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A simple fluorescent method for detecting mismatched DNAs using a MutS–fluorophore conjugate
- Source :
- Biosensors and Bioelectronics. 22:1376-1381
- Publication Year :
- 2007
- Publisher :
- Elsevier BV, 2007.
-
Abstract
- A fluorescent method was developed for the detection of unpaired and mismatched DNAs using a MutS-fluorophore conjugate. The fluorophore, 2-(4'-(iodoacetoamido)anilino) naphthalene-6-sulfonic acid (IAANS), was site-specifically attached to the 469 position of Thermus aquaticus (Taq.) MutS mutant (C42A/T469C). The fluorophore labeled residue located at the dimer interface of the protein undergoes a drastic conformational change upon binding with mismatched DNA. The close proximity of the two identical fluorescent molecules presumably causes the self-quenching of the fluorophore, since fluorescence emission of the biosensor decreases with increasing concentrations of mismatched DNA. The order of binding affinity for each unpaired and mismatched DNA obtained by this method was DeltaT (Kd=52 nM)>GT (62 nM)>DeltaC (130 nM)>CT (160 nM)>DeltaG (170 nM)>DeltaA (250 nM)>CC (720 nM)>AT (950 nM). This order is comparable to the previous results of the gel mobility shift assay. Thus, this method can be a simple, useful tool for elucidating the mechanism of DNA mismatch repair as well as a novel probe for detecting of genetic mutation.
- Subjects :
- Conformational change
Fluorophore
Dimer
Biomedical Engineering
Biophysics
Biosensing Techniques
chemistry.chemical_compound
Naphthalenesulfonates
Electrochemistry
Point Mutation
Electrophoretic mobility shift assay
Thermus
Fluorescent Dyes
Thermus aquaticus
biology
DNA
General Medicine
biology.organism_classification
Molecular biology
Fluorescence
MutS DNA Mismatch-Binding Protein
chemistry
Biotechnology
Conjugate
Subjects
Details
- ISSN :
- 09565663
- Volume :
- 22
- Database :
- OpenAIRE
- Journal :
- Biosensors and Bioelectronics
- Accession number :
- edsair.doi.dedup.....5b787f09159adfda794f4d1f42b4c65c