Method and oligonucleotide for detecting fgfr3 G380R site mutation

Abstract

The invention discloses a method and oligonucleotide for detecting FGFR3 gene G380R site mutation, and relates to a pair of specific amplification primers SEQ NO 1 and SEQ NO 2, and a pair of specific detection probes SEQ NO 3 and SEQ NO 4. By adopting the method and oligonucleotide, congenital achondroplasia can be rapidly diagnosed and identified, and the method and oligonucleotide have the advantages of being short in detection cycle, good in specificity, high in accuracy, high in sensitivity, less in condition dependence, low in pollution risk and the like.

Description

technical field [0001] The invention belongs to the field of gene mutation detection, in particular to a method and oligonucleotide for highly sensitive detection of gene FGFR3G380R site mutation. Background technique [0002] Fibroblast growth factor receptor 3 (FGFR3), belonging to the fibroblast growth factor receptors (FGFRs) family, is a transmembrane protein that regulates development and other functions, and plays an important role in bone development . The FGFR3 gene is located on human chromosome 4p1613 and contains 806 amino acid residues. It is a tyrosine kinase receptor and consists of an extracellular ligand-binding region modified by glycosylation, a hydrophobic transmembrane region and a Composition of the catalytic domain of intracellular tyrosine kinases. The FGFR3 gene is about 1615kb in length, with 19 exons and 18 introns, of which the 10th exon encodes the transmembrane region of FGFR3. [0003] Achondroplasia (ACH) is a common hereditary disease of t...

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Problems solved by technology

Among them, SYBR Green I is not as specific as the double-hybrid probe method and Taqman probe method because it is an unsaturated dye, and its specificity must be judged by observing the melting curve. In addition, it cannot recognize a specific double-stranded DNA sequence, as long as it is double-stranded DNA The sequence will be combined with luminescence, so the background is usually high, and false positives may occur when used clinically

The two-hybridization probe method involves two specific probes that are complementary to the template and adjacent (distance 1-5bp), the 3' end of the upstream probe is labeled with a donor fluorophore, and the 5' end of the adjacent downstream probe is End-labeled Red640 acceptor fluorophore, the cost is relatively expensive

The molecular beacon method involves a stem-loop double-labeled oligonucleotide probe in a hairpin structure. The nucleic acid sequences at both ends of the probe are complementary paired, and a fluorescent group labeled at one end is labeled with a quencher group at the other end. The biggest disadvantage of this method is that the probe cannot be completely combined with the template during hybridization, so the stability is poor, and the labeling process is more complicated when the probe is synthesized.

The Taqman probe method involves a gene-specific probe (usually 20-30bp long) complementary to the template. The 5' end and the 3' end of the probe are respectively labeled with a reporter fluorophore and a quencher fluorophore, but The longer probe sequence in this method makes the distance between the reporter fluorophore and the quencher fluorophore at both ends of the probe farther away, resulting in incomplete fluorescence quenching, and the quencher group will also produce fluorescence of different wavelengths. This will make the background higher

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