SSCP analysis of long DNA fragments in low pH gel.
Sensitivity of single-strand conformation polymorphism analysis of PCR products (PCR-SSCP analysis) is known to be decreased when the DNA fragments are longer than 300 bp. We examined effects of buffer ions in an attempt to extend the length limit of the analysis. It has been noted that addition of glycerol to the gel containing Tris-borate buffer enhances the sensitivity, but the effects of glycerol have been left unexplained. We found that the effects of glycerol are caused by the reduction of pH of the buffer by the reaction of glycerol and borate ion. We further extended these observations and found that sensitivity of SSCP can be greatly improved by running the electrophoresis in low pH buffer systems. Using a new buffer system and running the electrophoresis at a fixed temperature, we detected 27 of 31 known mutations of factor IX gene in six different sequence contexts ranging in length from 300 to 800 bp.
Kukita
Y
Y
Division of Genome Analysis, Institute of Genetic Information, Kyushu University, Japan.
Tahira
T
T
Sommer
S S
SS
Hayashi
K
K
D002021:Buffers; D053938:DNA Fragmentation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D014407:Tumor Cells, Cultured
D002021:Buffers; D005990:Glycerol