Detection of point mutation and insertion mutations in DNA using a quartz crystal microbalance and MutS, a mismatch binding protein.
MutS protein is a mismatch binding protein that recognizes mispaired and unpaired base(s) in DNA. In this study, we incorporate the MutS protein-based mutation recognition into quartz crystal microbalance (QCM) measurements for DNA single-base substitution mutation and 1-4 base(s) insertion (or deletion) mutation detection. The method involves the immobilization of single-stranded probe DNA on a QCM surface, the hybridization of target DNA to form homoduplex or heteroduplex DNA, and finally the application of MutS protein for the mutation recognition. By measuring the MutS binding signal, DNA containing a T:G mismatch or unpaired base(s) is(are) discriminated against perfectly matched DNA at target concentrations ranging from 1nM to 5 microM. Furthermore, the QCM damping behavior upon MutS-DNA complex formation is studied using a Network Analyzer. The measured motional resistance changes per coupled MutS unit mass (deltaR/deltaf) are found to be indicative of the viscoelastic or structural properties of the bound protein, corresponding to different binding mechanisms. In addition, the deltaR/deltaf values vary remarkably when the MutS protein binds at different distances away from the QCM surface. Thus, these values can be used as a "fingerprint" for MutS mismatch recognition and also used to quantitatively locate the mutation site.
Su
Xiaodi
X
Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602. xd-su@imre.a-star.edu.sg
Robelek
Rudolf
R
Wu
Yingju
Y
Wang
Guangyu
G
Knoll
Wolfgang
W
D000251:Adenosine Triphosphatases / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D020137:Base Pair Mismatch / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods; D001710:Biotin / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D051719:MutS DNA Mismatch-Binding Protein; D016254:Mutagenesis, Insertional; D009154:Mutation; D009693:Nucleic Acid Hybridization / Q000235:genetics; D017354:Point Mutation; D011791:Quartz / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D013824:Thermus / Q000201:enzymology; D014159:Transducers
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D015342:DNA Probes; D004268:DNA-Binding Proteins; D011791:Quartz; D012710:Serum Albumin, Bovine; D001710:Biotin; D006046:Gold; D004247:DNA; D019809:Streptavidin; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein