Automated band mapping in electrophoretic gel images using background information

Some popular methods for polymorphism and mutation discovery involve ascertainment of novel bands by the examination of electrophoretic gel images. Although existing strategies for mapping bands work well for specific applications, such as DNA sequencing, these strategies are not well suited for novel band detection. Here, we describe a general strategy for band mapping that uses background banding patterns to facilitate lane calling and size calibration. We have implemented this strategy in GelBuddy, a user-friendly Java-based program for PC and Macintosh computers, which includes several utilities to assist discovery of mutations and polymorphisms. We demonstrate the use of GelBuddy in applications based on single-base mismatch cleavage of heteroduplexed PCR products. Use of software designed to facilitate novel band detection can significantly shorten the time needed for image analysis and data entry in a high-throughput setting. Furthermore, the interactive strategy implemented in GelBuddy has been successfully applied to DNA fingerprinting applications, such as AFLP. GelBuddy promises to make electrophoretic gel analysis a viable alternative to DNA resequencing for discovery of mutations and polymorphisms.     

Performance evaluation of amino acid substitution matrices

Several choices of amino acid substitution matrices are currently available for searching and alignment applications. These choices were evaluated using the BLAST searching program, which is extremely sensitive to differences among matrices, and the Prosite catalog, which lists members of hundreds of protein families. Matrices derived directly from either sequence-based or structurebased alignments of distantly related proteins performed much better overall than extrapolated matrices based on the Dayhoff evolutionary model. Similar results were obtained with the FASTA searching program. Improved performance appears to be general rather than family-specific, reflecting improved accuracy in scoring alignments. An implementation of a multiple matrix strategy was also tested. While no combination of three matrices performed as well as the single best matrix, BLOSUM 62, good results were obtained using a combination of sequence-based and structure-based matrices. This hybrid set of matrices is likely to be useful in certain situations. Our results illustrate the importance of matrix selection and value of a comprehensive approach to evaluation of protein comparison tools. © 1993 Wiley-Liss, Inc.