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Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels
Journal of Biochemical and Biophysical Methods, 9 (1984) 153-161 Elsevier
153
BBM 00408
Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels * T h o m a s L.J. Boehm and D u s a n D r a h o v s k y Zentrum der Biologischen Chemic der Universiti~t Frankfurt am Main, Theodor Stern -Kai 7, D -6000 Frankfurt am Main 70. F.R. G.
~Received 16 April 1983) (Accepted 23 January 1984~
Summa~ We have studied with a number of bacterial restriction enzymes the conditions for digestion of D N A in agarose and polyacrylamide gels. The restriction endonucleases HpalI. MspI, HaeIII. HindIII. TaqI, HhaI. Alul. BamHI. EcoRI and SalI are capable of digesting D N A in agarose gels of low electroendosmosis and low sulfate concentration. All enzymes, except BamHI. are also capable of digesting D N A in polyacrylamide gels. With this method, rapid two-dimensional restriction mapping of genomes with low and high sequence complexity is possible. Key words: DNA; restriction mapping; two-dimensional electrophoresis.
Introduction Work on genome structure and function by use of recombinant DNA techniques requires the isolation and analysis of defined fragments. Electrophoresis in agarose and polyacrylamide gels is a simple, high resolution method of separating specific DNA fragments on the basis of molecular size and conformation [1,2]. The preparative isolation of DNA from gels is difficult, especially if only a limited amount of material is available. We have therefore tested the feasibility of digesting DNA with * Dedicated to Professor A. Wacker on the occasion of his 65th birthday. Abbreviations: HEEO agarose, agarose with high electroendosmosis ( - M r = 0.29) and high sulfate concentration (0.3%); MEEO agarose, agarose with medium electroendosmosis ( - M r = 0.15) and medium sulfate concentration (0.15%); LEEO agarose, agarose with low electroendosmosis ( - M r = 0.01) and low sulfate concentration (0.03%); Mr, coefficient of electroendosmosis; kb, kilobase pairs. 0165-022X/84/$03.00 © 1984 Elsevier Science Publishers B.V.
154 bacterial restriction enzymes in agarose and polyacrylamide gels. In this paper, we show that this is indeed possible. Several factors which may affect the digestion process and the optimized conditions for ten restriction enzymes are discussed. An exampIe for the application of this method is also given.
Materials and Methods
Description of the method Three different preparations of agarose were used wbSch differed in their electroendosmosis properties. Coefficients of electroendosmosis (Mr) were obtained from the manufacturer and represent the cathodal movement of a neutral molecule (dextran) relative to the migration of standard polyanion (albumin). Agarose of high endosmosis ( M r = 0.29) (HEE©) has a sulfate concentration of about 0.3%, agarose of medium electroendosmosis ( - M r = 0.15) (MEEO) has a sulfate concentration of about 0,15%, and agarose of low electroendosmosis ( M r = 0.0t) (LEEO) is a high gelling agarose -with a sulfate concentration of about 0.03%. H E E © and LEEO were obtained from Serva (Heidelberg, F,R.G.), MEEO was purchased from Bethesda Research Laboratories INeu-Isenburg, F.R.G.) as were viral DNAs (~X174. SV40 and X). Gels were prepared an a vertical slab gel apparatus, the thickness being 3 m m for agarose gels and 1.5 m m for polyacrylamide gels. respectively. Under standard conditions. 0.7% agarose gels and 4% polyacrylamide (acrylam~de/methylenebisacrylamide. 19 : 1) gels were used. Gel slices comaming 1 ~g of D N A were cut from the gels in the dimensions of 4 m m × 3 m m x 2 m m for agarose gels, and 4 m m × 1.5 m m × 2 m m for polyacrylamide gels. The slices were equilibrated in 50 vols. of appropriate reaction buffer containing 100 ~ g / m l nuclease-free bovine serum albumin for 6 - 8 h at 4°C with buffer changes every 2 h. The ge] slices were then soaked in 5 vols. of reaction buffer containing 20 units/m1 of the appropriate enzyme for about 12 h at 4°C and then incubated for 16 h at 37°C. The slices were then equilibrated in TBE-buffer (90 m M T r i s / 9 0 m M boric acid/2.5 m M EDTA. p H 8.2) for 60 rain and sealed into wells (5 m m × 3 m m × 4 ram) with hot agarose and polyacrylamide. The electrophoresis is then performed as usual and the gels are stained with ethidium bromide. The D N A is visualized under UV (254 nm) illumination. In experiments where the minimal concentration of restriction enzymes required for complete digestion was evaluated, the conditions were the same as above, except that the enzyme concentration was varied. When specific fragments of D N A were to b e analysed by a second digestion with restriction enzymes, the following procedure was followed. An aliquot of the digestion mixture is run in a parallel lane. cut away from the gel, stained with ethidium bromide, again aligned and illuminated under UV light. The fragments are then cut from the unstained lane at the position of the marker DNA. Alternatively, the mixture of D N A fragments is electrophoresed in wide (about 10 mm) sample wells. One half of the lane is cut away and prepared as above. The desired fragments are then cut off.
133
It was of interest to determine the lower limit of size of DNA fragments, which allowed an analysis by the procedure outlined above. The prolonged incubations in solution could cause loss of DNA by diffusion, and we have thus measured the amount of DNA in the gel before and after the incubation periods. Less than 20% of DNA of a length of about 800 bp is lost from agarose gels under standard conditions: for polyacrylamide the limit is about 80 bp. Thus. under the conditions employed here. fragments in the size range of approximately 30 0O0 to 80 bp can be analysed. In adaption of this method, one should be aware that the quality of agarose preparations is quite variable from batch to batch. It is advisable therefore to test a number of agarose preparations before using them in large scale experiments. We usually screen the suitability of agarose by digestion of linearized. 32P-end-labelled pBR322 DNA with HpaII and HaelII. The presence of two spots in autoradiographs indicates complete cleavage.
Other procedures DNA fragments to be end-labelled were dephosphorylated with alkaline phosphatase from calf intestine (Boehringer Mannheim) in 50 mM Tris-HC1, pH 8.2. After removal of the enzyme, the DNA was phosphorylated with [y-32P]ATP (New England Nuclear) using T4 polynucleotide kinase (Boehringer Mannheim) in 50 mM Tris-HC1, pH 8.2/10 mM MgC12/0.1 mM EDTA/5 mM DTT/0.1 mM spermidine. The DNA was purified by a spun-column procedure using Sephadex G-50 equilibrated in 10 mM Tris-HC1, pH 8.0/1 mM EDTA, and repeated ethanol precipitations. After electrophoresis of end-labelled fragments in agarose gels, the gels were vacuum dried using a Bio-Rad slab gel dryer and were exposed to Kodak X-Omat films using DuPont lightning plus intensifying screens. The films were developed manually with Agfa G-150 developer (1 : 3 diluted with water).
Results and Discussion
Digestion of DNA in agarose gels In initial attempts to digest DNA in agarose gels with the restriction enzymes HaeIII and HindIII, respectively, we found that HaeIII but not HindIII was active. We have therefore analysed whether the preparation of the agarose itself may affect the digestion process. Therefore, we have compared three different preparations of agarose, differing in their electroendosmosis properties and sulfate concentrations. With all enzymes tested, the low electroendosmosis agarose which also has a low sulfate concentration was found to give the best results. Table 1 summarizes the results obtained with three different preparations of agarose and ten restriction enzymes. HindIII, BamHI and EcoRI, respectively, were not active in HEEO agarose; however, all were active in LEEO. The effects of electroendosmosis and sulfate concentration could not be distinguished in these experiments, although we suspect the latter to be of critical importance for enzymatic activity. Table 2 shows the minimal concentrations of enzymes sufficient for complete digestion of DNA in
I56 TABLE 1 CAPABILITIES OF RESTRICTION ENZYMES OF DIGESTING ACRYLAMIDE GELS UNDER STANDARD CONDITIONS
D N A IN A G A R O S E A N D P O L Y -
S t a n d a r d c o n d i t i o n s as d e f i n e d in M a t e r i a l s a n d M e t h o d s w e r e used. + . C o m p l e t e d i g e s t i o n o f D N A : no digestion of DNA. Endo R
Gel Agarose HEEO
HpaII MspI HaeIII HindIII Taqt HhaI AluI BamHI EcoR[ SalI
-,
. . . . ~+ +
Agarose MEEO
. . .
. . .
. . .
.
.
