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Gene expression amount comparing analysis method

A technology of gene expression and analysis method, which is applied in the field of simultaneous determination of gene expression from various sources by bioluminescence method, can solve the problem that gene expression cannot be directly compared, and achieves the effects of low price, convenient operation and high sensitivity

Inactive Publication Date: 2003-02-26
周国华 +1
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0009] However, this method cannot be directly used to compare gene expression levels from various sources.

Method used

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  • Gene expression amount comparing analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1, the comparison of gene expression in two individuals:

[0033] This example describes the method of cutting cDNA fragments with restriction endonucleases and connecting them with DNA adapters, and then mixing equal amounts of cDNA from source-A and source-B for PCR amplification. The determination process is as follows: figure 2 shown. Taking the human P53 gene as an example, the mRNA was extracted using Gibico TRI20LLS-Reagent TM Standard method of the kit.

[0034] 1. Preparation of cDNA samples (cDNA samples from each source were prepared separately)

[0035] Take 1 μg source-A or source-B mRNA sample, add 0.5 μg Oligo(dT) 12.18 , 5.5 μl H 2 O, and mixed evenly, cDNA first-strand synthesis kit (Gibico Super Script TM Preamplification System for First Strand cDNA Synthesis), after incubation at 0°C for 10 min, add the pre-prepared mixture (4 μl of 5-fold buffer (I), 2 μl of 0.1M dithiothreitol, 1 μl of 10 mM dNTPs, 2 μl h 2 O and 1 μl of 200 U / μl r...

Embodiment 2

[0054] Example 2, using a 96-well reaction plate to simultaneously measure the expression levels of 96 different genes:

[0055] In this embodiment, a common 96-well reaction plate is mainly used to simultaneously measure the expression levels of 96 different genes between the disease group and the healthy group.

[0056] 1. Preparation of Assay Samples

[0057]According to the method of Example 1, extract the mRNA from source A and source B, and make double-stranded cDNA, then cut with restriction enzyme MobI, then add two kinds of DNA adapters representing source A and B respectively, and connect them with ligase Finally, in each well of the 96-well reaction plate, add 1 to 5 μl of the above-mentioned solution as the substrate for PCR amplification, and finally add MP-1, MP-2 and the corresponding GSP primers of each gene for PCR amplification. Amplification reaction, the 5' end of the GSP primer is modified with biotin, and the single-stranded DNA is prepared by the same m...

Embodiment 3

[0061] Example 3, the comparison of gene expression levels from six different sources:

[0062] Usually, microarray chips can only measure the expression of genes from two different sources. If one source is added, a different fluorescent marker needs to be added, which increases the cost of the assay, and usually does not use more than four excitations at the same time. Fluorescent markers with different wavelengths. Since the present invention uses a sequencing method to measure gene expression levels from different sources, and the determined base sequences represent different gene sources, increasing sample sources will not increase any measurement cost.

[0063] Since dATP is an analog of ATP, the background is high, which seriously interferes with the determination. Although it can be replaced by dATPαs, an analog of dATP, the cost of the assay increases. The present invention avoids the use of dATP. When using this method to compare the expression levels of genes from...

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Abstract

An analytical method for quantitative gene expression amount determination and comparison has great significance for disease-related gene screening and in clinical early diagnosis and development of specific medicine. The present invention utilizes quantitative characteristic of biological luminance process is sequencing to compare the gene expression amount of the same gene in different individuals or samples and to search disease-related gene. The specific steps includes: making mRNA with different sources by using a proper method and mixing in equal amount to form PCR substrate; PCR amplification with the primer corresponding to the gene of different sources and gene specific primer; and determining sequence by using bioluminescence analysis process with base variety to replace gene of different source.

Description

technical field [0001] The invention relates to a method for quantitatively measuring the content of each DNA fragment in a mixture of DNA fragments, specifically a method for simultaneously measuring the expression of each source gene by a bioluminescence method, which can be used for comparative analysis of the expression of each source gene. Background technique [0002] In recent years, with the advancement of molecular biology, the genome sequences of dozens of organisms have been determined, and the Human Genome Project is about to be completed. [1，2] . The first step of the genome project is to complete the structural analysis of the genome, and the second step is to analyze the gene function, that is, to understand the distribution of gene transcription product mRNA and the existence and distribution of mRNA translation product protein in cells and organs. and function. By comparing the mRNA expression of gene transcription products, it is possible to infer the fun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/6851C12Q1/6858
CPCC12Q1/6851C12Q1/6858
Inventor 周国华
Owner 周国华
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