CN1438325A - Gene-group DNA micro-array chip, its preparation and using method - Google Patents

Abstract

The invention relates to a genome DNA microarray chip and its preparation and use methods. Its features: (1) prepare genomic DNA samples from a group of cells of different biological individuals, and (2) fix these genomic DNA samples on different positions of the same carrier to form a genomic DNA microarray chip. Preparation method (1) Genomic DNA extracted from cells; (2) Genomic DNA is digested by restriction endonuclease to obtain the sum of DNA fragments as a genomic DNA sample, and the recognition site of the restriction endonuclease does not contain the The detected mutation or SNP site; (3) the genomic DNA sample is mixed with soluble polymer material to form a complex; (4) it is spotted on the carrier to make a chip. The method of use is to add DNA probes related to the detected gene loci, nucleotide monomers with markers and polymerase on the microarray chip, and use genomic DNA as a template to extend the probes and detect .

Description

Genomic DNA microarray chip and its preparation and use method

technical field

The present invention relates to a kind of genome DNA microarray chip and its preparation and using method. Microarray chips are formed on glass, silicon wafers, gels, plastics, rubber, ceramics and microspheres, etc.). The genomic DNA microarray chip can contain the genomic DNA of hundreds or thousands of different individuals, and can simultaneously analyze mutations and single nucleotide polymorphisms (SNPs) of a large number of samples to find biological functions or disease-related Gene polymorphic sites, discover important biological functions and disease-related genes. The genomic DNA microarray chip has the characteristics of convenience, practicality, reliability, high-throughput detection and the like.

technical background

With the progress of the genome project, discovering the biological functions of genes and disease-related genes has become a research hotspot in life sciences. By comparing the differences in the genomes of different individuals, analyzing the information of many gene mutations and single nucleotide polymorphism (SNP) sites in the genome is an important method for studying gene functions. Gene mutation is of great significance for elucidating the molecular mechanism of tumors and genetic diseases and early diagnosis of diseases. The single nucleotide polymorphism (SNP) helps to explain the phenotypic differences of individuals, the susceptibility of different groups and individuals to diseases, especially complex diseases, and the tolerance to various drugs and the environment. factor response. Therefore, finding and studying mutations and single nucleotide polymorphisms has become one of the research contents and goals of the post-human genome project.

The biological function research of gene mutation and polymorphism generally needs to analyze the genomic DNA of a large number (hundreds to tens of thousands) of individuals. Currently, mutation and SNP detection methods are based on established technologies such as DNA sequencing, restriction length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), and allelic Specific oligonucleotide hybridization (ASO), etc. Although these technologies can complete the detection of mutations or nucleotide polymorphisms, they must analyze and detect each individual's genomic DNA one by one, including: extraction of genomic DNA, PCR amplification, sequencing analysis, etc. Not only is it time-consuming and the workload is heavy, but it also consumes a lot of research expenses. Therefore, the development of rapid, efficient, and automated technologies that can be used for multiple genome polymorphisms and mutations detection is the goal that people are pursuing at present.

Gene chip is a high-tech developed rapidly in the field of life science in recent years. The so-called gene chip is a silicon chip, a glass slide, or a plastic sheet on which a large number of gene probes (oligonucleotide probes or cDNA probes) are fixed in a specific arrangement. Gene chip technology is the main means to efficiently obtain relevant biological information on a large scale. At present, the application fields of this technology mainly include gene expression profile analysis, new gene discovery, gene mutation and polymorphism analysis, genome library mapping, disease diagnosis and prediction, drug screening, gene sequencing, etc. In the early 1990s, the research and development of various biochips began mainly in the United States. In less than ten years, chip technology has developed rapidly and reached a peak of development. However, in the current chip detection, the pre-treatment of the sample is extremely cumbersome (such as DNA extraction, polymerase chain reaction amplification, fluorescent labeling, etc.). Gene chip technology has also received people's attention in the research and detection of gene polymorphisms. For example, the American Affymetrix company and others have developed a gene chip technology that can be used for the detection of thousands of single nucleotide polymorphisms (SNPs) in the genome, and has become the leader in the biochip industry. However, the current biochip technology cannot simultaneously analyze the genomes of a large number of different individuals, but can only perform high-throughput analysis and detection of multiple gene polymorphic sites in a certain genome. Genome research is very important.

