure 6. Solution pH versus formamide concentration for several buffer systems. Buffers are as follows: curve A, 0.02 M sodium tetraborate; B, 0.015 M Tris-HCl; C, 0.02 M sodium phosphate buffer; D, 0.02 M NaH2PO4; E, 0.02 M NaH2PO4 + HC1 to pH 2.8; F, 0.02 M sodium acetate buffer; G, 0.04 M sodium acetate buffer. 3545
Nucleic Acids Research
Melting temperature versus sodium ion concentration for DNA in The buffers and solution pHs were as follows: E. coli DNA in 0.04 M sodium acetate pH 5.0 ( 0 ), 0.03 M sodium acetate pH 6.0 ( U ), 0.03 M NaH2PO pH 7.0 ( 0 ), 0.015 M Tris HCl pH 8.6 ( 0 ) and Cl. perfringens DNA in 8.015 M Tris-HCl pH 8.6
Figure 7.
50% formamide at several different solution pHs.
Table 2.
Dependence of Tm on Na+ concentration for Cl. perfringens and
E. coli DNA in 40% DMS0 and 50% formamide. Solution pHs were measured before and after Tm determinations to assure that no significant drifting occurred. Solution 50% formamide It
it
50% formamide it
40% DMS0 40% DMSO aqueous
if
DNA
pH
5.0 6.0 7.0 8.6 8.6 8.0
E. coli " " " " E. coli
Cl.
perfringens
8.0
" E. coli
8.2
Cl. perfringens
dTm/d log Na+ (OC) 17.3 13.4 12.8 9.5 14.2 10.0 10.0 15.0
The dependence of dTm/d log Na+ on solution pH for 50% formamide was found to be similar to that observed for aqueous DNA solutions25 and is therefore consistent with the theoretical considerations of Record, Woodbury and Lohman26. Since Record10 did not report the buffer system used or the solution pH for
his determination of the 20.20C value, the difference could conceivably be accounted for if the formamide solutions used in that determination were inadequately buffered such that they either had pHs that were below 5, or had pHs that drifted to values less than 5 during the determination. This possi3546
Nucleic Acids Research bility is not unreasonable, since it is reported below that the pH of an inadequately buffered formamide solution can decrease to less than 5 on heat-
ing. To eliminate the possibility that the DNA preparation I used was in some way peculiar, three sources of 50% GC DNA were used: E. coli and bacteriophage lambda DNAs that were prepared by me as described in methods and E.
coli DNA prepared by Sigma Chemical Co. The same values of dTm/d log Na+ were obtained for each of the three DNA sources. To further eliminate the
possibility of the difference being due to some nonapparent difference in our experimental systems the value of dTm/d log Na+ was determined for 40% dimethylsulfoxide, another solvent system studied by Record1O. The value of 100C was obtained for DNA in 40% DMSO in agreement with the results reported by Record. In conclusion, I cannot unequivocally account for the discrepancy between the values of dTm/d log Na+ for DNA in 50% formamide reported by Record and those reported in this study. The difference in GC content of the DNAs used may account for 1 to 2 degrees difference since Cl. perfringens DNA (26.5% GC) has a greater dTm/d log Na+ in 50% formamide than does E. coli DNA (50% GC) as indicated in Table 2, but this certainly could not account for a 10 to 11 degree difference. The remaining 9 degree difference could conceivably be accounted for if the 50% formamide solutions studied by Record had low pHs of say 5 or less. (d) Renaturation rate of DNA in aqueous solutions of formamide The kinetics of DNA renaturation in formamide was studied as a function of formamide concentration, temperature and NaCl concentration. Figure 8 presents the rate-temperature profiles obtained for DNA at several different formamide concentrations. The same general bell-shaped curve is obtained for the formamide solutions as is obtained in the absence of formamide. In each case the optimal rate occurs at Tm-25+50C with a relatively flat temperature
response in this range. Figure 9 represents the optimal renaturation rates obtained for several
formamide concentrations graphed as a function of formamide concentration. Increasing formamide concentration decreases the renaturation rate by 1.1% per percent formamide. Each point in figure 9 represents the average value obtained from a set of 4 to 9 independent determinations of the rate at a given formamide concentration. The error bars represent + one standard deviation obtained for each set of determinations. The linear curve drawn through the points in figure 9 represents the "predicted" rate versus formamide profile indicated by the data of Table 3 if the only effect that forma3547
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oc
Figure 8. Relative renaturation rate versus renaturation temperature. All solutions contained 0.20 M NaCl, 0.015 M Tris pH 8.2, 5 x 10-4 M EDTA and 140 ug/ml DNA. Formamide concentrations were as follows: 03, 0%; 0, 20%, U , 30%; 0 , 40%; and A , 50%. DNA single strand molecular weight was 7 x 105 d. I
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RATE .4
0
30 PERCENT
I
60
Figure 9. Optimal renaturation rate versus percent formamide. Each data point represents the average value obtained from a set of 4 to 9 independent determinations of the rate. The error bars represent + one standard deviation obtained for the set of determinations. DNA single strand molecular weight was 5 x 105 d. All solutions contained 0.20 M NaCl, 0.015 M Tris-HGl pH 8.2, 5 x 10-4 M EDTA and percent formamide (v/v) as indicated.
mide has on the optimal renaturation rate is due to the microscopic viscosity of the solution20. As is obvious from figure 9, the observed results are in excellent agreement with those predicted from the solution viscosities. 3548
Nucleic Acids Research Table 3. Viscosities of formamide solutions at Tm-25+50C for E. coli DNA. All solutions contain 0.20 M NaCl, 0.015 M Tris pH 8.2, 5x10-4 M EDTA. The viscosities were measured using an Ostwald-Fenske viscometer submerged in an equilibrated H20 bath regulated at the indicated temperature. 4 F95 0 10 20 30 40 50 60
'~/qHqO at 0.499 0.568 0.648 0.756 0.880 1.073 1.284
250C
Temperature (0C)
~~~~~~~~~Predicted Rate: formamide at 660C) solution at T
no
660C 600C 540C 480C
1.00 0.88 0.77 0.66 0.57 0.47 0.39
430C 360C
310C
The rate-temperature profiles obtained for DNA in formamide solutions containing several different NaCl concentrations are presented in figures 10 and 11. Figure 10 illustrates the profiles obtained for 50 percent formamide at four different NaCl concentrations and figure 11 illustrates the profiles obtained for solutions of 60 percent formamide at five different NaCl concentrations, along with two representative profiles for 70 percent formamide solutions. Similar bell-shaped profiles were obtained for other salt concentrations as well (data not shown). The salt dependence of DNA renaturation in 50, 60 and 70 percent formamite solutions is illustrated in figure 12. In 70 percent formamide at 250C the renaturation rate of DNA is essentially independent of NaCl at concentrations above 0.6 molar. In 50 percent formamide at 380C and in 60 percent formamide at 340C and 250C, the renaturation rate is only weakly dependent on NaCl at concentrations above 0.8 molar.
