ABSTRACT
A sensitive and pyrosequencing-compatible method for determining the copy number of the short tandem repeat (STR) is presented in this study. When Escherichia coli ligase catalyzes the ligation of primer and probes complementary to the proper sites of the target DNA template, it converts nicotinamide adenine dinucleotide to adenosine monophosphate (AMP) and nicotinamide. The AMP release level is proportional to the copy number of the STR and can be measured using adenylate kinase, pyruvate kinase, and luciferase. Unlike current standard methods based on electrophoresis, the present assay is sensitive to the point mutation. Furthermore, after determination of the copy number of the tandem repeat using the proposed method, the DNA templates, primer and probes immobilized onto super paramagnetic beads can be washed and pyrosequencing can be applied for the remaining DNA sequencing. This assay is specially efficient to handle a large number of samples because massively parallel tests could be executed in a microplate photometer. Furthermore, it can work with the pyrosequencing for further sequencing like genome sequencing.
Subject(s)
DNA Copy Number Variations/genetics , DNA Ligases/metabolism , Microsatellite Repeats/genetics , NAD/metabolism , Sequence Analysis, DNA/methods , Adenosine Monophosphate/analysis , Adenosine Triphosphate/analysis , Adenylate Kinase/metabolism , Base Sequence , DNA/genetics , DNA/metabolism , DNA Primers/genetics , Escherichia coli/enzymology , Humans , Luciferases/metabolism , Point Mutation/genetics , Pyruvate Kinase/metabolismABSTRACT
In this study, the tested microRNA and the detection probe perfectly match with the capture probe instead of the traditional sandwich methods in which the tested oligonucleotide matches with the detection and capture probes. To avoid non-specific signals, mung-bean nuclease, a single-strand-specific nuclease, catalyzes the degradation of the capture probe if there is no tested miRNA in the samples. The gold nanoparticles conjugate the thiol-DNA as the detection probe and the biotin-single strand DNA serves as the capture probe. The avidin-biotin-Au-sample complex is captured by the anti-avidin antibody immobilized on a flow strip. The detection and quantification of the gold nanoparticle signal indicate the existence and quantity of the target miRNA. One fmol and five amol of the synthetic microRNA were detected without and with the silver enhancement, respectively. This highly sensitive and specific assay takes about 70 min after the RNA purification and preparation. It is simple, convenient, fast, and suitable for point-of-care.
