Construction Of Novel Molecular Marker For Locked Nucleic Acid And Its Application In Detection Of Serum UHRF1 DNA In Patients With Breast Cancer

Molecular beacons are stem-loop hairpin-structured fluorescence probes with a fluorescent dye at 5’ end and a fluorescence quencher at 3’ end. The loop is consist of 15～30 bases, the stem is consist of 5-7 bases. When a target complementary sequence is absent, the molecular beacons do not fluoresce, because the formation of the hairpin structure brings the quencher and fluorophore into close proximity, whereby fluorescence is quenched with high efficiency. When a target molecule is present, the hybridization between the target and the loop sequence of the molecular beacon results in the spatial separation of the fluorophore and quencher, which opens the stem-loop structure of molecular beacons to emit fluorescence. Due to the resonance energy transfer mechanism, molecular beacons can detect target segments without the separation of the unbound probes, and the flouresence intensity increases in proportion to the concentration of targets. Hence, molecular beacons has an enormous application range, such as gene screening, the constraction of gene chip, the detection of single-nucleotide polymorphism and so on. However, molecular beacon is also a DNA segment, it can digest by enzyme in the clinical samples, with restrict its applications in clinic. Locked nucleic acid is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. It possesses excellent binding affinity to nucleic acid, high biostability and resists to nuclease degradation. Due to the excellent sensitivity and high specificity, the combination of the molecular beacons and locked nucleic acid has aroused wide concern.We introduce the LNA into the construction of novel molecular beacons, which apparently improved the property of the probe. The main research is the design of molecular beacon and the application of molecular beacon in the detection of circulating DNA. In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. Compared to normal MB, LNA-MB has a higher singal to noise ratio; co-incubation with DNase I for 2 h, the florescence intensity has no changes, which suggest that LNA-MB has a resistance of DNase I; Besides, LNA-MB has a excellent abilty of mismatched bases recognition because the signal can be distinguished from that of the fully complementary target because it is nearly 50% less intense. The sequence of the UHRF1 DNA was obtained from the nucleotide database on the website of the National Center for Biotechnology Information (NCBI). A 27-base DNA recognition sequence was chosen for target and studied by extensive BLAST search analysis to evaluate specificity as the serum DNA target. The loop section of the hairpin DNA was designed according to the complementary base-pair specificity. The detection procedure is simple and can be completed within 1 h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11 nM (S/N1/43) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring.