Increased detection of polymorphism among randomly amplified wheat DNA fragments using a modified temperature sweep gel electrophoresis (TSGE) technique

Random amplified polymorphic DNA analysis of the wheat genome has been hampered because low levels of polymorphism are detected. Recently, the use of a denaturing gradient to separate RAPD fragments on acrylamide gels (DGGE) has been shown to increase the frequency of polymorphism detection in wheat. The inclusion of denaturant facilitates the resolution of double stranded DNA on the basis of sequence as well as size. We found that DGGE analysis had two major drawbacks. One, pouring of denaturant gradient gels was time-consuming and subject to technical error. Two, the amount of denaturant that individual DNA fragments were exposed to was dependent on their size. Small fragments migrated to the bottom of the gradient before large fragments encountered sufficient amounts of denaturant to cause mobility differentiation based on sequence. These small fragments would be eluted from the bottom of the gel if the large fragments were to be run further into the gradient. To overcome these difficulties we modified the temperature sweep gel electrophoresis (TSGE) technique of Yoshino et al. (1991) such that separation conditions were optimal for double stranded DNA.