5 Single-Strand Conformation Polymorphism (SSCP) Analysis
85
• Gel matrix composition
The pore size of the acrylamide gel is an important determinant for sensitivity of SSCP. The ratio of acrylamide to bisacrylamide should be 49:1.
Recently, a special gel matrix (MDE, AT Biochemical) reportedly allows
better resolution of molecules with subtle conformational differences.
• Electrophoresis conditions
Some single-stranded conformations can be remarkably sensitive to glycerol concentration and temperaturein gels. The electrophoresis in SSCP
analysis is thus routinely performed using gels comprised of different
glycerol concentrations and run at different temperatures. There is almost an infinite combination of possible running conditions. Generally,
one starts with two conditions:
- lOo/o (v/v) glycerol and electrophoresis at room temperature
- no glycerol and electrophoresis at 4°C
One can enhance the sensitivity by trying additional conditions which differ
in the glycerol concentration of the gelandin the temperature of the buffer/
gel during the run.
Furtherparameters affecting SSCP sensitivity include the sequence of the
fragment, the nature of the base pair change, and the base position of the
nucleotide substitution with the fragment.
References
Orita M, Iwahara H, Kanazawa H, Hayashi K, and Sekiya T (1989a) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation
polymorphisms. Proc Natl Acad Sei USA 86: 2766-2770
Orita M, Suzuki Y, Sekiya T, and Hayashi K {1989b) Rapid and sensitive detection of
point mutations and DNA polymorphisms using the polymerase chain reaction.
Genomics 5: 874-879
Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW and Stone EM {1993) The sensitivity of
single-stranded conformation polymorphism analysis for the detection of single base
substitution. Genomics 16:325-332
85
• Gel matrix composition
The pore size of the acrylamide gel is an important determinant for sensitivity of SSCP. The ratio of acrylamide to bisacrylamide should be 49:1.
Recently, a special gel matrix (MDE, AT Biochemical) reportedly allows
better resolution of molecules with subtle conformational differences.
• Electrophoresis conditions
Some single-stranded conformations can be remarkably sensitive to glycerol concentration and temperaturein gels. The electrophoresis in SSCP
analysis is thus routinely performed using gels comprised of different
glycerol concentrations and run at different temperatures. There is almost an infinite combination of possible running conditions. Generally,
one starts with two conditions:
- lOo/o (v/v) glycerol and electrophoresis at room temperature
- no glycerol and electrophoresis at 4°C
One can enhance the sensitivity by trying additional conditions which differ
in the glycerol concentration of the gelandin the temperature of the buffer/
gel during the run.
Furtherparameters affecting SSCP sensitivity include the sequence of the
fragment, the nature of the base pair change, and the base position of the
nucleotide substitution with the fragment.
References
Orita M, Iwahara H, Kanazawa H, Hayashi K, and Sekiya T (1989a) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation
polymorphisms. Proc Natl Acad Sei USA 86: 2766-2770
Orita M, Suzuki Y, Sekiya T, and Hayashi K {1989b) Rapid and sensitive detection of
point mutations and DNA polymorphisms using the polymerase chain reaction.
Genomics 5: 874-879
Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW and Stone EM {1993) The sensitivity of
single-stranded conformation polymorphism analysis for the detection of single base
substitution. Genomics 16:325-332