One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA.
A novel, universal method for mutation detection utilising the ability of MutS protein to recognise DNA incomplementarities is proposed. The examined and reference DNA fragments are PCR amplified. The PCR products are purified, mixed, heated and cooled to form heteroduplexes. In the case of mutation the heteroduplex DNA containing mismatch is protected against exonuclease digestion by MutS, while the DNA without mismatches is degraded. The protection effect is visualised by the direct addition of a highly sensitive fluorescent dye (SYBR-Gold) selectively binding DNA. The Thermus thermophilus recombined His-tagged MutS and 3'-5' exonuclease activity of T4 DNA polymerase were used in the assay.
Sachadyn
P
P
Technical University of Gdañsk, Department of Microbiology, ul. Narutowicza 11/12, 80-952 Gdañsk, Poland. psach@altis.chem.pg.gda.pl
Stanislawska
A
A
Kur
J
J
D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins / Q000378:metabolism*; D020137:Base Pair Mismatch; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D051719:MutS DNA Mismatch-Binding Protein; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016963:Thermus thermophilus; D014764:Viral Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009692:Nucleic Acid Heteroduplexes; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli