Nucleic Acids Research, Vol. 20, No. 20 5491
Simplified DGGE in a horizontal electrophoresis system Ingolf Russ and Ivica Medjugorac Lehrstuhl fOr Tierzucht der Technischen Universitat Munchen, D-8050 Freising-Weihenstephan, Germany Submitted July 20, 1992 Based on the sequence-dependent melting behaviour of doublestranded DNA molecules, denaturing gradient gel electrophoresis (DGGE) (1) separates fragments of 100-1000 bp according to sequence composition. Thus, point mutations may be revealed. However, DGGE performed according to the original protocol requires an aquarium with heated buffer into which gel holders are placed. Here we report the method accomplished on a cooling (heating) plate connected to a thermostatic circulator. The use of thin foil-stabilized gels guarantees high sensitivity, continuous temperature across the gel, convenient handling without danger of disturbance and use of low acrylamide concentrations. The method is performed in a Mulfiphor II (Pharmacia-LKB) apparatus, but any horizontal electrophoresis apparatus equipped with a cooling plate can be substituted. Denaturing gradient gels [110/180 x250 mm] were cast using a two chamber-mixer (2). The casting cassette included a glass plate to which a GelBond PAG film (FMC) was fixed with water, a 0.5 mm thick U-shaped rubber spacer and a silanized glass plate carrying 24 slot-formers (0.2 x 3 x7 mm) cut from a strip of label tape (Dymo) placed 1.5 cm from the top of the gel. The gel was fixed with water on the cooling plate of the electrophoresis apparatus and the electrode wicks were applied so that they overlapped the gel by 10-15 mm on each side. The space between slots and anodic wick was covered with GelBond PAG film. After application of 5 to 10 1d of PCR products, the wicks were weighted with a glass plate and electrophoresis performed at 150 V (11 cm) and 300 V (18 cm) at 60°C for 15 min (up to 45 min for large fragments). Subsequently, the cathodic wick was moved forward to protect the exposed application area from drying out and electrophoresis was continued. The gels were silver stained (3), soaked in 10% glycerol for 2-10 min and dried for storage.
B
A BB
AB
AA
T 75 E M P 70 E R A 66 T
R E 60O 0
50
100
200 260 SEQUENC E
150
300
360
FIgmue 2. Melting map of the bovine x-CN fragnt. Arrows indicate polymorphic sites.
A 351 bp fragment of the bovine x-casein (x-CN) gene (4) was used for demonstration ofthe system. This fragment contains two polymorphic nucleotides, C to T at position 99 and A to C at position 135, coding for the protein variants A and B, respectively. Results of separation with DGGE are shown in Figure 1A and B. The difference in melting behaviour between allele A and B is most likely a result of the A to C substitution at position 135 since the other polymorphic site is located in the highest melting domain (Figure 2). Due to the fact that more than 80% of the double-stranded DNA is disassociated before melting at position 135 occurs, resulting in the apparent change in electrophoretic mobility, we would conclude that the resolution of this system is very high.
ACKNOWLEDGEMENTS We are grateful to Dr L.S.Lerman for making computer programs available to us, to P.Schlee for providing us with the PCR-products and to J.Beever for helpful comments on the manuscript.
REFERENCES t
13%
7 70%
+
Figure 1. (A) Part of the perpendicular DGG 0-80%, 7% acrylamide (29.1:0.9 acrylamide:bisacrylamide), 18 cm. Electrophoresis: 300 V, 3.5 h, 60°C. Sample: 3 Il PCR-amplified x-CN AA. Silver Staining. (B) Part of an analytical DGG 38-50%, 7% acrylamide, 11 cm. Electrophoresis: 150 V, 7 h, 60°C. Samples: 5 IA x-CN AA, AB, BB. Silver staining.
Myers,R.M., Maniatis,T. and Lerman,L.S. (1987) In: Methods in Enzwology 155, 501-527. 2. Gorg,A., Postel,W., Westermeier,R., Gianazza,E. and Righetti,P.G. (1980) 1.
J.Biochem. Biophys. Meth. 3, 273-284. 3. Blum,H., Brier,H. and Gross,H.J. (1987) Electrophoresis 8, 93-99. 4. Medrano,J.F. and Aguilar-Cordova,E. (1990) Biotechnology 8, 144-146.
Simplified DGGE in a horizontal electrophoresis system Ingolf Russ and Ivica Medjugorac Lehrstuhl fOr Tierzucht der Technischen Universitat Munchen, D-8050 Freising-Weihenstephan, Germany Submitted July 20, 1992 Based on the sequence-dependent melting behaviour of doublestranded DNA molecules, denaturing gradient gel electrophoresis (DGGE) (1) separates fragments of 100-1000 bp according to sequence composition. Thus, point mutations may be revealed. However, DGGE performed according to the original protocol requires an aquarium with heated buffer into which gel holders are placed. Here we report the method accomplished on a cooling (heating) plate connected to a thermostatic circulator. The use of thin foil-stabilized gels guarantees high sensitivity, continuous temperature across the gel, convenient handling without danger of disturbance and use of low acrylamide concentrations. The method is performed in a Mulfiphor II (Pharmacia-LKB) apparatus, but any horizontal electrophoresis apparatus equipped with a cooling plate can be substituted. Denaturing gradient gels [110/180 x250 mm] were cast using a two chamber-mixer (2). The casting cassette included a glass plate to which a GelBond PAG film (FMC) was fixed with water, a 0.5 mm thick U-shaped rubber spacer and a silanized glass plate carrying 24 slot-formers (0.2 x 3 x7 mm) cut from a strip of label tape (Dymo) placed 1.5 cm from the top of the gel. The gel was fixed with water on the cooling plate of the electrophoresis apparatus and the electrode wicks were applied so that they overlapped the gel by 10-15 mm on each side. The space between slots and anodic wick was covered with GelBond PAG film. After application of 5 to 10 1d of PCR products, the wicks were weighted with a glass plate and electrophoresis performed at 150 V (11 cm) and 300 V (18 cm) at 60°C for 15 min (up to 45 min for large fragments). Subsequently, the cathodic wick was moved forward to protect the exposed application area from drying out and electrophoresis was continued. The gels were silver stained (3), soaked in 10% glycerol for 2-10 min and dried for storage.
B
A BB
AB
AA
T 75 E M P 70 E R A 66 T
R E 60O 0
50
100
200 260 SEQUENC E
150
300
360
FIgmue 2. Melting map of the bovine x-CN fragnt. Arrows indicate polymorphic sites.
A 351 bp fragment of the bovine x-casein (x-CN) gene (4) was used for demonstration ofthe system. This fragment contains two polymorphic nucleotides, C to T at position 99 and A to C at position 135, coding for the protein variants A and B, respectively. Results of separation with DGGE are shown in Figure 1A and B. The difference in melting behaviour between allele A and B is most likely a result of the A to C substitution at position 135 since the other polymorphic site is located in the highest melting domain (Figure 2). Due to the fact that more than 80% of the double-stranded DNA is disassociated before melting at position 135 occurs, resulting in the apparent change in electrophoretic mobility, we would conclude that the resolution of this system is very high.
ACKNOWLEDGEMENTS We are grateful to Dr L.S.Lerman for making computer programs available to us, to P.Schlee for providing us with the PCR-products and to J.Beever for helpful comments on the manuscript.
REFERENCES t
13%
7 70%
+
Figure 1. (A) Part of the perpendicular DGG 0-80%, 7% acrylamide (29.1:0.9 acrylamide:bisacrylamide), 18 cm. Electrophoresis: 300 V, 3.5 h, 60°C. Sample: 3 Il PCR-amplified x-CN AA. Silver Staining. (B) Part of an analytical DGG 38-50%, 7% acrylamide, 11 cm. Electrophoresis: 150 V, 7 h, 60°C. Samples: 5 IA x-CN AA, AB, BB. Silver staining.
Myers,R.M., Maniatis,T. and Lerman,L.S. (1987) In: Methods in Enzwology 155, 501-527. 2. Gorg,A., Postel,W., Westermeier,R., Gianazza,E. and Righetti,P.G. (1980) 1.
J.Biochem. Biophys. Meth. 3, 273-284. 3. Blum,H., Brier,H. and Gross,H.J. (1987) Electrophoresis 8, 93-99. 4. Medrano,J.F. and Aguilar-Cordova,E. (1990) Biotechnology 8, 144-146.
