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Gene chip without nucleic acid marking and application thereof

A gene chip and nucleic acid labeling technology, which is applied in the field of gene chips without nucleic acid labeling, can solve problems such as complicated operation, and achieve the effects of simple operation, high throughput and low cost.

Inactive Publication Date: 2007-03-14
SHANGHAI BIOCHIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although nucleic acid amplification or cascade amplification can improve the fluorescent signal to a certain extent, these techniques cannot fundamentally solve these problems, and the operation is relatively complicated

Method used

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  • Gene chip without nucleic acid marking and application thereof
  • Gene chip without nucleic acid marking and application thereof
  • Gene chip without nucleic acid marking and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 probe design and preparation of gene chip

[0034]1. Probe design: The design criteria of the probe are as follows: 1) The sequence flanking the selected gene or SNP site is blasted to exclude gene sequences or SNPs with poor sequence specificity, so as not to cause non-specific hybridization; 2) SNP site It is in the RNA sequence and located in the middle part of the probe; 3) The annealing temperature of the probe is between 48-60 ° C, and the annealing temperature of the DNA part at both ends is at least 15 ° C lower than that of the entire probe, so that the probe after enzyme digestion The fragment is separated from the target DNA sequence; 4) The self-complementary sequence of the probe does not exceed 3 base pairs, and the dimer should not be formed between the probes, especially the RNA sequence, so as to avoid high fluorescent background; 5) Perform SNP Blast, Make sure that the probe sequence is outside the target SNP site, and there are no other ...

Embodiment 2

[0041] 1. Target gene amplification

[0042] (1) Primer sequence:

[0043] Primer 1F 5'-gtcgcaacctggtgcctataa-3' (SEQ ID NO: 1);

[0044] Primer 1R 5'-tgctataagccagctgagagattt-3' (SEQ ID NO: 2);

[0045] Primer 2F 5'-aagccaaggctatgacattct-3' (SEQ ID NO: 3);

[0046] Primer 2R 5'-aattcccggagaacttgtgct-3' (SEQ ID NO: 4).

[0047] (2) Probe sequence: CY3 is a fluorescent group, ECLIPSE is a quencher group, and the uppercase base sequence in the middle bracket is the RNA sequence

[0048] rs13431727-A

[0049] 5'-NH2-C18-ggcctt(Cy3)(ATAG)g(ECLIPSE)ggaattta-3' (SEQ ID NO: 5);

[0050] rs13431727-T

[0051] 5'-NH2-C18-ggcctta(Cy3)(TTGG)g(ECLIPSE)gaattta-3'(SEQ ID NO:6);

[0052] rs13431727-C

[0053] 5'-NH2--C18-ggcctta(Cy3)(TCGG)g(ECLIPSE)gaattta-3'(SEQ ID NO:7);

[0054] rs1146808-A

[0055] 5'-NH2-C18-ggaaatgt(Cy3)(TATC)a(ECLIPSE)aattatctg-3' (SEQ ID NO: 8);

[0056] rs1146808-C

[0057] 5'-NH2--C18-ggaaatgt(Cy3)(TCTC)a(ECLIPSE)aattatct-3'(SEQ ID NO:9);

[0058] rs11...

Embodiment 3

[0076] 1. Asymmetric PCR

[0077] Utilize the primer and probe sequence of embodiment 2 to carry out asymmetric PCR amplification, carry out in 60 μ l reaction system, reaction system is 0.3mM dNTP, 10mM Tris-HCl, 50mM KCl, 2mM MgCl 2 , 20% Q solution (Qiagen), upstream primer concentration 0.16 μM, upstream primer concentration 0.008 μM, genomic DNA 60 ng, Taq enzyme 0.6 U (Takara). Apply Touch-down PCR reaction procedure [Don RH, Cox PT, WainwrightBJ, Baker K, Mattick JS. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991, 19: 4008]: Denaturation at 94°C for 5 min; 94 Denaturation at ℃ for 40s, annealing at 64℃ for 1min, each cycle lowered by 0.5℃, extension at 72℃ for 50s, a total of 10 cycles; then denaturation at 94℃ for 40s, annealing at 59℃ for 40s, extension at 72℃ for 40s, a total of 30 cycles; finally 72℃ Extend for 5 minutes. The PCR product was purified with QIAquick PCR Purification Kit (Qiagen, Cat. No. 28106), and...

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Abstract

The present invention discloses one kind of gene chip without need of nucleic acid label, and the gene chip includes DNA-RNA-DNA probe fixed on a solid carrier. The present invention also discloses the detection method with gene chip and the application of the gene chip in clinical detection and diagnosis. The gene chip of the present invention has high efficiency signal amplification effect, easy direct detection of genome DNA and greatly raised low expression gene detecting rate.

Description

technical field [0001] The invention relates to a gene chip, in particular to a gene chip without nucleic acid labeling; moreover, the invention also relates to a detection method using the gene chip. Background technique [0002] The gene chip is the most valuable scientific research achievement brought about by the Human Genome Project, which integrates the latest technologies in various disciplines such as life sciences, chemistry, microelectronics technology, computer science, statistics and life informatics. With the development of micro-processing technology, the highest density of high-density oligonucleotide chips can reach millions of probes. Therefore, the throughput level of the gene chip is not restricted by the chip technology itself, but by the processing of samples and the design of the gene chip. Method limitations. In the human genome, there are more than 10 million common single nucleotide polymorphism (single nucleotide polymorphism, SNP) sites, accountin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 盛海辉肖华胜
Owner SHANGHAI BIOCHIP