Fluorescence detection of single-nucleotide polymorphisms using a thymidine-based molecular beacon.
We have developed a universal molecular beacon (T(7)-MB-T(7)) for the detection of single-nucleotide polymorphisms (SNPs). The beacon, which contains a 19-mer loop and a stem comprising a pair of seven thymidine (T) bases, forms double-stranded structures with target DNA molecules, leading to increases in the fluorescence of ethidium bromide (EthBr) as a result of intercalation. The interactions of the beacon with perfectly matched (DNA(pm)) and single-base mismatched (DNA(mm)) DNA strands are stronger and weaker, respectively, than those with Hg(2+) ions. As a result, the fluorescence of a solution containing T(7)-MB-T(7), DNA(pm), EthBr, and Hg(2+) is higher than that of a corresponding solution containing T(7)-MB-T(7), DNA(mm), EthBr, and Hg(2+), because the former has a greater number of intercalation sites for EthBr. Under the optimal conditions (100 nM T(7)-MB-T(7), 20 mM NaCl, 5.0 microM Hg(2+), and 300 nM EthBr in 5.0 mM Tris-HCl solution, pH 7.4), the plot of the fluorescence intensity against the concentration of DNA(pm) was linear over the range 5.0-100 nM (R(2)=0.98). A similar probe, T(7)-MB(t)-T(7), is sensitive and selective for the detection of a gene associated with hereditary tyrosinemia type I. Relative to conventional MBs, our new probe offers the advantages of higher selectivity toward DNA, less nonspecific binding toward single-stranded-DNA-binding protein, greater resistance to nuclease digestion, and low cost; therefore, we suspect that this system holds great potential for practical studies of SNPs.
Liu
Chi-Wei
CW
Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 106, Taiwan.
Lin
Yang-Wei
YW
Huang
Chih-Ching
CC
Chang
Huan-Tsung
HT
D015374:Biosensing Techniques / Q000295:instrumentation; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation*; D015342:DNA Probes / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015336:Molecular Probe Techniques / Q000295:instrumentation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*; D013936:Thymidine / Q000737:chemistry / Q000235:genetics*
D015342:DNA Probes; D004247:DNA; D013936:Thymidine