. + + +
Agarose LEEO
Polyacrylamide
±
-
+ + + +
+ + +
agarose gels under standard conditions. The data dearly show that the method presented here does not require very large amounts of restriction enzymes. In a typical assay under standard conditions, 1 b~g of D N A in an agarose gel slice (volume approximately 25 #1) is digested in a volume of 125 #l of reaction buffer containing about 2-10 units of enzymes. A number of factors besides the agarose preparation affect the digestion of D N A TABLE 2 ENZYME CONCENTRATIONS POLYACRYLAMIDE GELS
REQUIRED
FOR COMPLETE
DIGESTION
IN AGAROSE
AND
S t a n d a r d c o n d i t i o n s as d e f i n e d in M a t e r i a l s a n d M e t h o d s w e r e u s e d except t h a t the e n z y m e c o n c e n t r a t i o n w a s varied. Endo R
Enzyme concentration (units/ml) Agarose LEEO
HpaII MspI HaeIII HindlII TaqI HhaI AIM BamHI EcoRI SalI
20 20 20 50 20 20 25 100 50 25
Polyacrylamide 25 25 25 100 25 25 25 100 a 100 50
A t this e n z y m e c o n c e n t r a t i o n , the d i g e s t i o n w a s a p p r o x i m a t e l y 50% c o m p l e t e , as j u d g e d b y d e n s i t o m e t ric analysis.
157
in agarose gels. It is of interest that a nonlinear relationship exists between the enzyme concentration required for complete digestion and the amount of D N A in the gel slice. Up to 10/xg of DNA per slice can be digested with the two-fold enzyme concentrations indicated in Table 2. The standard conditions described in Material and Methods have been optimized with respect to handling time. since the gel concentration as well as the thickness of gel obviously determine the length of time periods required for buffer equilibration and the enzyme soak. Complete digestion in 0.7% LEEO agarose with the enzyme concentrations listed in Table 2 can be achieved with the following time periods: (i) buffer equilibration: three times for 60 min each. (ii) enzyme soak: 12 h, (iii) digestion: 6 h. It was of interest to study a possible alteration in sequence specificity df the restriction enzymes under the conditions of digesting DNA in agarose. This was examined by comparing the restriction patterns of DNAs digested in agarose and solution, respectively. No altered sequence specificity, especially of EcoRI and BamHI. was observed (data not shown).
1
23
Fig. 1. Specificity and completeness of digestion with HaeIII of X DNA in agarose and polyacrylamlde gels: X D N A digested with HaelII in solution and run on a 1% analytical agarose gel (lane 1), X DNA digested with HaelII in agarose (lane 2) and polyacrylamide (lane 3) gels under the conditions given in Table 2.
158
Digestion of DNA in polyacrylamide gels The concentration of the polyacrylamide as well as the degree of cross-linking was adjusted so that similar experimental protocols could be followed for both agarose and polyacrylamide gels. From Table 2, it is evident that polyacrylamide gels required a slightly larger enzyme concentration for complete digestion of DNA than in agarose gels. The only enzyme which failed t o cleave satisfactorily under these conditions was BamHI. At an enzyme concentration of 100 units/rot, only incomplete digestion was ObserVed. We have attempted to optimize t h e conditions for BamHI cleavage further b y lowering the percentage of polyacrylamide and the degree of cross-linking, lengthening the time periods for the equilibration with reaction buffer, etc,; however, with little success. Fig, 1 shows the cleavage pattern obtained under the conditions detailed in Table 2 for the digestion with HaeIH of )t DNA in solution (lane 1), in agarose (lane 2) and polyacrylamide (lane 3). It is evident that a specific and complete digestion is obtained.
b
a
21,7
i~
1
,98 ] ,90
t ] ~
1,59
!
~" ~,37
t j,-
3 - •
,,
~
2
3
4
5,15 5,0 4,27 •e- 3,48
O~, 2 ~
1
~
<"
O 0
0,94
0,56
Fig. 2, Two-dimensional restriction mapping of DNA fragments with HaeIII. X D N A was digested with EcoRI and HindlII. end-labelled with T4 polynucleotide kinase and run in a 0.7% agarose gel (a). The indicated region was cut out and processed for digestion with HaelII as described in Material and Methods. This region was chosen since there are two fragments containing HaeIII sites and two without. ~o show the appearance of a complete digestion pattern. The two-dimensional display (bY indicates that the fragments numbered 1 and 2 do contain HaeIII sites.