(e)
Nucleation rate constants The nucleation rate constant, KN, for DNA renaturation can be determined for each of the solvent systems studied. Some of these are listed in Table 4. KN is as defined by Hutton and Wetmur17 where KN K2 K2 is the experimentally determined second order rate constant, N is the complexity in base pairs of the DNA under study and L is the size in bases of the single stranded DNA segments in the renaturation solution. KN of 6.2 x 104 for DNA in aqueous 0.20 M NaCl was calculated from the rate versus monovalent cation concentration curve of Britten27 and the nucleation rate constant of 1.7 x 105 for DNA in 0.40 M NaC117. This same value for KN was also determined experimentally by comparing the optimal renaturation rates of sonicated E. coli DNA in 0.20 and 0.40 M NaCl solutions. The values of KN for each of the or=
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Nucleic Acids Research I
2.01-
I-.
ml1.0
Ie*N U37oI
I 40
,ft
I
OC
7
60
Figure 10. Renaturation rate versus renaturation temperature for DNA solutions containing 50% formamide. Each solution contained 0.015 M Tris pH 8.2, 5 x 10-4 M EDTA, 50% (v/v) formamide and sodium chloride concentrations as , 0.20 M. , 0.38 M; andE 0.78 M; follows: * , 1.58 M; Q,
2.0
0
°( 20 0~~~~~~~~~~6 30 60 so Renaturation rate versus renaturation temperature for DNA solutions containing 60 or 70% formamide. Each solution contained 0.015 M Tris pH 8.2, 5 x 10-4 M EDTA, and formamide and sodium chloride concentrations as follows: For 60% formamide solutions - 0 1.08M; 0 0.78 M; * 0.58 M; A, 0.40 M; and 0 , 0.30 M; and for 70% formamide solutions - V , 1.47 M; and v, 0.39 M.
Figure 11.
,
,
,
ganic solvent systems were calculated directly from the experimentally determined rates by the simple relation KN = 6.2 x 104 x (K2,organic system/ K2, 0.2 M aqueous). TR is the renaturation temperature used for the determination of KN.
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Nucleic Acids Research
0.5
1.0
MOLE S/L I TER
Figure 12.
Renaturation rate versus sodium chloride concentration for the formamide concentrations and renaturation temperatures indicated below. All solutions contained 0.015 M Tris pH 8.2 and 5 x 10-4 M EDTA. The formamide concentrations and renaturation temperatures were as follows: 0 , 50% formamide at 38°C; * , 60% formamide at 340C; *, 60% formamide at 250C; and O3, 70% formamide at 250C. Table 4. Optimal nucleation rate constant for DNA renaturation in aqueous solutions of formamide and urea.
(NaCl)
(Urea)
Z Formamide
0.20M
0 4M 6M
0 0 0 0 40 50
0.20M 0.20M
0.20M
8A
0.20M 0.20M
0 0
0.38M 0.78M
0 0 0 0 0 0 0 0
1.58M 0.20M 0.40M
0.78M 1.08M 0.40M 0.70M 1.47M
0 0
50
50 50 60 60 60 60 70 70 70
Tm(OC) 92.0 84.0 80.0 75.0 67.0 61.5 64.5 66.0 66.0 54.5 57.8 58.6
TR(0C) 65 56 55 48 45
41 42 45 45 35 35 35
58.6
35
51.7
25
52.0
25
52.0
25
KN
6.2x104 4.3x104 3.4x104 2.5x104 3.5x104 2.8x104
6.4x104 1.2x105 1.4x105 2.3x104 6.2x104 1.2x105
l.3x105 4.3x104 5.5x104 5.7x104
(ff%J AA---*---ao nf 1i nlliA t l l rq t4 an nc J.Wj. for 'nWA W-L for:L.Fwqxx r--:l Jr_L1C&L.%AJ.C&L..JLWLLLW&U&C"UJLUW_C;PW_L"L..LW&1.3 Xi r&%&V"L&U=&r_
fragment size stability. If aqueous-formamide-salt solutions are to provide an advantage over aqueous salt solutions for DNA renaturation studies, the slower nucleation rate constants of the formamide systems must be more than compensated for by a reduced rate of degradation of single-stranded DNA in the formamide system 3551
Nucleic Acids Research as compared to that of the aqueous system at their respective optimal renaturation temperatures. Studier has demonstrated that the infectivity of tobacco mosaic virus RNA is not altered by incubation in formamide at room temperature for 24 hours6 and Sinsheimer has observed similar results for the infectivity of 0x174 DNA (reported in reference 6). It was therefore concluded by Studier that formamide does not chemically degrade nucleic acids. McConaughy, Laird and McCarthy7 have also demonstrated that eukaryotic DNAs
(D. melanogaster and mouse) can be successfully renatured in 48% formamide solutions at 370C. However, these studies give no quantitative indication of the stability of the primary structure of DNA in formamide at renaturation temperatures over the prolonged periods of time needed in renaturation studies of highly complex genomes. In other words, it has not actually been shown that the formamide solutions provide an advantage over aqueous salt systems for renaturation studies of complex genomes in spite of the apparent advantage that the reduced renaturation temperatures would be expected to afford. It probably goes without saying that prior to use any solvent system to be used for DNA thermal stability or renaturation studies should be checked for stability at the temperatures to be used. This is especially true for
formamide. When this study was begun, two brands of formamide were tried: Matheson, Coleman and Bell 99% formamide (a brand popular with many nucleic acid electron microscopists) and Fisher's F-95. The pH of a 50 percent MC/B formamide solution initially buffered with 0.01 M Tris-HCl pH 8.2 would fall below 5.0 within a few minutes of heating at 950C. Even if the solution contained 0.10 M Tris buffer, the pH would fall to 5.3 after 30 minutes at 950C. In this solvent system the DNA was degraded to such an extent by the time it was melted that reliable renaturation kinetics could not be obtained. On the other hand, the pH of a 50 percent solution of F-95 formamide buffered with 0.03 to 0.10 M Tris-HCl would not change significantly after 8 hours of incubation at 950C. The pH of a 50 percent F-95 solution buffered with 0.01 M Tris-HCl dropped to 7.3 after 8 hours at 950C. This latter solvent system proved sufficiently stable for prolonged renaturation studies and was therefore used in the renaturation experiments reported in this study. To determine whether or not formamide solutions provide an advantage
over aqueous solutions for DNA renaturation studi-es, the relative single stranded DNA scission rates as well as the nucleation rate constants must be known for both systems. The nucleation rate constants are available from the preceeding section of this report. The relative scission rates are now needed
3552
Nucleic Acids Research to make possible a critical comparison of the two systems. The study presented in figure 13 was performed in order to provide this information.
Inspection of curve D of figure 13 indicates that the fragment size L of E. coli DNA decreases at the same rate in 60% formamide as in aqueous salt solution when incubated at 1000C. Therefore, it appears that at a given temperature the primary structure of single stranded DNA is equally stable in the two systems. Comparison of the chain scission rates for single stranded DNA at Tm + 80C for each of the two systems should provide a reasonable ap-
proximation to the relative rates of chain scission for single stranded DNA at Tm - 250C in these systems. Comparison of curves C and D indicates that 17 days of incubation in the 60% formamide system at Tm + 80C results in the same extent of DNA degradation as 6 hours at Tm + 80C in the aqueous salt system. That is, the single stranded DNA fragmentation rate is approximately 68 times as great for the aqueous salt solution as for the 60% formamide so-
lution when incubated at Tm + 80C. Curves A and B indicate the relative stabilities of the single stranded fragment size of duplex DNA at the optimal
1.0
A.
