Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis.
We have studied 57 different mutations within three beta-globin gene promoter fragments with sizes 52 bp, 77 bp, and 193 bp by fluorescent capillary electrophoresis CE-SSCP analysis. For each mutation and wild type, energetically most-favorable predicted secondary structures were calculated for sense and antisense strands using the MFOLD DNA-folding algorithm in order to investigate if any correlation exists between predicted DNA structures and actual CE migration time shifts. The overall CE-SSCP detection rate was 100% for all mutations in three studied DNA fragments. For shorter 52 bp and 77 bp DNA fragments we obtained a positive correlation between the migration time shifts and difference in free energy values of predicted secondary structures at all temperatures. For longer 193 bp beta-globin gene fragments with 46 mutations MFOLD predicted different secondary structures for 89% of mutated strands at 25 degrees C and 40 degrees C. However, the magnitude of the mobility shifts did not necessarily correlate with their secondary structures and free energy values except for the sense strand at 40 degrees C where this correlation was statistically significant (r = 0.312, p = 0.033). Results of this study provided more direct insight into the mechanism of CE-SSCP and showed that MFOLD prediction could be helpful in making decisions about the running temperatures and in prediction of CE-SSCP data patterns, especially for shorter (50-100 bp) DNA fragments.
Glavac
Damjan
D
Laboratory of Molecular Genetics, Institute of Pathology, Ljubljana, Slovenia.
Potocnik
Uros
U
Podpecnik
Darja
D
Zizek
Teofil
T
Smerkolj
Sava
S
Ravnik-Glavac
Metka
M
D000465:Algorithms; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics; D019075:Electrophoresis, Capillary; D005914:Globins / Q000235:genetics*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D018807:Polymorphism, Single-Stranded Conformational; D011401:Promoter Regions, Genetic / Q000235:genetics; D013696:Temperature
D004277:DNA, Single-Stranded; D005914:Globins; D004247:DNA