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Flight mass spectrometry genotyping detection method

A genotyping and flight mass spectrometry technology, which is applied in the field of flight mass spectrometry genotyping and detection, can solve problems such as weak signals of high molecular weight products, and achieve the effects of improving detection resolution, high throughput and high resolution

Inactive Publication Date: 2020-04-21
博淼生物科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In multiplex PCR reactions, the high signal intensity of low molecular weight primers will lead to weak signals of high molecular weight products to some extent.

Method used

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  • Flight mass spectrometry genotyping detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Designing 4 probes related to the risk of type 2 diabetes

[0043] Type 2 diabetes (type2diabetes, T2D) is a polygenic genetic disease. Its clinical diagnosis is mainly evaluated by the corresponding indicators of glycosylated hemoglobin, fasting blood glucose and glucose tolerance. It cannot be detected and diagnosed as early as possible, thereby affecting early intervention and treatment by mistake, and bringing greater pain and economic loss to patients. If the etiology of the disease is combined with personalized molecular testing, early diagnosis of T2D can be realized. Combined with domestic and foreign genome-wide association and candidate gene methods, the T2D single nucleotide polymorphic sites reported are relatively high in the Chinese population. 4 SNPs highly correlated with T2D susceptibility were selected.

[0044] For the selected 4 SNPs, the upstream and downstream flanking sequences of the SNP sites were obtained from the 1000Genomes websit...

Embodiment 2

[0046] Example 2: Oligonucleotide Ligation Reaction (OLA) and Enrichment of Ligation Products

[0047] Fragment the sample genomic DNA, adjust the sample genomic DNA concentration to 100ng / μl with 1×TE (10mM Tris-HCL PH8.0,0.1mM EDTA), take out 15μl, 99℃ for 6 minutes, so that the DNA fragments are distributed in 2-7kb between.

[0048] The reaction system for configuring OLA is 0.3μl OLA buffer (10×), 0.3μl DTT (25mM), 0.04μl TaqDNA Ligase (40U / μl), 0.03μl ASO Oligo Mix, 0.03μl LSO Oligo Mix, 2.3μl ddH2O. And take 3 μ l of the above reaction system and divide it into the centrifuge tube that has added the fragmented sample, and carry out the oligonucleotide ligation reaction. The reaction conditions were: denaturation at 95°C for 5 minutes, 3 cycles (95°C for 30s and 50°C for 25 minutes), and storage at 4°C.

[0049] Use general-purpose PCR primers Forword-(5 / -CCCAGTCACGACGTTGTAAAACG-3 / ), Reverse (5 / -AGCGGATAACAATTTCACACAGG-3 / ) to amplify the above ligation products. The PC...

Embodiment 3

[0050] Example 3: Restriction enzyme reaction and purification of digested products

[0051] According to different restriction endonucleases, the reaction system and reaction conditions will be different. In this example, DpnI restriction endonuclease and its corresponding buffer were added to the above PCR product for digestion. The digestion system was: 15 μl of PCR product, 2 μl of Buffer (10×), 1 μl of DpnI, and 4 μl of ddH2O. After digestion at 37°C for 4 hours, the PCR product will be cut into multiple fragments, and the fragments carrying Tags are biotin-labeled, so they can be purified using streptavidin-coated beads to retain biotin-coated beads. Modified Tags sequence. Take an equal volume of the digested product and add it to the resuspended streptavidin magnetic beads, shake well, shake gently at room temperature for 30 minutes, put the centrifuge tube on the magnetic stand, absorb for about 30 seconds, and discard the supernatant. Remove the centrifuge tube fro...

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Abstract

The invention discloses a flight mass spectrometry genotyping detection method. The method comprises the following steps: (1) designing three oligonucleotide probes for a SNP site, wherein the 3 / endsof a first probe and a second probe are respectively two allelic genes, i.e., allele-specific oligos (ASOs) of a pre-selected SNP site, (2) when the sequence is completely matched with the target sequence, preparing a connection product, (4) carrying out enzyme digestion on the PCR product by using restriction endonuclease, and purifying the product by using streptavidin-coated beads, and (5) transferring the purified product to a 384-well chip. SNP site typing is carried out by adopting MALDI-TOF mass spectrometry, and SNP typing is provided by adopting the method which is simple to operate,low in cost and high in resolution ratio.

Description

technical field [0001] The present invention relates to the use of time-of-flight mass spectrometry (matrix-assisted laser desorption-ionization-time-offlight (MALDI-TOF) mass spectrometry (MS)) analysis of single nucleotide polymorphisms (SNP, Single Nucleotide Polymorphisms) in biological nucleic acid molecules, In particular, it relates to mass spectrometry genotyping detection methods on the fly. Background technique [0002] SNP refers to a single nucleotide variation on the genome, including changes such as transitions, transversions, deletions, and insertions, and the frequency of occurrence in the population is not less than 1%. It has been linked to differences in susceptibility and drug response to many diseases. SNP has the characteristics of large number, wide distribution, and genetic stability. In the human genome, there is an average of one SNP for every 1,000 bases, and the total number of SNPs in the human genome is about 3 million. At the same time, there...

Claims

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Application Information

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IPC IPC(8): C12Q1/6872C12Q1/6858G01N27/62
CPCC12Q1/6872C12Q1/6858G01N27/628
Inventor 梁海咏
Owner 博淼生物科技(北京)有限公司
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