~~~~~~~~~~~~~~~~~~~~~~~142 125
.100
.2
0
5
DAYS
128
10
Figure 13. Single strand molecular weight of DNA as a function of incubation time in 60% formamide and aqueous renaturation solutions. Solutions of 75 ug/ml E. coli DNA with L = 2 x 104 nucleotides were prepared containing either 60% formamide (v/v), 1.2 M NaCl, 0.04 M Tris 8.2 and 4 x 10- M EDTA, or 0.20 M NaCl, 0.04 M Tris pH 8.2 and 4 x 10-4 M EDTA. The solutions in sealed tubes were incubated at 1000C for 5 minutes and then incubated at either 34, 65 or 1000C for the indicated times. The renaturation rates on the left ordinate are reported relative to the renaturation rate of an aliquot of the solution taken before incubation. The rates for the 60% formamide solutions were determined at 340C and those for 0.20 M NaCl were determined at 650C. The values of L1/2 for the DNA calculated from the renaturation rates are indicated on the right ordinate. The incubation temperatures were as follows: 60% formamide at 340C ( A ; curve A), 650C ( * ; curve C) and 1000C ( 0 ; curve D) and aqueous 0.20 M NaCl at 650C ( 0 ; curve B) and 1000C ( O ; curve D).
_H
3553
Nucleic Acids Research renaturation temperatures of the 60% formamide system and the aqueous salt system, respectively. It is obvious from these studies that aqueous-formamide-salt solutions do provide an advantage for long term renaturation studies, provided the pH of the system does not drift appreciably. The nucleation rate constants of
the two systems studied in figure 13 are approximately 1.3x105 and 6.2x104 for the formamide and the aqueous systems respectively. From these and the ratio of 68 for the chain scission rates it can be determined that at a given DNA concentration the DNA in the formamide system could have a complexity, N, approximately 140 times as great as that of the DNA in the aqueous system and both could be renatured to the same extent with the same amount of DNA degradation occurring. Of course, 68 times as much renaturation time would be required for the formamide system. In other words, the DNA from organisms having genomes two orders of magnitude more complex can be renatured in formamide solutions than those that can be renatured in aqueous salt solutions. In conclusion, formamide solutions can be more advantageous than aqueous salt solutions for renaturation studies of complex genomes. The advantage gained will depend on the precise systems considered, but can be greater than two orders of magnitude. Since many considerations must be taken into account to determine the most desirable conditions for any given renaturation study (e.g. convenient times, DNA quantitives available, etc.), a general recommendation as to "the most suitable" system is not reasonable and will not be attempted. However, systems with 50 to 70% formamide, 1.0 M NaCl, 0.03 to 0.10 M Tris (solution pH 8), and 0.2 to l.Ox10-3 M EDTA seem to be reasonable choices.
ACKNOWLEDGEMENTS This work was supported by Marquette COR grant 5640. REFERENCES 1. 2. 3.
4. 5.
6. 7.
8. 9. 3554
Wetmur, J.G. and Davidson, N. (1968). J. Molec. Biol. 31, 349-370. Lindahl, T. and Nyberg, B. (1972). Biochemistry 11, 3610-3618. Lindahl, T. and Andersson, A. (1972). Biochemistry 11, 3618-3623. Britten, R.J., Graham, D.E. and Neufeld, B.R. (1974) in Methods in Enzymology, Vol. XXIXE, pp. 363-406, Academic Press, New York. Wetmur, J.G. (1976) in Annual Review of Biophysics and Bioengineering, Vol. 5, pp. 337-361, Annual Reviews Inc., California. Studier, F.W. (1963). Doctoral Dissertation, California Institute of Technology, Pasadena. McConaughy, B.L., Laird, C.D., and McCarthy, B.J. (1969). Biochemistry
8, 3289-3297. Bluthmann, H., Bruck, D., Hubner, L. and Schoffski, A. (1973). Biochem. Biophys. Res. Comm. 50, 91-97. Hutton, J.R. and Wetmur, J.G. (1975). Biochem. Biophys. Res. Comm. 66, 942-948.
Nucleic Acids Research 10. 11.
12. 13. 14. 15. 16. 17. 18. 19.
Record, M.T., Jr. (1967). Biopolymers 5, 975-992. Record, M.T., Jr. (1967). Doctoral Dissertation, University of California, San Diego. Record, M.T., Jr. and Zimm, B.H. (1972). Biopolymers 11, 1435-1484. Elson, E.L. and Record, M.T., Jr. (1974). Biopolymers 13, 797-824. Kallay, M. and Record, M.T., Jr. (1974). Biopolymers 13, 825-841. Mandell, J.D. and Hershey, A.D. (1960). Anal. Biochem. 1, 66. Hutton, J.R. and Wetmur, J.G. (1973). Biochemistry 12, 558-563. Hutton, J.R. and Wetmur, J.G. (1973). J. Mol. Biol. 77, 495-500. Kingsbury, D.T. (1969). J. Bacteriology 98, 1400-1401. Gillis, M., DeLey, J. and DeCleene, M. (1970). Eur. J. Biochem. 12,
143-153. 20. 21. 22. 23. 24.
25. 26. 27.
Chang, C.-T., Hain, T.C., Hutton, J.R. and Wetmur, J.G. (1974). Biopolymers 13, 1847-1858. Hamaguchi, K. and Geiduschek, E.P. (1962). J. Amer. Chem. Soc. 84,
1329-1338. Schildkraut, C. and Lifson, S. (1965). Biopolymers 3, 195-208. Gruenwedel, D.W. and Hsu, C.-H. (1969). Biopolymers 7, 557-570. Gruenwedel, D.W., Hsu, C.-H. and Lu, D.S. (1971). Biopolymers 10, 47-68. Record, M.T., Jr. (1967). Biopolymers 5, 993-1008. Record, M.T., Jr., Woodbury, C.P. and Lohman, T.M. (1976). Biopolymers 15, 893-915. Britten, R.J. (1968). Yearbook Carnegie Inst. 67, 334.
3555
Nucleic Acids Research
Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea
James R. Hutton
Department of Biology, Marquette University, Milwaukee, WI 53233, USA Received 18 July 1977
ABSTRACT This paper reports the results of a systematic study of the effects of formamide and urea on the thermal stability and renaturation kinetics of DNA. Increasing concentrations of urea in the range 0 to 8 molar lowers the Tm by 2.250C per molar, and decreases the renaturation rate by approximately 8 percent per molar. Increasing concentrations of formamide in the range from 0 to 50 percent lowers the Tm by 0.600C per percent formamide for sodium chloride concentrations ranging from 0.035M to 0.88M. At higher salt concentrations the dependence of Tm on percent formamide was found to be slightly greater. Increasing formamide concentration decreases the renaturation rate linearly by 1.1% per percent formamide such that the optimal rate in 50% formamide is 0.45 the optimal rate in an identical solution with no formamide. The effects of urea and formamide on the renaturation rates of DNA are explained by consideration of the viscosities of the solutions at the renaturation temperatures.
INTRODUCTION
The complex genomic DNAs of higher organisms require a considerable length of time, sometimes weeks, for a large fraction of their unique sequences to renature when using reasonable DNA concentrations. Since the optimal temperatures for renaturation to occur in aqueous salt solutions are fairly high (65 to 750C, depending on the monovalent cation concentrationl), a considerabile amount of DNA degradation can result from an extended exposure to
those temperatures2'3. In an effort to minimize such thermally induced degradation while still maintaining stringent reaction criteria4, investigators have frequently resorted to studying renaturation rates in aqueous solutions of organic reagents. For excellent reviews see Wetmur5 and Britten, et al.4. These solvent systems allow reasonable renaturation rates and criteria to be achieved at relatively low temperatures. Studier6 has shown that formamide does not chemically degrade nucleic acids at room temperature, and at the lower renaturation temperatures DNA degradation is thought to be much less of
X Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
3537
Nucleic Acids Research a problem.
Two organic reagents that have been used for this purpose are
formamide and urea7'4. Several investigators have reported limited studies on the effect of formamide on the thermal stability of DNA7-12 and on the renaturation kinetics of DNA7. Several studies of the helix-coil transition of DNA have also been performed in aqueous formamide solutions (e.g. 6, 11-14) in an effort to minimize DNA degradation problems. The reported values for the lowering of Tm per percent formamide in solution range from 0.60 to 0.720C. Although the need for a systematic study of the effect of formamide on renaturation
kinetics has previously been expressed in the literature4, no such studies have been reported for this reagent or for urea. This study endeavors to systematically determine the effects of the variables temperature, monovalent cation concentration, and organic reagent concentration on the thermal stability and the second order renaturation rate of DNA. The results of this study will provide the information that is necessary to critically compare the results of renaturation studies that are performed in aqueous solutions of these organic reagents with the results of similar studies performed in aqueous salt solutions. This should allow conditions to be selected for a given experiment that will provide the desired renaturation criteria with minimal DNA degradation problems.
METHODS
(a)
DNA and Reagents E. coli W3110 was grown in nutrient broth, collected by centrifugation,
washed and resuspended in 0.03 M Tris HC1, 0.01 M EDTA, pH 8.0. The suspension was adjusted to 20 ug/ml with lysozyme (Sigma, Grade I) and incubated
for 20 min. at 240C. An equal volume of 2% sarcosyl, 0.01 M Tris HC1, 0.03 M EDTA, pH 8.0 was added to lyse the cells. The mixture was then adjusted to 200 ug/ml with pronase (Calbiochem, Grade B) and incubated at 370C with gentle shaking for 12 hours. The NaCl concentration was adjusted to 0.15 M and 2 volumes of ice cold ethanol were added to precipitate the DNA. The DNA was spun out of solution on glass rods and resuspended in 0.15 M NaCl, 0.03 M Tris, 0.01 M EDTA, pH 8.0. The solution was adjusted to 20 ug/ml with RNase A (Sigma, type IA) and stirred gently at 240C for 30 min. It was treated with pronase as before and extracted twice with equal volumes of freshly distilled phenol 15 . The aqueous phase was dialyzed exhaustively against 0.50 M NaCl, 10-3 M EDTA, 0.03 M Tris pH 8.2. After dialysis the DNA was fragmented by sonic irradiation with a Biosonik sonicator. The sonicated DNA
3538
Nucleic Acids Research was dialyzed against 0.40 M NaCl, 10-3 M EDTA, 0.03 M Tris, pH 8.2 and used
in the following studies. Cl. perfringens DNA and an alternative source of E. coli DNA were purchased from Sigma. Bacteriophage lambda DNA was prepared as previously described16. The single-stranded fragment size L of E. coli DNA was determined from the second order rate constant K2 obtained under reaction conditions of known DNA nucleation rate constant KN1,l7. L was calculated from the relation
L1/2=K2NKNj1, where the kinetic complexity N of E. coli is taken as 4.0x106 base pairs 18,19 and KN is 1.7x105 for 0.40 M NaC117. The single-strand fragment sizes of the DNA preparations used in the following studies range from 1,600 nucleotides to 20,000 nucleotides. Unless otherwise indicated, the results reported for the following studies were independent of the DNA fragment size used within this range of L. All chemicals used were reagent grade. Formamide was Fisher F-95 and Urea was obtained from Sigma. (b) Melting Temperatures The methods used to measure the DNA melting temperatures were essentially those previously describedl6. Sonicated E. coli DNA was adjusted to 0.015 M Tris, pH 8.2, 5x10-4 M EDTA and the desired concentrations of NaCl and organic reagent. The samples were placed in cuvettes and overlaid with mineral oil to prevent evaporation. The absorbance at 275 nm was measured in a recording Gilford model 2400 spectrophotometer equipped with a reference compensator and a temperature programmer to monitor the absorbance of four cells and the temperature of the cell compartment. A recirculating Haake bath was equipped with a motor to rotate the thermostat control of the contact thermometer. The rate of temperature increase was 0.50C per minute. Absorbance at 275 nm was used to monitor the denaturation and renaturation reactions in order to minimize the contribution of formamide to the absorbance of the solution. E. coli, Cl. perfringens and bacteriophage/N DNAs each exhibited a hyperchromicity of 40% at 275 nm for each of the solutions studied. (c)
Renaturation Kinetics Solutions with DNA concentrations ranging from 30 to 150 ug/ml were placed in cuvettes and overlaid with mineral oil. The absorbance at 275 nm was determined for a native DNA solution at the desired renaturation temperature using a Gilford 2400 spectrophotometer. This A275 was taken as A . The cell chamber of the Gilford was then switched to a 1000C Haake bath and the chamber temperature raised to Tm+100C or greater. The absorbance of the
3539
Nucleic Acids Research fully denatured DNA at Tm+100C was taken as AO. The chamber was then switched back to the Haake bath regulated at the temperature desired for renaturation. Absorbance at 275 nm and temperature were recorded as a function of time. After the renaturation reaction had been monitored, the chamber was switched back to the 1000C Haake bath to recheck AO. The rate data were treated according to the procedure of Wetmur and Davidsonl. Only the first
half of a reaction, during which simple second order kinetics are followed, was used to determine the rate for a given sample. DNA solutions that demonstrated Tms greater than 800C were placed in a water-jacketed fused silica spectrophotometer cell that was connected directly to a recirculating Haake bath. The melting profiles and renaturation curves were then determined as previously described16.
All renaturation rates reported in this study are expressed relative to the optimal rate for the same DNA in an aqueous 0.20 M sodium chloride solution. That is, each reported rate is obtained by dividing the rate determined for a given set of conditions by the rate determined for the same DNA
preparation at the same concentration in 0.20 M NaCl at 650C. RESULTS AND DISCUSSION
(a)
Thermal Stability of DNA in aqueous solutions of urea Solutions of sonicated E. coli DNA were adjusted to 0.20 M NaCl, 0.015 M Tris HC1, 5x10-4 M EDTA and the desired urea concentration with a final pH of 7.7. The DNAs were melted as described in the methods section. The temperature at which half the cooperative absorbance change had occurred (Tm) is plotted in figure 1 as a function of the molarity of urea in the solution. As can be seen from figure 1, the melting temperature of the DNA has a linear dependence on the concentration of urea. Increasing concentrations of urea
in the range studied lowers the Tm by 2.250C per molar added urea.
100__
oc
Figure 1. 3540
0 2.0 4.0 60 8.0 MOLES/LITER Melting temperature versus urea concentration.