159
Two-dimensional restriction mapping The digestion of DNA in agarose and polyacrylamide makes possible two-dimensional restriction mapping of DNA without the need of isolating DNA fragments before the second digestion. An example for such a two-dimensional analysis is given in Fig. 2. D N A was double-digested with HindIII and EcoRI, and end-labelled with T 4 kinase. The D N A fragments were then separated in a 0.8% agarose gel (Fig. 2a), a region of interest was cut out. and processed for the digestion with HaelII in the second dimension. After electrophoresis in the second-dimension gel, the gel was autoradiographed. Fig. 2b shows that no more than two spots per original fragment appear on the autoradiograph. This strongly suggests that the digestion is complete under the described conditions. The results also indicate that the fragments numbered 3 and 4 do not contain HaeIII sites. Although the resolved bands in the two-dimensional procedure are not as sharp as in the conventional analysis, it is possible to assign fairly precise sizes to each fragment in the second electrophoresis by comparing to markers run in parallel We also wish to illustrate the application of this technique to the study of complex genomes. Our laboratory has a long-standing interest in the process of enzymatic DNA methylation. When analysing the DNA methylation pattern in fetal and adult liver tissues of C 5 7 B L / 6 × DBA/2F~ mice with the 5-methylcytosine-sensitive restriction enzymes HpaII and MspI, we noted in both distinct bands in MspI digests as shown in Fig. 3a. We were interested whether these bands are related to a
b
lq
l
2
-, .t,
..e.
•- ' ' 4-'-
- 1,8 1.2 1.1 - 0,8 --
C
E? 9 ~.,~
? j/~
?
~
5.o
~
I~
3.3
Fig. 3. Two-dimensional analysis of repetitive elements generated in MspI digests of C57BL/6 x D B A / 2 F 1 mouse DNA. Digestion of mouse liver DNA with MspI generates three bands, 5.0 kb, 3.3 kb, and 0.6 kb in length. When the region containing the two larger bands is analysed in the second dimension digestion with HaeIII (b), a number of spots is detectable. A schematic description of their arrangement is given in c, emphasizing the possible relationship between these two repetitive elements, The largest fragment (1.8 kb) is missing in the 3.3 kb fragments. The scale is 1 kb. O, HaeIIl site; D, Mspl site.
160 each other or represent different repetitive elements sharing only MspI sites at their ends. To this end. we have used the above described two-dimensional restriction mapping procedure to study this question. The region containing the 5.0 kb and the 3°3 kb bands generated in MspI digests was cut out, and digested with HaelII. In the second-dimension gel (Fig. 3b). four spots were seen corresponding to the 5.0 kb band. and three for the 3.3 kb band. Incomplete digestions suggest the arrangemenl of sites indicated in Fig. 3c. From the two-dimensional analysis it can be suggested that the two MspI-generated bands are related as indicated in the diagram of Fig. 3c. We are now studying by molecular cloning whether this is indeed the case. The 0.6 kb band in Msp! digests does not contain H a e I I I sites/not shown). Recently, a sequential digestion technique for separation and two-dimensional display of restriction segments has been developed [3 5]. In this method, the D N A is cleaved with one restriction enzyme and then fractionated according to size on an electroelution device. The D N A in each fraction is then subsequently digested with another restriction enzyme and analysed on a slab gel. Two-dimensional agarose gel electrophoresis employing low-melting agarose has also been developed [6]. After excision of discrete bands of the gel, the agarose is melted, and buffers and restriction enzymes for the second dimension can be added. The main disadvantage of these techniques is thal they require the simultaneous manipulation of many samples. The method described here circumvents the isolation of individual fragments from the first-dimension gel prior to digestion with the second enzyme and allows the simultaneous digestion of D N A fragments trapped in either agarose or polyacrylamide gels under conditions which are economical of the second dimension enzyme. The method is therefore applicable to the restriction mapping of both large (by using agarose in the first dimension) and small (by using polyacrylamide in the first dimension) D N A fragments and it further offers the possibility to analyse complex genomes in two-dimensional displays, since it is possible to manipulate complete lanes from the gels rather than discrete regions as required by the other methods so far developed. In addition, we have shown here that most of the restriction enzymes may be capable of working with this svs~em, which should allow a wide application for restriction enzyme mapping of almost any DNA, especially in concert with the methods in current use. After completion of this manuscript, we became aware that another group has experimented with a two-dimensional mapping procedure [7]. However. in their experiments, the m a n y variables of this method are not dealt with. and no proof for complete digestion was given.
Simplified description of the method and its applications This work describes the conditions for complete digestion of DNA trapped in agarose and polyacrylamide gels with various restriction endonucleases. In two-dimensional mapping procedures of genomes with both low and high sequence complexities,this method circumvents the need for isolation of DNA fragments from the first dimension gel. The method is both rapid and economicalwith respect to the second-dimensionenzyme.
161
Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft (grant Dr 104/6-7).
References I 2 3 4 5 6 7
Johnson, P.H. and Grossman, L.I. (1977) Biochemistry 16, 4217-4225 Johnson, P.H., Miller, M.J. and Grossman, L.I. (1980) Anal. Biochem. 102, 159-162 Potter, S.S. and Newbold, J.E. (1976) Anal. Biochem. 71, 452-458 Potter, S.S. and Thomas, C.A. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 1023-1031 Smith, S.S. and Thomas, C.A. (1981) Gene 13, 395-408 Parker, R.C. and Seed, B. (1980) Methods Enzymol. 65, 358-363 Yee, T. and Inouye, M. (1982) J. Mol. Biol. 154, 181-196
153
BBM 00408
Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels * T h o m a s L.J. Boehm and D u s a n D r a h o v s k y Zentrum der Biologischen Chemic der Universiti~t Frankfurt am Main, Theodor Stern -Kai 7, D -6000 Frankfurt am Main 70. F.R. G.
~Received 16 April 1983) (Accepted 23 January 1984~
Summa~ We have studied with a number of bacterial restriction enzymes the conditions for digestion of D N A in agarose and polyacrylamide gels. The restriction endonucleases HpalI. MspI, HaeIII. HindIII. TaqI, HhaI. Alul. BamHI. EcoRI and SalI are capable of digesting D N A in agarose gels of low electroendosmosis and low sulfate concentration. All enzymes, except BamHI. are also capable of digesting D N A in polyacrylamide gels. With this method, rapid two-dimensional restriction mapping of genomes with low and high sequence complexity is possible. Key words: DNA; restriction mapping; two-dimensional electrophoresis.
Introduction Work on genome structure and function by use of recombinant DNA techniques requires the isolation and analysis of defined fragments. Electrophoresis in agarose and polyacrylamide gels is a simple, high resolution method of separating specific DNA fragments on the basis of molecular size and conformation [1,2]. The preparative isolation of DNA from gels is difficult, especially if only a limited amount of material is available. We have therefore tested the feasibility of digesting DNA with * Dedicated to Professor A. Wacker on the occasion of his 65th birthday. Abbreviations: HEEO agarose, agarose with high electroendosmosis ( - M r = 0.29) and high sulfate concentration (0.3%); MEEO agarose, agarose with medium electroendosmosis ( - M r = 0.15) and medium sulfate concentration (0.15%); LEEO agarose, agarose with low electroendosmosis ( - M r = 0.01) and low sulfate concentration (0.03%); Mr, coefficient of electroendosmosis; kb, kilobase pairs. 0165-022X/84/$03.00 © 1984 Elsevier Science Publishers B.V.
154 bacterial restriction enzymes in agarose and polyacrylamide gels. In this paper, we show that this is indeed possible. Several factors which may affect the digestion process and the optimized conditions for ten restriction enzymes are discussed. An exampIe for the application of this method is also given.
Materials and Methods
Description of the method Three different preparations of agarose were used wbSch differed in their electroendosmosis properties. Coefficients of electroendosmosis (Mr) were obtained from the manufacturer and represent the cathodal movement of a neutral molecule (dextran) relative to the migration of standard polyanion (albumin). Agarose of high endosmosis ( M r = 0.29) (HEE©) has a sulfate concentration of about 0.3%, agarose of medium electroendosmosis ( - M r = 0.15) (MEEO) has a sulfate concentration of about 0,15%, and agarose of low electroendosmosis ( M r = 0.0t) (LEEO) is a high gelling agarose -with a sulfate concentration of about 0.03%. H E E © and LEEO were obtained from Serva (Heidelberg, F,R.G.), MEEO was purchased from Bethesda Research Laboratories INeu-Isenburg, F.R.G.) as were viral DNAs (~X174. SV40 and X). Gels were prepared an a vertical slab gel apparatus, the thickness being 3 m m for agarose gels and 1.5 m m for polyacrylamide gels. respectively. Under standard conditions. 0.7% agarose gels and 4% polyacrylamide (acrylam~de/methylenebisacrylamide. 19 : 1) gels were used. Gel slices comaming 1 ~g of D N A were cut from the gels in the dimensions of 4 m m × 3 m m x 2 m m for agarose gels, and 4 m m × 1.5 m m × 2 m m for polyacrylamide gels. The slices were equilibrated in 50 vols. of appropriate reaction buffer containing 100 ~ g / m l nuclease-free bovine serum albumin for 6 - 8 h at 4°C with buffer changes every 2 h. The ge] slices were then soaked in 5 vols. of reaction buffer containing 20 units/m1 of the appropriate enzyme for about 12 h at 4°C and then incubated for 16 h at 37°C. The slices were then equilibrated in TBE-buffer (90 m M T r i s / 9 0 m M boric acid/2.5 m M EDTA. p H 8.2) for 60 rain and sealed into wells (5 m m × 3 m m × 4 ram) with hot agarose and polyacrylamide. The electrophoresis is then performed as usual and the gels are stained with ethidium bromide. The D N A is visualized under UV (254 nm) illumination. In experiments where the minimal concentration of restriction enzymes required for complete digestion was evaluated, the conditions were the same as above, except that the enzyme concentration was varied. When specific fragments of D N A were to b e analysed by a second digestion with restriction enzymes, the following procedure was followed. An aliquot of the digestion mixture is run in a parallel lane. cut away from the gel, stained with ethidium bromide, again aligned and illuminated under UV light. The fragments are then cut from the unstained lane at the position of the marker DNA. Alternatively, the mixture of D N A fragments is electrophoresed in wide (about 10 mm) sample wells. One half of the lane is cut away and prepared as above. The desired fragments are then cut off.
133
It was of interest to determine the lower limit of size of DNA fragments, which allowed an analysis by the procedure outlined above. The prolonged incubations in solution could cause loss of DNA by diffusion, and we have thus measured the amount of DNA in the gel before and after the incubation periods. Less than 20% of DNA of a length of about 800 bp is lost from agarose gels under standard conditions: for polyacrylamide the limit is about 80 bp. Thus. under the conditions employed here. fragments in the size range of approximately 30 0O0 to 80 bp can be analysed. In adaption of this method, one should be aware that the quality of agarose preparations is quite variable from batch to batch. It is advisable therefore to test a number of agarose preparations before using them in large scale experiments. We usually screen the suitability of agarose by digestion of linearized. 32P-end-labelled pBR322 DNA with HpaII and HaelII. The presence of two spots in autoradiographs indicates complete cleavage.
Other procedures DNA fragments to be end-labelled were dephosphorylated with alkaline phosphatase from calf intestine (Boehringer Mannheim) in 50 mM Tris-HC1, pH 8.2. After removal of the enzyme, the DNA was phosphorylated with [y-32P]ATP (New England Nuclear) using T4 polynucleotide kinase (Boehringer Mannheim) in 50 mM Tris-HC1, pH 8.2/10 mM MgC12/0.1 mM EDTA/5 mM DTT/0.1 mM spermidine. The DNA was purified by a spun-column procedure using Sephadex G-50 equilibrated in 10 mM Tris-HC1, pH 8.0/1 mM EDTA, and repeated ethanol precipitations. After electrophoresis of end-labelled fragments in agarose gels, the gels were vacuum dried using a Bio-Rad slab gel dryer and were exposed to Kodak X-Omat films using DuPont lightning plus intensifying screens. The films were developed manually with Agfa G-150 developer (1 : 3 diluted with water).
Results and Discussion
Digestion of DNA in agarose gels In initial attempts to digest DNA in agarose gels with the restriction enzymes HaeIII and HindIII, respectively, we found that HaeIII but not HindIII was active. We have therefore analysed whether the preparation of the agarose itself may affect the digestion process. Therefore, we have compared three different preparations of agarose, differing in their electroendosmosis properties and sulfate concentrations. With all enzymes tested, the low electroendosmosis agarose which also has a low sulfate concentration was found to give the best results. Table 1 summarizes the results obtained with three different preparations of agarose and ten restriction enzymes. HindIII, BamHI and EcoRI, respectively, were not active in HEEO agarose; however, all were active in LEEO. The effects of electroendosmosis and sulfate concentration could not be distinguished in these experiments, although we suspect the latter to be of critical importance for enzymatic activity. Table 2 shows the minimal concentrations of enzymes sufficient for complete digestion of DNA in
I56 TABLE 1 CAPABILITIES OF RESTRICTION ENZYMES OF DIGESTING ACRYLAMIDE GELS UNDER STANDARD CONDITIONS
D N A IN A G A R O S E A N D P O L Y -
S t a n d a r d c o n d i t i o n s as d e f i n e d in M a t e r i a l s a n d M e t h o d s w e r e used. + . C o m p l e t e d i g e s t i o n o f D N A : no digestion of DNA. Endo R
Gel Agarose HEEO
HpaII MspI HaeIII HindIII Taqt HhaI AluI BamHI EcoR[ SalI
-,
. . . . ~+ +
Agarose MEEO
. . .
. . .
. . .
.
.
. + + +
Agarose LEEO
Polyacrylamide
±
-
+ + + +
+ + +
agarose gels under standard conditions. The data dearly show that the method presented here does not require very large amounts of restriction enzymes. In a typical assay under standard conditions, 1 b~g of D N A in an agarose gel slice (volume approximately 25 #1) is digested in a volume of 125 #l of reaction buffer containing about 2-10 units of enzymes. A number of factors besides the agarose preparation affect the digestion of D N A TABLE 2 ENZYME CONCENTRATIONS POLYACRYLAMIDE GELS
REQUIRED
FOR COMPLETE
DIGESTION
IN AGAROSE
AND
S t a n d a r d c o n d i t i o n s as d e f i n e d in M a t e r i a l s a n d M e t h o d s w e r e u s e d except t h a t the e n z y m e c o n c e n t r a t i o n w a s varied. Endo R
Enzyme concentration (units/ml) Agarose LEEO
HpaII MspI HaeIII HindlII TaqI HhaI AIM BamHI EcoRI SalI
20 20 20 50 20 20 25 100 50 25
Polyacrylamide 25 25 25 100 25 25 25 100 a 100 50
A t this e n z y m e c o n c e n t r a t i o n , the d i g e s t i o n w a s a p p r o x i m a t e l y 50% c o m p l e t e , as j u d g e d b y d e n s i t o m e t ric analysis.
157
in agarose gels. It is of interest that a nonlinear relationship exists between the enzyme concentration required for complete digestion and the amount of D N A in the gel slice. Up to 10/xg of DNA per slice can be digested with the two-fold enzyme concentrations indicated in Table 2. The standard conditions described in Material and Methods have been optimized with respect to handling time. since the gel concentration as well as the thickness of gel obviously determine the length of time periods required for buffer equilibration and the enzyme soak. Complete digestion in 0.7% LEEO agarose with the enzyme concentrations listed in Table 2 can be achieved with the following time periods: (i) buffer equilibration: three times for 60 min each. (ii) enzyme soak: 12 h, (iii) digestion: 6 h. It was of interest to study a possible alteration in sequence specificity df the restriction enzymes under the conditions of digesting DNA in agarose. This was examined by comparing the restriction patterns of DNAs digested in agarose and solution, respectively. No altered sequence specificity, especially of EcoRI and BamHI. was observed (data not shown).
1
23
Fig. 1. Specificity and completeness of digestion with HaeIII of X DNA in agarose and polyacrylamlde gels: X D N A digested with HaelII in solution and run on a 1% analytical agarose gel (lane 1), X DNA digested with HaelII in agarose (lane 2) and polyacrylamide (lane 3) gels under the conditions given in Table 2.
158
Digestion of DNA in polyacrylamide gels The concentration of the polyacrylamide as well as the degree of cross-linking was adjusted so that similar experimental protocols could be followed for both agarose and polyacrylamide gels. From Table 2, it is evident that polyacrylamide gels required a slightly larger enzyme concentration for complete digestion of DNA than in agarose gels. The only enzyme which failed t o cleave satisfactorily under these conditions was BamHI. At an enzyme concentration of 100 units/rot, only incomplete digestion was ObserVed. We have attempted to optimize t h e conditions for BamHI cleavage further b y lowering the percentage of polyacrylamide and the degree of cross-linking, lengthening the time periods for the equilibration with reaction buffer, etc,; however, with little success. Fig, 1 shows the cleavage pattern obtained under the conditions detailed in Table 2 for the digestion with HaeIH of )t DNA in solution (lane 1), in agarose (lane 2) and polyacrylamide (lane 3). It is evident that a specific and complete digestion is obtained.
b
a
21,7
i~
1
,98 ] ,90
t ] ~
1,59
!
~" ~,37
t j,-
3 - •
,,
~
2
3
4
5,15 5,0 4,27 •e- 3,48
O~, 2 ~
1
~
<"
O 0
0,94
0,56
Fig. 2, Two-dimensional restriction mapping of DNA fragments with HaeIII. X D N A was digested with EcoRI and HindlII. end-labelled with T4 polynucleotide kinase and run in a 0.7% agarose gel (a). The indicated region was cut out and processed for digestion with HaelII as described in Material and Methods. This region was chosen since there are two fragments containing HaeIII sites and two without. ~o show the appearance of a complete digestion pattern. The two-dimensional display (bY indicates that the fragments numbered 1 and 2 do contain HaeIII sites.
159
Two-dimensional restriction mapping The digestion of DNA in agarose and polyacrylamide makes possible two-dimensional restriction mapping of DNA without the need of isolating DNA fragments before the second digestion. An example for such a two-dimensional analysis is given in Fig. 2. D N A was double-digested with HindIII and EcoRI, and end-labelled with T 4 kinase. The D N A fragments were then separated in a 0.8% agarose gel (Fig. 2a), a region of interest was cut out. and processed for the digestion with HaelII in the second dimension. After electrophoresis in the second-dimension gel, the gel was autoradiographed. Fig. 2b shows that no more than two spots per original fragment appear on the autoradiograph. This strongly suggests that the digestion is complete under the described conditions. The results also indicate that the fragments numbered 3 and 4 do not contain HaeIII sites. Although the resolved bands in the two-dimensional procedure are not as sharp as in the conventional analysis, it is possible to assign fairly precise sizes to each fragment in the second electrophoresis by comparing to markers run in parallel We also wish to illustrate the application of this technique to the study of complex genomes. Our laboratory has a long-standing interest in the process of enzymatic DNA methylation. When analysing the DNA methylation pattern in fetal and adult liver tissues of C 5 7 B L / 6 × DBA/2F~ mice with the 5-methylcytosine-sensitive restriction enzymes HpaII and MspI, we noted in both distinct bands in MspI digests as shown in Fig. 3a. We were interested whether these bands are related to a
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Fig. 3. Two-dimensional analysis of repetitive elements generated in MspI digests of C57BL/6 x D B A / 2 F 1 mouse DNA. Digestion of mouse liver DNA with MspI generates three bands, 5.0 kb, 3.3 kb, and 0.6 kb in length. When the region containing the two larger bands is analysed in the second dimension digestion with HaeIII (b), a number of spots is detectable. A schematic description of their arrangement is given in c, emphasizing the possible relationship between these two repetitive elements, The largest fragment (1.8 kb) is missing in the 3.3 kb fragments. The scale is 1 kb. O, HaeIIl site; D, Mspl site.
160 each other or represent different repetitive elements sharing only MspI sites at their ends. To this end. we have used the above described two-dimensional restriction mapping procedure to study this question. The region containing the 5.0 kb and the 3°3 kb bands generated in MspI digests was cut out, and digested with HaelII. In the second-dimension gel (Fig. 3b). four spots were seen corresponding to the 5.0 kb band. and three for the 3.3 kb band. Incomplete digestions suggest the arrangemenl of sites indicated in Fig. 3c. From the two-dimensional analysis it can be suggested that the two MspI-generated bands are related as indicated in the diagram of Fig. 3c. We are now studying by molecular cloning whether this is indeed the case. The 0.6 kb band in Msp! digests does not contain H a e I I I sites/not shown). Recently, a sequential digestion technique for separation and two-dimensional display of restriction segments has been developed [3 5]. In this method, the D N A is cleaved with one restriction enzyme and then fractionated according to size on an electroelution device. The D N A in each fraction is then subsequently digested with another restriction enzyme and analysed on a slab gel. Two-dimensional agarose gel electrophoresis employing low-melting agarose has also been developed [6]. After excision of discrete bands of the gel, the agarose is melted, and buffers and restriction enzymes for the second dimension can be added. The main disadvantage of these techniques is thal they require the simultaneous manipulation of many samples. The method described here circumvents the isolation of individual fragments from the first-dimension gel prior to digestion with the second enzyme and allows the simultaneous digestion of D N A fragments trapped in either agarose or polyacrylamide gels under conditions which are economical of the second dimension enzyme. The method is therefore applicable to the restriction mapping of both large (by using agarose in the first dimension) and small (by using polyacrylamide in the first dimension) D N A fragments and it further offers the possibility to analyse complex genomes in two-dimensional displays, since it is possible to manipulate complete lanes from the gels rather than discrete regions as required by the other methods so far developed. In addition, we have shown here that most of the restriction enzymes may be capable of working with this svs~em, which should allow a wide application for restriction enzyme mapping of almost any DNA, especially in concert with the methods in current use. After completion of this manuscript, we became aware that another group has experimented with a two-dimensional mapping procedure [7]. However. in their experiments, the m a n y variables of this method are not dealt with. and no proof for complete digestion was given.
Simplified description of the method and its applications This work describes the conditions for complete digestion of DNA trapped in agarose and polyacrylamide gels with various restriction endonucleases. In two-dimensional mapping procedures of genomes with both low and high sequence complexities,this method circumvents the need for isolation of DNA fragments from the first dimension gel. The method is both rapid and economicalwith respect to the second-dimensionenzyme.
161
Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft (grant Dr 104/6-7).
References I 2 3 4 5 6 7
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