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Method for detecting target DNA sequence, gene chip adopting same and application of gene chip

A DNA sequence and gene chip technology, applied in the field of application of the method, can solve problems such as false negatives, amplification contamination, and difficulty in meeting detection requirements, so as to avoid false negative and false positive results, reduce nucleic acid pollution, and improve detection accuracy. rate effect

Inactive Publication Date: 2012-12-26
深圳博睿祥晖企业管理咨询有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Detection by PCR amplification can easily lead to false negative (PCR amplification failure) or false positive (PCR amplification contamination) and other results, thus affecting the accuracy and efficiency of gene sequence detection
In addition, the amount of target gene required for PCR amplification is large, and it is difficult to meet the detection requirements in the case of scarce samples.

Method used

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  • Method for detecting target DNA sequence, gene chip adopting same and application of gene chip
  • Method for detecting target DNA sequence, gene chip adopting same and application of gene chip
  • Method for detecting target DNA sequence, gene chip adopting same and application of gene chip

Examples

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Effect test

Embodiment 1

[0041] The present embodiment uses the gene chip of the present invention to detect Shigella, wherein:

[0042]The target DNA is from the genome of Shigella, which is double-stranded DNA and consists of SEQ ID NO: 1;

[0043] The RNA probe is artificially synthesized and consists of SEQ ID NO: 2. The sequence shown in the 1-22 positions of SEQ ID NO: 2 is the detection sequence, and the sequence shown in the 23-42 positions is the marker sequence; the detection of the RNA probe The sequence is identical to the sequence shown in positions 478-499 of SEQ ID NO: 1, and is complementary to the complementary strand of the sequence;

[0044] Marked DNA is artificially synthesized and consists of SEQ ID NO: 3, in which the 5' end is labeled with biotin;

[0045] The specific experimental method is as follows:

[0046] 1. Genomic DNA extraction;

[0047] Transfer 1.5ml of the culture into a centrifuge tube and centrifuge at 12,000×g for 3min;

[0048] Discard the supernatant, susp...

Embodiment 2

[0066] In this embodiment, the gene chip of the present invention is used to detect the DNA of the transgenic component Cry1A(b) of the transgenic corn Bt11, wherein:

[0067] The target DNA is from transgenic corn Bt11, which is double-stranded DNA and consists of SEQ ID NO: 5;

[0068] The RNA probe is artificially synthesized and consists of SEQ ID NO: 6. The sequence shown in the 1-21 positions of SEQ ID NO: 6 is the detection sequence, and the sequence shown in the 22-41 positions is the marker sequence; the detection of the RNA probe The sequence is identical to the sequence shown in the 214-234 positions of SEQ ID NO: 1, and is complementary to the complementary strand of the sequence;

[0069] The marked DNA is artificially synthesized and consists of SEQ ID NO: 7, in which the 5' end is labeled with biotin;

[0070] The specific experimental method is as follows:

[0071] 1. Genomic DNA extraction: (extract composition: Tris-HCl pH7.5, 150mM; NaCl 100mM; EDTA pH8.0,...

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Abstract

The invention discloses a method for detecting a target DNA sequence. The method comprises the following steps: 1) an RNA probe contacts with a target DNA to be annealed, wherein one end of the RNA probe is fixed on a substrate, the RNA probe contains a detection sequence close to the fixed end and a marking sequence close to the free end and the target DNA has a segment which is complimentary to the detection sequence in the RNA probe; 2) ribonuclease is used to hydrolyze the obtained DNA after annealling, wherein the RNA component in the RNA heteroduplex releases the target DNA and the marking sequence of the RNA probe; and 3) the free target DNA and marking sequence are removed through washing, the marking DNA with a marker is bound with the RNA probe of the detection sequence to perform sequence analysis on the target DNA, wherein the RNA probe is not hydrolyzed and is fixed on the substrate, and the marking DNA is complimentary to the marking sequence of the RNA probe. By adopting the method to detect the target DNA, the target gene does not require PCR amplification and the target DNA is directly detected, thus avoiding adopting the false negative and false positive results of the PCR amplification and increasing the detection accuracy rate.

Description

technical field [0001] The invention relates to a molecular biological detection method of a gene sequence, a gene chip for detecting a gene sequence using the method, and an application of the method. Background technique [0002] At present, the detection of genetic background-related genes (disease-causing genes, abnormal tumor genes), bacteria, viruses, and transgenic components of transgenic animals and plants is mainly through the design of primers and PCR amplification of the corresponding DNA fragments, and then through the subsequent detection platform. achieve the purpose of testing. Detection by PCR amplification can easily lead to false negative (PCR amplification failure) or false positive (PCR amplification contamination) and other results, thereby affecting the correctness and efficiency of gene sequence detection. In addition, the amount of target genes required for PCR amplification is large, and it is difficult to meet the detection requirements in the cas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 汪朝晖杨祥朱中伟胡春凌张国宁张伟红欧阳辉柴宝玲张珮颖
Owner 深圳博睿祥晖企业管理咨询有限公司