Two dimensional single-strand conformation polymorphism analysis: a useful tool for the detection of mutations in long DNA fragments.
A new two-dimensional gel system for the analysis of single strand conformational polymorphisms has been developed to identify point mutations, deletions and insertions in long DNA fragments (e.g. 2.7 kb) generated by the polymerase chain reaction. In this procedure, such DNA fragments are first restricted with frequent-cutter enzymes. The resulting small fragments are then separated in the first dimension according to their size by electrophoresis under denaturing conditions; these single stranded DNA fragments are subsequently fractionated in the second dimension by electrophoresis on a non denaturing slab gel based on their fold-back conformation which is completely sequence-dependent. The method was tested on three previously characterized pH 4.5 resistant mutants of HRV14 and was then used to determine changes in three further mutants.
Kovar
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Children's Cancer Research Institute, St Anna Kinderspital, Vienna, Austria.
Jug
G
G
Auer
H
H
Skern
T
T
Blaas
D
D
D001483:Base Sequence; D002872:Chromosome Deletion; D004252:DNA Mutational Analysis; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012229:Rhinovirus / Q000235:genetics*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004262:DNA Restriction Enzymes