.:) 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 11 3153
Temperature sweep gel electrophoresis: a simple method to detect point mutations Kenji Yoshino, Koichi Nishigaki and Yuzuru Husimi* Department of Environmental Chemistry, Saitama University, Urawa 338, Japan Submitted April 5, 1991 The detection of point mutations in the specified region of DNA is required in the various fields of biotechnology including newly emerging evolutionary molecular engineering (1). It has generally been realized through the use of the denaturant gradient get electrophoresis (DGGE) (2, 3, 6) or the temperature gradient gel electrophoresis (TGGE) (4, 5). The principle of these techniques is based on the following properties of DNA. Namely, the electrophoretic mobility of DNA fragment slows down at the melting point of its local cooperatively melting region and the melting point is shifted by the point mutation. Introduction of GC-clamp into a sample DNA fragment through PCR method has made these techniques widely used (6). Making a precise gradient gel plate, however, requires a skilled hand or a sophisticated instrument. And this situation may limit the number of mutants to be detected. Here we propose a simpler version of TGGE, a temperature sweep gel electrophoresis (TSGE). In TGGE the temperature gradient is established in the spaceaxis before electrophoresis, but in TSGE the temperature of gel plate is raised gradually and uniformly during electrophoresis. The gradient of TGGE is linear, but the sweep speed of TSGE is easily varied during electrophoresis to give any temperature history to DNA samples. Theoretically the linear TSGE gives almost same results as the parallel TGGE, in which DNA moves parallel to the gradient. In TSGE the slowing-down of sweep speed in the middle of the run will give better resolution for specified mutants group. A demerit of TSGE is absence of any mode corresponding to the vertical TGGE. Our instrument of TSGE consists of a normal thermojacketed vertical slab-gel apparatus (hand-made), a circulating thermobath (Tokyo Rika, UA-10, precision i 0.1 0C) and a magnetic stirrer. The gel plate is immersed in the bottom buffer, which is intensively stirred in order to maintain evenness of the temperature. Unevenness gives artifacts in the band pattern. The gene VmI of coliphage Ff family was cloned by PCR using a 20-mer primer and a 50-mer primer having 5' GC-clamp region of 29 bp. All the cloned DNA fragments are 289 bp. TSGE gel used was made with 8% acrylamide/8 M urea. The acrylamide:bisacrylamide ratio was 159:1. TEMED (0.04%) and ammonium persulfate (0.04%) were added. 2.0,l of PCR product was mixed with dye solution, and then loaded into a well at the top of the gel. Electrophoresis was performed in TAE buffer (40 mM Tris-acetate, pH 8.0, 20 mM sodium acetate, 1 mM EDTA) at 300 V. The temperature of the thermojacket of electrophoretic plate was 45.0°C before the run. The temperature
*
To whom correspondence should be addressed
was raised continuously at the rate of 1.0°C/5 min from 45.0°C through 63.0°C during the run. The gel was stained with silver and photographed (Figure 1). The number of base substitution between M13 and fl in the gene VIII is one (# 1403 site is A for M13 and G for fl). TSGE detected the point mutation. Vertical and parallel TGGE for these DNA fragments were also performed. The results supported the data from TSGE. When TSGE gel was made with 4% or 6% acrylamide/8 M urea and the acrylamide:bisacrylamide ratio was 19:1, the band resolution was poor for these DNA samples. It suggests the pore size and/or the pore flexibility of the gel is important to discrimination of the DNA conformations.
REFERENCES 1. Husimi,Y. (1989) Adv. Biophys. 25, 1-43. 2. Fischer,S.G. and Lerman,L.S. (1979) Cell 16, 191-200. 3. Nishigaki,K., Husimi,Y. and Tsubota,M. (1986) J. Biochem. (Tokyo) 99, 663-671. 4. Po,T., Steger,G., Rosenbaum,V., Kaper,J. and Riesner,D. (1987) Nucl. Acids Res. 15, 5069-5083. 5. Steger,G., Po,T., Kaper,J. and Riesner,D. (1987) ibid, 5085-5103. 6. Sheffield,V.C., Cox,D.R., Lerman,L.S. and Myers,R.M. (1989) Proc. Natl. Acad. Sci. USA 86, 232-236.
1 2:
-*
M 309 313
1 75 158
Figure 1. Detection of point mutation by TSGE (45.0°C - 63.0°C). Gene Vm of coliphage Ff family was cloned through PCR/GC-clamp method. Lanes 1, 2 and 3 are from fl, fd-U1l1 (mutant of fd), and M13, respectively. Lane M is from HaeIII digests of M13 ssDNA. The arrow locates an asymmetric PCR product (289-mer ssDNA).
