Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples.
MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 microg of Thermus thermophilus his6-MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations.
Stanisławska-Sachadyn
Anna
A
Gdańsk University of Technology, Microbiology Department, Narutowicza 11/12, 80952 Gdańsk, Poland. annast@altis.chem.pg.gda.pl
Paszko
Zygmunt
Z
Kluska
Anna
A
Skasko
Elzbieta
E
Sromek
Maria
M
Bałabas
Aneta
A
Janiec-Jankowska
Aneta
A
Wiśniewska
Alicja
A
Kur
Józef
J
Sachadyn
Paweł
P
D018797:Cell Cycle Proteins / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D019398:Genes, BRCA1; D018814:Multiple Endocrine Neoplasia Type 2b / Q000235:genetics; D051718:MutS Homolog 2 Protein; D009687:Nuclear Proteins / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D004247:DNA; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051718:MutS Homolog 2 Protein