Nucleic Acids Research (b)
Renaturation of DNA in aqueous solutions of urea
The renaturation rate of sonicated E. coli DNA in 0.20 M NaCl was studied as a function of temperature and of urea concentration. Figure 2 presents the rate-temperature profiles obtained for solutions containing 0, 4.0, 6.0 and 8.0 M urea. Figure 3 depicts the dependence of the renaturation rate
at the optimum renaturation temperature (i.e. the temperature that allows the fastest renaturation rate) on the concentration of urea in the solution. As indicated by the slope of the linear curve, the renaturation rate decreases by approximately 8 percent for each added mole/liter of urea. I
I
I
I
I
1.0 .8 .6 < .4
.2 _0
I
I
I
I
40
50
60
70
%
I
80
oc Figure 2. Relative renaturation rate versus renaturation temperature. All M 0.015 M Tris pH 7.7)5 x 10-4 M EDTA and 140 ug 0.20 were solutions NaCl, DNA/ml. 03: no urea; 0: 4.0 M urea; *: 6.0 M urea; A: 8.0 M urea. I
I
I
I
1.0i_ .8
.4
.2 N0
I
I
2.0
40
I
60
MOLES/LITER
I
8.0
Optimal renaturation rate versus urea molarity. The maximum renaturation rates obtained from rate-temperature profiles such as those presented in figure 2 are plotted as a function of urea concentration. All solutions contained 0.20 M NaCl, 0.015 M Tris pH 7.7 and 5 x 10-4 M EDTA.
Figure 3.
3541
Nucleic Acids Research The effect of urea on the renaturation rate of DNA can be explained by considering the viscosities of the renaturation solutions. The nucleation rate for DNA renaturation has been shown to be inversely proportional to the microscopic viscosity of the reaction solutionl20. The linear curve in figure 3 can be derived from the measured viscosities of the urea solutions at
Tm-250C (e.g. the viscosities of 0 M urea at 660C, 4.0 M urea at 520C and 6.0 M urea at 520C relative to H20 at 250C are respectively 0.499, 0.76 and 0.88) by simply graphing the ratio of the viscosity of the 0 M urea solution at 660C to the viscosity of the renaturation solution as a function of urea concentration. From the above studies it is obvious that urea has a rather severe effect on the renaturation rate of DNA relative to its effect on the Tm. Therefore, this reagent was considered to be of little probable value in minimizing thermally induced DNA degradation and was not studied further. (c) Thermal stability of DNA in aqueous solutions of formamide The thermal stability of sonicated E. coli DNA was studied as a function of formamide concentration and of sodium chloride concentration. Figure 4 illustrates the dependence of the Tm of E. coli DNA on the percent formamide contained in the solution for several different sodium chlodride concentrations. From consideration of the curves in figure 4 and similar curves (not shown) obtained for other sodium chloride concentrations, it can be concluded that for DNA solutions containing NaCl concentrations ranging from 0.035 to 0.88 M increasing the formamide concentration in the range studied lowers the
100s _
1
U
I
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_
0
80
60
0
20 40 PERCENT FORMAMIDE
60
Figure 4. Melting temperature versus percent formamide. DNA solutions contained 0.015 M Tris pH 8.2, 5 x 10-4 M EDTA, the indicated percent (volume/ volume) of formamide and sodium chloride to give 1.12 M,V ; 0.20 M, A ; 0.10 M, O ; and 0.035 M,O . DNA single strand molecular weight was 7 x 105 daltons. 3542
Nucleic Acids Research Tm by 0.60+0.010C per percent formamide added. At higher NaCl concentrations the dependence of the Tm on percent formamide was found to be slightly greater (e.g. dTm/d percent formamide equals -0.66 for 1.12 M NaCl) with the exact linear slope depending on the NaCl concentration. The reason for the greater dependence at higher salt concentrations is the shift of the Tm plateau region to lower NaCl concentrations with increasing formamide concentration as indicated in figure 5. Figure 5 presents the results obtained for the dependence of the Tm of E. coli DNA on the NaCl concentration for several different formamide concentrations and for Cl.perfringens DNA at zero percent formamide. It has
long been known that at high salt concentrations the salt dependence of the Tm of DNA will level off and even reverse signl,2l-24. The data in figure 5 indicates that this is also true for DNA in aqueous-formamide solutions. As is evident from figure 5, the NaCl concentration at which the salt dependence of Tm levels off decreases with increasing formamide. For each of the formamide concentrations studied the data for NaCl concentrations less than 2.0 M are fit quite well by two intersecting straight lines. The intersection point (i.e. the minimum NaCl concentration above which the Tm of E. coli DNA will no longer increase with additonal NaCl) can be estimated from figure 5
for each of the formamide concentrations studied. If these estimated minimum NaCl concentrations listed in Table 1 are graphed as a function of percent
oc~~~~~~~~~~~~~
60
F |l
.04
.10
MO LAR ITY
1.0
5.0
Melting temperatures of E. coli (curves B-G) and Cl. perfringens (curve A) DNA versus sodium chloride concentration for several different concentrations of formamide. DNA single strand molecular weight was 7 x 105 daltons. Formamide concentrations are - curve A, 0%; B, 20%, C, 30%; D, 40%; E, 50%; F, 60%; and G, 70%.
Figure 5.
3543
Nucleic Acids Research
Table 1. NaCl concentrations above which the thermal stability of DNA in formamide solution no longer increases with additional NaCl. The values for zero percent formamide are for Clostridium perfringens DNA (26.5% GC) and all other values are for E. coli DNA (50% GC).
20
Maximum Tm (OC) 91.0 85.0
30 40 50 60 70
78.0 72.0 66.0 58.5 52.0
% Formamide 0
NaCl (moles/liter) 1.06 .85
.75 .70 .62
.46 .40
formamide, a reasonable fit is obtained to a curve that can be defined by the linear equation NaCl molarity = -0.0096 x percent formamide + 1.06. Within the range of formamide concentrations studied, this equation can be used to obtain an estimate of the minimum NaCl concentration for a given formamide concentration above which the Tm no longer increases with additional NaCl (i.e. the minimum NaCl concentration that will yield the maximum DNA Tm for the formamide solution). If the maximum Tms for E. coli DNA listed in Table 1 are graphed as a function of percent formamide, a linear curve is obtained that is defined by the equation
Tm(maximum) = -0.66 x percent formamide + 98.50C. These equations suggest that E. coli DNA should have a maximum Tm of
98.50C in aqueous NaCl solutions at pH 8.2, and that this maximum should occur at a minimum NaCl concentration of 1.06 M. As seen from curve A of figure 5, the minimum NaCl concentration at which Cl. perfringens DNA demonstrates a maximum Tm of 910C is approximately 1.06 M.
If the value of 33
is taken as dTm/dXGC for DNA in 1 M NaC124 where XGC is the mole fraction of GC composing the DNA, the Tm of 910C for Cl. perfringens DNA with XGC=0.265 can be converted to the equivalent Tm of 98.80C for E. coli DNA with XGc= 0.50 in excellent agreement with the equations. The slopes of the low salt regions of the curves in figure 5 are contrary to previously reported results for formamide solutions. Record1O has reported a dTm/d log Na+ value of 20.20C for calf thymus DNA in 50% formamide. Even if the counter ion contributions of the buffer and formamide degradation products are taken into consideration, the maximum value of dTm/d 3544
Nucleic Acids Research log Na+ that can be obtained from the data of figure 5 for E. coli DNA in 50% formamide is 9.50C.