Nucleic Acids Research, Vol. 19, No. 11 3153
Temperature sweep gel electrophoresis: a simple method to detect point mutations Kenji Yoshino, Koichi Nishigaki and Yuzuru Husimi* Department of Environmental Chemistry, Saitama University, Urawa 338, Japan Submitted April 5, 1991 The detection of point mutations in the specified region of DNA is required in the various fields of biotechnology including newly emerging evolutionary molecular engineering (1). It has generally been realized through the use of the denaturant gradient get electrophoresis (DGGE) (2, 3, 6) or the temperature gradient gel electrophoresis (TGGE) (4, 5). The principle of these techniques is based on the following properties of DNA. Namely, the electrophoretic mobility of DNA fragment slows down at the melting point of its local cooperatively melting region and the melting point is shifted by the point mutation. Introduction of GC-clamp into a sample DNA fragment through PCR method has made these techniques widely used (6). Making a precise gradient gel plate, however, requires a skilled hand or a sophisticated instrument. And this situation may limit the number of mutants to be detected. Here we propose a simpler version of TGGE, a temperature sweep gel electrophoresis (TSGE). In TGGE the temperature gradient is established in the spaceaxis before electrophoresis, but in TSGE the temperature of gel plate is raised gradually and uniformly during electrophoresis. The gradient of TGGE is linear, but the sweep speed of TSGE is easily varied during electrophoresis to give any temperature history to DNA samples. Theoretically the linear TSGE gives almost same results as the parallel TGGE, in which DNA moves parallel to the gradient. In TSGE the slowing-down of sweep speed in the middle of the run will give better resolution for specified mutants group. A demerit of TSGE is absence of any mode corresponding to the vertical TGGE. Our instrument of TSGE consists of a normal thermojacketed vertical slab-gel apparatus (hand-made), a circulating thermobath (Tokyo Rika, UA-10, precision i 0.1 0C) and a magnetic stirrer. The gel plate is immersed in the bottom buffer, which is intensively stirred in order to maintain evenness of the temperature. Unevenness gives artifacts in the band pattern. The gene VmI of coliphage Ff family was cloned by PCR using a 20-mer primer and a 50-mer primer having 5' GC-clamp region of 29 bp. All the cloned DNA fragments are 289 bp. TSGE gel used was made with 8% acrylamide/8 M urea. The acrylamide:bisacrylamide ratio was 159:1. TEMED (0.04%) and ammonium persulfate (0.04%) were added. 2.0,l of PCR product was mixed with dye solution, and then loaded into a well at the top of the gel. Electrophoresis was performed in TAE buffer (40 mM Tris-acetate, pH 8.0, 20 mM sodium acetate, 1 mM EDTA) at 300 V. The temperature of the thermojacket of electrophoretic plate was 45.0°C before the run. The temperature
*
To whom correspondence should be addressed
was raised continuously at the rate of 1.0°C/5 min from 45.0°C through 63.0°C during the run. The gel was stained with silver and photographed (Figure 1). The number of base substitution between M13 and fl in the gene VIII is one (# 1403 site is A for M13 and G for fl). TSGE detected the point mutation. Vertical and parallel TGGE for these DNA fragments were also performed. The results supported the data from TSGE. When TSGE gel was made with 4% or 6% acrylamide/8 M urea and the acrylamide:bisacrylamide ratio was 19:1, the band resolution was poor for these DNA samples. It suggests the pore size and/or the pore flexibility of the gel is important to discrimination of the DNA conformations.
REFERENCES 1. Husimi,Y. (1989) Adv. Biophys. 25, 1-43. 2. Fischer,S.G. and Lerman,L.S. (1979) Cell 16, 191-200. 3. Nishigaki,K., Husimi,Y. and Tsubota,M. (1986) J. Biochem. (Tokyo) 99, 663-671. 4. Po,T., Steger,G., Rosenbaum,V., Kaper,J. and Riesner,D. (1987) Nucl. Acids Res. 15, 5069-5083. 5. Steger,G., Po,T., Kaper,J. and Riesner,D. (1987) ibid, 5085-5103. 6. Sheffield,V.C., Cox,D.R., Lerman,L.S. and Myers,R.M. (1989) Proc. Natl. Acad. Sci. USA 86, 232-236.
1 2:
-*
M 309 313
1 75 158
Figure 1. Detection of point mutation by TSGE (45.0°C - 63.0°C). Gene Vm of coliphage Ff family was cloned through PCR/GC-clamp method. Lanes 1, 2 and 3 are from fl, fd-U1l1 (mutant of fd), and M13, respectively. Lane M is from HaeIII digests of M13 ssDNA. The arrow locates an asymmetric PCR product (289-mer ssDNA).
