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Method for digitally detecting micro-mutation by using micro-emulsion clone amplified bound water gel microsphere chip

A technology of microemulsion and hydrogel, applied in the field of medical biology, to solve the problem of low detection throughput and increase the positive detection rate

Inactive Publication Date: 2010-06-16
HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, there is currently no method to combine microemulsion clonal expansion with hydrogel microsphere chip digital detection to improve the sensitivity of micromutation detection

Method used

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  • Method for digitally detecting micro-mutation by using micro-emulsion clone amplified bound water gel microsphere chip
  • Method for digitally detecting micro-mutation by using micro-emulsion clone amplified bound water gel microsphere chip
  • Method for digitally detecting micro-mutation by using micro-emulsion clone amplified bound water gel microsphere chip

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Experimental program
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Embodiment 1

[0045] combine Figure 1~4 , the specific description is as follows:

[0046] 1. Preparation of fragmented DNA template

[0047] Firstly, the genomic DNA of the sample was extracted, extracted according to the phenol / chloroform method, and then dissolved in TE buffer solution, and its concentration and purity were determined by ultraviolet spectrophotometry, and the appropriate final concentration was adjusted with TE buffer solution. Using the extracted DNA molecule as a template, add MLPA probes for ligation (design corresponding ligation probes for each target gene), use three probes for each mutation site, one left probe and two sites Specific right probe. like figure 2 As shown, the left probe consists of two parts: one is the same sequence as the public primer (such as forward primer) bound to the microsphere, and the other is complementary to the gene of appropriate length on the left side of the template mutation site (excluding the mutation site) The two right pr...

Embodiment 2

[0071] Embodiment 2: Microemulsion PCR realizes the conditional control of single molecule amplification

[0072] 1. Control each microcapsule to contain at most one molecule.

[0073] The realization of single-molecule amplification is based on the principle of dilution, that is, through the dilution of template molecules, a certain volume of solution finally contains at most one molecular template. In the process of preparing the microemulsion system, the size of the microcapsule volume is mainly affected by the following conditions: the ratio of the oil phase to the water phase, the ratio of the concentration of various ions in the water phase, and the stirring bar when forming the microemulsion. Size (commercially available stirring bars are usually used in the laboratory, and the general length does not exceed 13mm) and stirring speed. The inventor has determined the following microemulsion preparation process by experiment:

[0074] The microemulsion PCR amplification ...

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Abstract

The invention belongs to the field of medical biological technique and discloses a method for digitally detecting micro-mutation by using a micro-emulsion clone amplified bound water gel microsphere chip. The method detects the micro-mutation by combining the microsphere mediated micro-emulsion clone amplifying technique with the water gel chip fixing microsphere fluorescence detection and digitization analysis techniques. The method promotes the positive relevance ratio of micro-mutation, has low cost, high sensitivity and fine specificity and is applied to quantitatively measuring micro-mutation of various low abundance tumor relative genes.

Description

technical field [0001] The method belongs to the field of medical biotechnology, and relates to a microemulsion clonal amplification combined with a hydrogel microsphere chip digital detection method for micromutations. Background technique [0002] In recent years, micromutation detection has played an increasingly important role in the early diagnosis of diseases and has become an important means of disease diagnosis. In the early stage of the disease, the number of mutants in related genes is far less than that of normal DNA molecules, making it difficult to detect micromutants. Among some existing techniques for detecting micromutations, DNA sequencing is a relatively classic method, but this method cannot detect samples with less than 20% mutations. However, usually only a very small part of the DNA has mutations in the early stage of cancer. In order to detect this very few mutations, many methods have been developed. Currently, the methods used for micro-mutation det...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 周国华黄欢乞宗泰
Owner HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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