Several experiments were performed in an attempt to re-
solve this difference. The NaCl dependence of the Tm of E. coli DNA in 50% formamide was deter-
mined using three different sources of formamide: Matheson Coleman and Bell formamide, and Fisher Scientific Co. F95 and F82 formamides. The conductivities determined for 50% solutions of formamide in distilled H20 were 830, 1200 and 1380 micromhos for MC/B, F95 and F82, respectively. This is equivalent to the conductivity of 0.005 to 0.01 M NaCl. The same value of dTm!d log Na+ was obtained for each of the three formamides, so the formamide does
not appear to be the likely source of the difference. Since record25 has shown that the value of dTm/d log Na+ for DNA in aqueous solutions is a function of pH, this was considered a likely source for the discrepancy. The values of dTm/d log Na+ for 50% formamide were deter-
mined at several different solution pHs. Figure 6 depicts the dependence of the solution pH as measured with a glass combination electrode on the percent
formamide for several buffer systems. Figure 7 presents the graphs of the Tm versus sodium ion concentration for 50% formamide solutions of E. coli DNA at four different pHs and Cl. perfringens DNA at one pH. The results of figure 7 are summarized in Table 2 along with the values obtained for 40% DMSO
solutions.
I
I
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I'
I A
10
8
C
pH
D _
6
Ewt -
4~~~~~~~'
X -~~~
0
PERCENT
SO
Figure 6. Solution pH versus formamide concentration for several buffer systems. Buffers are as follows: curve A, 0.02 M sodium tetraborate; B, 0.015 M Tris-HCl; C, 0.02 M sodium phosphate buffer; D, 0.02 M NaH2PO4; E, 0.02 M NaH2PO4 + HC1 to pH 2.8; F, 0.02 M sodium acetate buffer; G, 0.04 M sodium acetate buffer. 3545
Nucleic Acids Research
Melting temperature versus sodium ion concentration for DNA in The buffers and solution pHs were as follows: E. coli DNA in 0.04 M sodium acetate pH 5.0 ( 0 ), 0.03 M sodium acetate pH 6.0 ( U ), 0.03 M NaH2PO pH 7.0 ( 0 ), 0.015 M Tris HCl pH 8.6 ( 0 ) and Cl. perfringens DNA in 8.015 M Tris-HCl pH 8.6
Figure 7.
50% formamide at several different solution pHs.
Table 2.
Dependence of Tm on Na+ concentration for Cl. perfringens and
E. coli DNA in 40% DMS0 and 50% formamide. Solution pHs were measured before and after Tm determinations to assure that no significant drifting occurred. Solution 50% formamide It
it
50% formamide it
40% DMS0 40% DMSO aqueous
if
DNA
pH
5.0 6.0 7.0 8.6 8.6 8.0
E. coli " " " " E. coli
Cl.
perfringens
8.0
" E. coli
8.2
Cl. perfringens
dTm/d log Na+ (OC) 17.3 13.4 12.8 9.5 14.2 10.0 10.0 15.0
The dependence of dTm/d log Na+ on solution pH for 50% formamide was found to be similar to that observed for aqueous DNA solutions25 and is therefore consistent with the theoretical considerations of Record, Woodbury and Lohman26. Since Record10 did not report the buffer system used or the solution pH for
his determination of the 20.20C value, the difference could conceivably be accounted for if the formamide solutions used in that determination were inadequately buffered such that they either had pHs that were below 5, or had pHs that drifted to values less than 5 during the determination. This possi3546
Nucleic Acids Research bility is not unreasonable, since it is reported below that the pH of an inadequately buffered formamide solution can decrease to less than 5 on heat-
ing. To eliminate the possibility that the DNA preparation I used was in some way peculiar, three sources of 50% GC DNA were used: E. coli and bacteriophage lambda DNAs that were prepared by me as described in methods and E.
coli DNA prepared by Sigma Chemical Co. The same values of dTm/d log Na+ were obtained for each of the three DNA sources. To further eliminate the
possibility of the difference being due to some nonapparent difference in our experimental systems the value of dTm/d log Na+ was determined for 40% dimethylsulfoxide, another solvent system studied by Record1O. The value of 100C was obtained for DNA in 40% DMSO in agreement with the results reported by Record. In conclusion, I cannot unequivocally account for the discrepancy between the values of dTm/d log Na+ for DNA in 50% formamide reported by Record and those reported in this study. The difference in GC content of the DNAs used may account for 1 to 2 degrees difference since Cl. perfringens DNA (26.5% GC) has a greater dTm/d log Na+ in 50% formamide than does E. coli DNA (50% GC) as indicated in Table 2, but this certainly could not account for a 10 to 11 degree difference. The remaining 9 degree difference could conceivably be accounted for if the 50% formamide solutions studied by Record had low pHs of say 5 or less. (d) Renaturation rate of DNA in aqueous solutions of formamide The kinetics of DNA renaturation in formamide was studied as a function of formamide concentration, temperature and NaCl concentration. Figure 8 presents the rate-temperature profiles obtained for DNA at several different formamide concentrations. The same general bell-shaped curve is obtained for the formamide solutions as is obtained in the absence of formamide. In each case the optimal rate occurs at Tm-25+50C with a relatively flat temperature
response in this range. Figure 9 represents the optimal renaturation rates obtained for several
formamide concentrations graphed as a function of formamide concentration. Increasing formamide concentration decreases the renaturation rate by 1.1% per percent formamide. Each point in figure 9 represents the average value obtained from a set of 4 to 9 independent determinations of the rate at a given formamide concentration. The error bars represent + one standard deviation obtained for each set of determinations. The linear curve drawn through the points in figure 9 represents the "predicted" rate versus formamide profile indicated by the data of Table 3 if the only effect that forma3547
Nucleic Acids Research
oc
Figure 8. Relative renaturation rate versus renaturation temperature. All solutions contained 0.20 M NaCl, 0.015 M Tris pH 8.2, 5 x 10-4 M EDTA and 140 ug/ml DNA. Formamide concentrations were as follows: 03, 0%; 0, 20%, U , 30%; 0 , 40%; and A , 50%. DNA single strand molecular weight was 7 x 105 d. I
I
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I
1.0
RATE .4
0
30 PERCENT
I
60
Figure 9. Optimal renaturation rate versus percent formamide. Each data point represents the average value obtained from a set of 4 to 9 independent determinations of the rate. The error bars represent + one standard deviation obtained for the set of determinations. DNA single strand molecular weight was 5 x 105 d. All solutions contained 0.20 M NaCl, 0.015 M Tris-HGl pH 8.2, 5 x 10-4 M EDTA and percent formamide (v/v) as indicated.