Contents of the invention

The present invention provides a genome DNA microarray chip and its preparation and use methods aiming at the above disadvantages. The chip is a group of genomic DNA samples extracted from the cells of different biological individuals. After appropriate digestion, the total DNA fragments of each individual are respectively fixed on different positions of the same carrier to form a chip containing multiple genomic DNAs. microarray chip.

Technical solution: The core of the present invention is how to achieve high-efficiency immobilization of genomic DNA and sequence detection of the immobilized genomic DNA. The present invention proposes to digest genomic DNA with appropriate restriction endonucleases to form shorter fragments, then mix with soluble high molecular polymers (such as polylysine, etc.), and cross-link through ultraviolet cross-linking equipment to form Molecular brush. Finally, the cross-linked genomic DNA is immobilized on a specially treated (such as aldehyde) solid substrate (such as glass, silicon wafer, gel, plastic, rubber, ceramics, and microspheres, etc.) to form a microarray through a spotting device. chip.

A genomic DNA microarray chip, characterized in that: (1) Genomic DNA samples are respectively prepared from a group of cells of different biological individuals; (2) These genomic DNA samples are respectively fixed on different positions of the same carrier to form Genomic DNA Microarray Chip.

A method for preparing a genome DNA microarray chip. It is characterized in that (1) the genomic DNA extracted from the cells; (2) the genomic DNA is digested by a restriction endonuclease to obtain the sum of DNA fragments as a genomic DNA sample, and the recognition site of the restriction endonuclease does not contain The mutation or single nucleotide polymorphism SNP site to be detected; (3) the genomic DNA sample is mixed with soluble polymer material to form a DNA complex; (4) the complex is made on the carrier Genomic DNA Microarray Chip. The soluble polymer material adopts polylysine, polyvinyl alcohol, polyethylene fluoride imine, polyallyl imine, etc. The carrier of the fixed genome sample is a solid substrate, using glass, silicon wafer, gel, plastic, rubber, ceramics. The carrier used in the preparation of the microarray chip is modified by special groups of aldehyde, sulfhydryl and amino groups.

Genomic DNA microarray chip is used by adding DNA probes related to the detected gene loci on the microarray chip, connecting or extending, so as to realize the difference analysis of a large number of individual samples in the genome of a certain species. The root is characterized in that T4 DNA ligase is used for connection, and DNA polymerase I large fragment (Klenow fragment) or Taq DNA polymerase is used for in-sheet extension. It is characterized in that fluorescent groups or biotin are used as markers incorporated in sheet extension. It is characterized in that fluorescence detection, chemiluminescence detection, enzymatic chemical method detection and colloidal gold detection are used for detection of chip use results.

Beneficial effects: the invention is a DNA chip with simple preparation, reliable results and convenient use and its preparation and use method. The genome DNA microarray chip can be used to analyze and detect certain sequence differences of genome DNA of tens of thousands of individuals in experiments, thereby realizing high efficiency and low cost of gene polymorphism detection. Through high molecular polymers (such as polylysine, etc.), genomic DNA can be fixed on solid substrates (such as glass, silicon wafers, gels, plastics, rubber, ceramics and microspheres) that have been specially treated (such as aldehyde groups). etc.) on a microarray chip. Finally, mutations and single nucleotide polymorphisms were analyzed using on-sheet extension technology. High-molecular polymers (such as polylysine, etc.) have a polymerization effect on genomic DNA. The method can detect mutations and single nucleotide polymorphisms without polymerase chain reaction amplification, which greatly simplifies the pre-treatment process of samples. Moreover, the genomic DNA microarray chip prepared by this method can contain the genomic DNA of hundreds of thousands of different biological individuals, and can simultaneously analyze mutations and single nucleotide polymorphisms (SNPs) of a large number of samples to find biologically functional ones. or disease-associated genes. This function cannot be achieved by any current technology, including DNA chip technology. Therefore, the genome DNA chip can be widely used in the detection of mutation and single nucleotide polymorphism (SNP).

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings.

Fig. 1 is a flowchart of the preparation of the genomic DNA microarray chip of the present invention.

Fig. 2 is a flow chart of on-sheet extension in the method of use of the present invention.

In the figure 1, genomic DNA; 2, the total DNA short fragment after endonuclease digestion; 3, high molecular polymer (such as polylysine, etc.); 4, carrier; 5, mutation or SNP site; 6. Oligonucleotide probes; 7. Fluorescence or biotin labels.

Detailed ways

With reference to accompanying drawing 1,2, a kind of genome DNA microarray chip is characterized in that: (1) prepare its genome DNA sample respectively from the cell of a group of different biological individuals, (2) fix these genome DNA sample respectively on the same Different positions of the carrier 4 constitute a genomic DNA microarray chip.

Referring to accompanying drawing 1, a kind of genomic DNA microarray chip preparation method. The details are as follows: 1. Extract genomic DNA 1; 2. Digest genomic DNA with appropriate restriction enzymes 2; 3. Combine the digested total DNA fragments with polymers (such as polylysine) 3. Mix and cross-link with an ultraviolet cross-linking instrument to form a complex; 4. Use a spotting device to immobilize the cross-linked genomic DNA complex on a solid phase carrier 4 that has been specially treated (such as an aldehyde group) on a solid substrate, (such as Glass off, silicon wafers, gels, plastics, rubber, ceramics and microspheres, etc.) constitute microarray chips. 5. In a humid environment, keep it airtight at room temperature overnight. Then, use 0.1% sodium dodecyl sulfate (SDS) to remove unbound cross-linked complexes, then thoroughly wash with double distilled water, and dry to prepare a genomic DNA microarray chip.

With reference to accompanying drawing 2 a kind of genomic DNA microarray chip usage method, specifically as follows:

1. According to the mutation or SNP site 5 to be detected, synthesize a complementary oligonucleotide probe 6, and design the mutation or SNP site at the 3' end; 2. Combine the oligonucleotide probe with the above micro Hybridize the array chip for several hours; 3. Remove unbound oligonucleotide probes, then thoroughly wash with double distilled water, and dry before use; 4. Use DNA polymerase I large fragment (Klenow fragment) or Taq DNA to polymerize Enzymes do in-sheet extension, incorporating fluorescent or biotin labels during extension7. Samples that contain mutations or SNPs will incorporate the marker, and vice versa. Detection by fluorescence or other methods can determine which samples on the chip contain relevant mutations or SNP sites.

Claims (9)

1, a kind of genomic dna micro-array chip, it is characterized in that: (1) prepares its genome DNA sample respectively from one group of different biology individual cell, (2) respectively these genome DNA samples are fixed on the different positions of identical carrier, constitute the genomic dna micro-array chip.

2, the described a kind of genomic dna process for preparing microarray chip of claim 1 is characterized in that the genomic dna that (1) extracts from cell; (2) genomic dna obtains the dna fragmentation summation as genome DNA sample after by digestion with restriction enzyme, and does not comprise sudden change to be measured or single nucleotide polymorphism SNP site in the recognition site of restriction enzyme; (3) macromolecule polymer material that mixes solubility in its genome DNA sample forms the DNA mixture; (4) compound object point is made the genomic dna micro-array chip on carrier.

3, a kind of genomic dna micro-array chip according to claim 2 and preparation method thereof is characterized in that the macromolecule polymer material of solubility adopts poly-lysine, poly-second fluorine imines, polyene propyl group imines.

4, according to claim 1 and 2 described a kind of genomic dna micro-array chips and preparation method thereof, the carrier that it is characterized in that preparing micro-array chip is a solid substrate, adopts glass, silicon chip, gel, plastics, rubber, pottery, microballoon.

5, a kind of genomic dna micro-array chip according to claim 1 and 2 and preparation method thereof, it is characterized in that preparing the used carrier of micro-array chip is to modify through aldehyde radical, sulfydryl, amino specific groups.

6, the described a kind of genomic dna micro-array chip of claim 1, its using method is for adding relevant dna probe with detected gene locus, connecting or extend by chemical reaction on micro-array chip, realization is to a large amount of individual specimen difference analysis of a certain species gene group.

7, the using method of a kind of genomic dna micro-array chip according to claim 5, what it is characterized in that ligation adopts is the T4DNA ligase enzyme, extending what adopted at sheet is big fragment of dna polymerase i (Klenow fragment) or TaqDNA polysaccharase.

8, a kind of genomic dna micro-array chip using method according to claim 5 is characterized in that extending the marker that is mixed at sheet adopts fluorophor or vitamin H.

9, a kind of genomic dna micro-array chip using method according to claim 5 is characterized in that chip uses the result to detect and adopts fluoroscopic examination, chemiluminescence detection, enzyme chemical method detection, Radioactive colloidal gold to detect.

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