© 1991 Oxford University Press Nucleic Acids Research, Vol. 19, No. 11 3153

Temperature sweep gel electrophoresis: a simple methodto 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 DNAis required in the various fields of biotechnology including newlyemerging evolutionary molecular engineering (1). It has generallybeen realized through the use of the denaturant gradient getelectrophoresis (DGGE) (2, 3, 6) or the temperature gradientgel electrophoresis (TGGE) (4, 5). The principle of thesetechniques is based on the following properties of DNA. Namely,the electrophoretic mobility of DNA fragment slows down at themelting point of its local cooperatively melting region and themelting point is shifted by the point mutation. Introduction ofGC-clamp into a sample DNA fragment through PCR methodhas made these techniques widely used (6). Making a precisegradient gel plate, however, requires a skilled hand or asophisticated instrument. And this situation may limit the numberof mutants to be detected. Here we propose a simpler versionof TGGE, a temperature sweep gel electrophoresis (TSGE).

In TGGE the temperature gradient is established in the space-axis before electrophoresis, but in TSGE the temperature of gelplate is raised gradually and uniformly during electrophoresis.The gradient of TGGE is linear, but the sweep speed of TSGEis easily varied during electrophoresis to give any temperaturehistory to DNA samples. Theoretically the linear TSGE givesalmost same results as the parallel TGGE, in which DNA movesparallel to the gradient. In TSGE the slowing-down of sweepspeed in the middle of the run will give better resolution forspecified mutants group. A demerit of TSGE is absence of anymode corresponding to the vertical TGGE. Our instrument ofTSGE consists of a normal thermojacketed vertical slab-gelapparatus (hand-made), a circulating thermobath (Tokyo Rika,UA-10, precision ±0.1°C) and a magnetic stirrer. The gel plateis immersed in the bottom buffer, which is intensively stirredin order to maintain evenness of the temperature. Unevennessgives artifacts in the band pattern.

The gene VUI of coliphage Ff family was cloned by PCR usinga 20-mer primer and a 50-mer primer having 5' GC-clamp regionof 29 bp. All the cloned DNA fragments are 289 bp. TSGE gelused was made with 8% acrylamide/8 M urea. Theacrylamide:bisacrylamide ratio was 159:1. TEMED (0.04%) andammonium persulfate (0.04%) were added. 2.0 jil of PCRproduct was mixed with dye solution, and then loaded into a wellat the top of the gel. Electrophoresis was performed in TAEbuffer (40 mM Tris-acetate, pH 8.0, 20 mM sodium acetate, 1mM EDTA) at 300 V. The temperature of the thermojacket ofelectrophoretic plate was 45.0°C before the run. The temperature

was raised continuously at the rate of 1.0°C/5 min from 45.0°Cthrough 63.0°C during the run. The gel was stained with silverand photographed (Figure 1).

The number of base substitution between M13 and fl in thegene VHI is one (# 1403 site is A for M13 and G for fl). TSGEdetected the point mutation. Vertical and parallel TGGE for theseDNA fragments were also performed. The results supported thedata from TSGE. When TSGE gel was made with 4% or 6%acrylamide/8 M urea and the acrylamiderbisacrylamide ratio was19:1, the band resolution was poor for these DNA samples. Itsuggests the pore size and/or the pore flexibility of the gel isimportant to discrimination of the DNA conformations.

REFERENCES1. 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)/ 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

Figure 1. Detection of point mutation by TSGE (45.0°C — 63.0°C). Gene VIIIof coliphage Ff family was cloned through PCR/GC-clamp method. Lanes 1,2 and 3 are from fl, fd-UlOl (mutant of fd), and M13, respectively. Lane Mis from Haelll digests of M13 ssDNA. The arrow locates an asymmetric PCRproduct (289-mer ssDNA).

* To whom correspondence should be addressed

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