mide has on the optimal renaturation rate is due to the microscopic viscosity of the solution20. As is obvious from figure 9, the observed results are in excellent agreement with those predicted from the solution viscosities. 3548
Nucleic Acids Research Table 3. Viscosities of formamide solutions at Tm-25+50C for E. coli DNA. All solutions contain 0.20 M NaCl, 0.015 M Tris pH 8.2, 5x10-4 M EDTA. The viscosities were measured using an Ostwald-Fenske viscometer submerged in an equilibrated H20 bath regulated at the indicated temperature. 4 F95 0 10 20 30 40 50 60
'~/qHqO at 0.499 0.568 0.648 0.756 0.880 1.073 1.284
250C
Temperature (0C)
~~~~~~~~~Predicted Rate: formamide at 660C) solution at T
no
660C 600C 540C 480C
1.00 0.88 0.77 0.66 0.57 0.47 0.39
430C 360C
310C
The rate-temperature profiles obtained for DNA in formamide solutions containing several different NaCl concentrations are presented in figures 10 and 11. Figure 10 illustrates the profiles obtained for 50 percent formamide at four different NaCl concentrations and figure 11 illustrates the profiles obtained for solutions of 60 percent formamide at five different NaCl concentrations, along with two representative profiles for 70 percent formamide solutions. Similar bell-shaped profiles were obtained for other salt concentrations as well (data not shown). The salt dependence of DNA renaturation in 50, 60 and 70 percent formamite solutions is illustrated in figure 12. In 70 percent formamide at 250C the renaturation rate of DNA is essentially independent of NaCl at concentrations above 0.6 molar. In 50 percent formamide at 380C and in 60 percent formamide at 340C and 250C, the renaturation rate is only weakly dependent on NaCl at concentrations above 0.8 molar.
(e)
Nucleation rate constants The nucleation rate constant, KN, for DNA renaturation can be determined for each of the solvent systems studied. Some of these are listed in Table 4. KN is as defined by Hutton and Wetmur17 where KN K2 K2 is the experimentally determined second order rate constant, N is the complexity in base pairs of the DNA under study and L is the size in bases of the single stranded DNA segments in the renaturation solution. KN of 6.2 x 104 for DNA in aqueous 0.20 M NaCl was calculated from the rate versus monovalent cation concentration curve of Britten27 and the nucleation rate constant of 1.7 x 105 for DNA in 0.40 M NaC117. This same value for KN was also determined experimentally by comparing the optimal renaturation rates of sonicated E. coli DNA in 0.20 and 0.40 M NaCl solutions. The values of KN for each of the or=
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Nucleic Acids Research I
2.01-
I-.
ml1.0
Ie*N U37oI
I 40
,ft
I
OC
7
60
Figure 10. Renaturation rate versus renaturation temperature for DNA solutions containing 50% formamide. Each solution contained 0.015 M Tris pH 8.2, 5 x 10-4 M EDTA, 50% (v/v) formamide and sodium chloride concentrations as , 0.20 M. , 0.38 M; andE 0.78 M; follows: * , 1.58 M; Q,
2.0
0
°( 20 0~~~~~~~~~~6 30 60 so Renaturation rate versus renaturation temperature for DNA solutions containing 60 or 70% formamide. Each solution contained 0.015 M Tris pH 8.2, 5 x 10-4 M EDTA, and formamide and sodium chloride concentrations as follows: For 60% formamide solutions - 0 1.08M; 0 0.78 M; * 0.58 M; A, 0.40 M; and 0 , 0.30 M; and for 70% formamide solutions - V , 1.47 M; and v, 0.39 M.
Figure 11.
,
,
,
ganic solvent systems were calculated directly from the experimentally determined rates by the simple relation KN = 6.2 x 104 x (K2,organic system/ K2, 0.2 M aqueous). TR is the renaturation temperature used for the determination of KN.
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Nucleic Acids Research
0.5
1.0
MOLE S/L I TER
Figure 12.
Renaturation rate versus sodium chloride concentration for the formamide concentrations and renaturation temperatures indicated below. All solutions contained 0.015 M Tris pH 8.2 and 5 x 10-4 M EDTA. The formamide concentrations and renaturation temperatures were as follows: 0 , 50% formamide at 38°C; * , 60% formamide at 340C; *, 60% formamide at 250C; and O3, 70% formamide at 250C. Table 4. Optimal nucleation rate constant for DNA renaturation in aqueous solutions of formamide and urea.
(NaCl)
(Urea)
Z Formamide
0.20M
0 4M 6M
0 0 0 0 40 50
0.20M 0.20M
0.20M
8A
0.20M 0.20M
0 0
0.38M 0.78M
0 0 0 0 0 0 0 0
1.58M 0.20M 0.40M
0.78M 1.08M 0.40M 0.70M 1.47M
0 0
50
50 50 60 60 60 60 70 70 70
Tm(OC) 92.0 84.0 80.0 75.0 67.0 61.5 64.5 66.0 66.0 54.5 57.8 58.6
TR(0C) 65 56 55 48 45
41 42 45 45 35 35 35
58.6
35
51.7
25
52.0
25
52.0
25
KN
6.2x104 4.3x104 3.4x104 2.5x104 3.5x104 2.8x104
6.4x104 1.2x105 1.4x105 2.3x104 6.2x104 1.2x105
l.3x105 4.3x104 5.5x104 5.7x104
(ff%J AA---*---ao nf 1i nlliA t l l rq t4 an nc J.Wj. for 'nWA W-L for:L.Fwqxx r--:l Jr_L1C&L.%AJ.C&L..JLWLLLW&U&C"UJLUW_C;PW_L"L..LW&1.3 Xi r&%&V"L&U=&r_
fragment size stability. If aqueous-formamide-salt solutions are to provide an advantage over aqueous salt solutions for DNA renaturation studies, the slower nucleation rate constants of the formamide systems must be more than compensated for by a reduced rate of degradation of single-stranded DNA in the formamide system 3551
Nucleic Acids Research as compared to that of the aqueous system at their respective optimal renaturation temperatures. Studier has demonstrated that the infectivity of tobacco mosaic virus RNA is not altered by incubation in formamide at room temperature for 24 hours6 and Sinsheimer has observed similar results for the infectivity of 0x174 DNA (reported in reference 6). It was therefore concluded by Studier that formamide does not chemically degrade nucleic acids. McConaughy, Laird and McCarthy7 have also demonstrated that eukaryotic DNAs
(D. melanogaster and mouse) can be successfully renatured in 48% formamide solutions at 370C. However, these studies give no quantitative indication of the stability of the primary structure of DNA in formamide at renaturation temperatures over the prolonged periods of time needed in renaturation studies of highly complex genomes. In other words, it has not actually been shown that the formamide solutions provide an advantage over aqueous salt systems for renaturation studies of complex genomes in spite of the apparent advantage that the reduced renaturation temperatures would be expected to afford. It probably goes without saying that prior to use any solvent system to be used for DNA thermal stability or renaturation studies should be checked for stability at the temperatures to be used. This is especially true for
formamide. When this study was begun, two brands of formamide were tried: Matheson, Coleman and Bell 99% formamide (a brand popular with many nucleic acid electron microscopists) and Fisher's F-95. The pH of a 50 percent MC/B formamide solution initially buffered with 0.01 M Tris-HCl pH 8.2 would fall below 5.0 within a few minutes of heating at 950C. Even if the solution contained 0.10 M Tris buffer, the pH would fall to 5.3 after 30 minutes at 950C. In this solvent system the DNA was degraded to such an extent by the time it was melted that reliable renaturation kinetics could not be obtained. On the other hand, the pH of a 50 percent solution of F-95 formamide buffered with 0.03 to 0.10 M Tris-HCl would not change significantly after 8 hours of incubation at 950C. The pH of a 50 percent F-95 solution buffered with 0.01 M Tris-HCl dropped to 7.3 after 8 hours at 950C. This latter solvent system proved sufficiently stable for prolonged renaturation studies and was therefore used in the renaturation experiments reported in this study. To determine whether or not formamide solutions provide an advantage
over aqueous solutions for DNA renaturation studi-es, the relative single stranded DNA scission rates as well as the nucleation rate constants must be known for both systems. The nucleation rate constants are available from the preceeding section of this report. The relative scission rates are now needed
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Nucleic Acids Research to make possible a critical comparison of the two systems. The study presented in figure 13 was performed in order to provide this information.
Inspection of curve D of figure 13 indicates that the fragment size L of E. coli DNA decreases at the same rate in 60% formamide as in aqueous salt solution when incubated at 1000C. Therefore, it appears that at a given temperature the primary structure of single stranded DNA is equally stable in the two systems. Comparison of the chain scission rates for single stranded DNA at Tm + 80C for each of the two systems should provide a reasonable ap-
proximation to the relative rates of chain scission for single stranded DNA at Tm - 250C in these systems. Comparison of curves C and D indicates that 17 days of incubation in the 60% formamide system at Tm + 80C results in the same extent of DNA degradation as 6 hours at Tm + 80C in the aqueous salt system. That is, the single stranded DNA fragmentation rate is approximately 68 times as great for the aqueous salt solution as for the 60% formamide so-
lution when incubated at Tm + 80C. Curves A and B indicate the relative stabilities of the single stranded fragment size of duplex DNA at the optimal
1.0
A.
~~~~~~~~~~~~~~~~~~~~~~~142 125
.100
.2
0
5
DAYS
128
10
Figure 13. Single strand molecular weight of DNA as a function of incubation time in 60% formamide and aqueous renaturation solutions. Solutions of 75 ug/ml E. coli DNA with L = 2 x 104 nucleotides were prepared containing either 60% formamide (v/v), 1.2 M NaCl, 0.04 M Tris 8.2 and 4 x 10- M EDTA, or 0.20 M NaCl, 0.04 M Tris pH 8.2 and 4 x 10-4 M EDTA. The solutions in sealed tubes were incubated at 1000C for 5 minutes and then incubated at either 34, 65 or 1000C for the indicated times. The renaturation rates on the left ordinate are reported relative to the renaturation rate of an aliquot of the solution taken before incubation. The rates for the 60% formamide solutions were determined at 340C and those for 0.20 M NaCl were determined at 650C. The values of L1/2 for the DNA calculated from the renaturation rates are indicated on the right ordinate. The incubation temperatures were as follows: 60% formamide at 340C ( A ; curve A), 650C ( * ; curve C) and 1000C ( 0 ; curve D) and aqueous 0.20 M NaCl at 650C ( 0 ; curve B) and 1000C ( O ; curve D).
_H
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Nucleic Acids Research renaturation temperatures of the 60% formamide system and the aqueous salt system, respectively. It is obvious from these studies that aqueous-formamide-salt solutions do provide an advantage for long term renaturation studies, provided the pH of the system does not drift appreciably. The nucleation rate constants of
the two systems studied in figure 13 are approximately 1.3x105 and 6.2x104 for the formamide and the aqueous systems respectively. From these and the ratio of 68 for the chain scission rates it can be determined that at a given DNA concentration the DNA in the formamide system could have a complexity, N, approximately 140 times as great as that of the DNA in the aqueous system and both could be renatured to the same extent with the same amount of DNA degradation occurring. Of course, 68 times as much renaturation time would be required for the formamide system. In other words, the DNA from organisms having genomes two orders of magnitude more complex can be renatured in formamide solutions than those that can be renatured in aqueous salt solutions. In conclusion, formamide solutions can be more advantageous than aqueous salt solutions for renaturation studies of complex genomes. The advantage gained will depend on the precise systems considered, but can be greater than two orders of magnitude. Since many considerations must be taken into account to determine the most desirable conditions for any given renaturation study (e.g. convenient times, DNA quantitives available, etc.), a general recommendation as to "the most suitable" system is not reasonable and will not be attempted. However, systems with 50 to 70% formamide, 1.0 M NaCl, 0.03 to 0.10 M Tris (solution pH 8), and 0.2 to l.Ox10-3 M EDTA seem to be reasonable choices.
ACKNOWLEDGEMENTS This work was supported by Marquette COR grant 5640. REFERENCES 1. 2. 3.
4. 5.
6. 7.
8. 9. 3554
Wetmur, J.G. and Davidson, N. (1968). J. Molec. Biol. 31, 349-370. Lindahl, T. and Nyberg, B. (1972). Biochemistry 11, 3610-3618. Lindahl, T. and Andersson, A. (1972). Biochemistry 11, 3618-3623. Britten, R.J., Graham, D.E. and Neufeld, B.R. (1974) in Methods in Enzymology, Vol. XXIXE, pp. 363-406, Academic Press, New York. Wetmur, J.G. (1976) in Annual Review of Biophysics and Bioengineering, Vol. 5, pp. 337-361, Annual Reviews Inc., California. Studier, F.W. (1963). Doctoral Dissertation, California Institute of Technology, Pasadena. McConaughy, B.L., Laird, C.D., and McCarthy, B.J. (1969). Biochemistry
8, 3289-3297. Bluthmann, H., Bruck, D., Hubner, L. and Schoffski, A. (1973). Biochem. Biophys. Res. Comm. 50, 91-97. Hutton, J.R. and Wetmur, J.G. (1975). Biochem. Biophys. Res. Comm. 66, 942-948.
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12. 13. 14. 15. 16. 17. 18. 19.
Record, M.T., Jr. (1967). Biopolymers 5, 975-992. Record, M.T., Jr. (1967). Doctoral Dissertation, University of California, San Diego. Record, M.T., Jr. and Zimm, B.H. (1972). Biopolymers 11, 1435-1484. Elson, E.L. and Record, M.T., Jr. (1974). Biopolymers 13, 797-824. Kallay, M. and Record, M.T., Jr. (1974). Biopolymers 13, 825-841. Mandell, J.D. and Hershey, A.D. (1960). Anal. Biochem. 1, 66. Hutton, J.R. and Wetmur, J.G. (1973). Biochemistry 12, 558-563. Hutton, J.R. and Wetmur, J.G. (1973). J. Mol. Biol. 77, 495-500. Kingsbury, D.T. (1969). J. Bacteriology 98, 1400-1401. Gillis, M., DeLey, J. and DeCleene, M. (1970). Eur. J. Biochem. 12,
143-153. 20. 21. 22. 23. 24.
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Chang, C.-T., Hain, T.C., Hutton, J.R. and Wetmur, J.G. (1974). Biopolymers 13, 1847-1858. Hamaguchi, K. and Geiduschek, E.P. (1962). J. Amer. Chem. Soc. 84,
1329-1338. Schildkraut, C. and Lifson, S. (1965). Biopolymers 3, 195-208. Gruenwedel, D.W. and Hsu, C.-H. (1969). Biopolymers 7, 557-570. Gruenwedel, D.W., Hsu, C.-H. and Lu, D.S. (1971). Biopolymers 10, 47-68. Record, M.T., Jr. (1967). Biopolymers 5, 993-1008. Record, M.T., Jr., Woodbury, C.P. and Lohman, T.M. (1976). Biopolymers 15, 893-915. Britten, R.J. (1968). Yearbook Carnegie Inst. 67, 334.
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