AKT1 gene mutation detection specificity primer and liquid chip thereof

A detection solution and specific technology, applied in the field of molecular biology, can solve problems such as inability to meet practical applications and cannot be used, and achieve the effects of improving detection accuracy, consistent detection effect, and good detection specificity

Active Publication Date: 2013-03-27
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a restriction endonuclease. Amplify the product and observe the size of the fragment by electrophoresis. This method is used to detect gene mutations with altered restriction sites, and can directly determine the genotype. However, this method cannot be used for the detection of gene mutations without new restriction sites.
Again, the above methods all have limitations in detection throughput, and can only detect one mutation type at a time, which cannot meet the needs of practical applications.

Method used

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  • AKT1 gene mutation detection specificity primer and liquid chip thereof
  • AKT1 gene mutation detection specificity primer and liquid chip thereof
  • AKT1 gene mutation detection specificity primer and liquid chip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0022] Embodiment 1 AKT1 gene mutation detection liquid chip mainly includes:

[0023] 1. ASPE Primers

[0024] Specific primer sequences were designed for the wild-type and mutant types of two common genotypes E17K and E49K of the AKT1 gene, respectively. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0025] ASPE primer sequence (tag sequence+specific primer sequence) of table 1 AKT1 gene

[0026]

[0027] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0028] 2. Microspheres coated with anti-tag sequences

[0029] ...

Embodiment 3

[0087] The detection of the AKT1 gene mutation site by the liquid chip of embodiment 3 different ASPE primers

[0088] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)

[0089] Taking the AKT1 gene E17K site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of E17K, and the tag sequence of the 5' end of the ASPE primer was selected from SEQ ID NO.1 - SEQ ID NO.4, correspondingly, the anti-tag sequence coated on the microspheres and complementary to the corresponding tag sequence is selected from SEQ ID NO.9-SEQ ID NO.12. The specific design is shown in the following table (Table 6). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0090] Table 6 Design of liquid phase chip preparation

[0091]

[0092] 2. ...

Embodiment 4A

[0098] The selection of embodiment 4AKT1 gene mutation detection specific primer sequence

[0099] 1. Design of liquid-phase chip preparation (selection of wild-type and mutant-specific primer sequences)

[0100] Taking the AKT1 gene E49K mutation site detection liquid chip as an example, using the forward or reverse complementary sequence of the target sequence where the mutation site is located as a template, the specific 3' end of the ASPE primer was designed for the wild type and mutant type of E49K, respectively. Primer sequences, including preferred specific primer sequences and 2 alternative specific primer sequences in Example 1 of the present invention, as shown in Table 8. in, Inner bases are mutation sites.

[0101] Table 8 specific primer sequence

[0102]

[0103] Taking the AKT1 gene E49K mutation site detection liquid chip as an example, different specific primer sequences were selected for E49K, while the tag sequence at the 5' end of the ASPE primer was...

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Abstract

The invention discloses an AKT1 gene mutation detection specificity primer and a liquid chip thereof. The liquid chip mainly comprises: an ASPE primer composed of a 5'-terminal tag sequence and 3'-terminal specificity primer sequences focused on target gene mutation sites, wherein the specificity primer sequences comprise SEQ ID NO.5 and SEQ ID NO.6 focused on an E17K site, and / or SEQ ID NO.7 and SEQ ID NO.8 focused on an E49K site; a microsphere coated by an anti-tag sequence; and an amplimer. The consistency between the detection result of the detection liquid chip provided by the invention and the detection result of a sequencing method is high to 100%, and the wild-type and mutant parallel detection of a plurality of mutation sites is realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for detecting AKT1 gene mutation and a liquid phase chip. Background technique [0002] AKT is the human homologous gene of the viral oncogene v-akt, which encodes serine threonine protein kinase, plays an important role in PI3K-related signaling pathways, affects cell survival, proliferation and invasion, and participates in cell apoptosis and glucose cellular processes including metabolism. [0003] Current studies have shown that in lung cancer and breast cancer, AKT gene activation is significantly associated with chemotherapeutic drug resistance, and elevated AKT1 gene expression is considered to be the cause of lung cancer cell resistance to cisplatin; while in breast cancer cell studies, AKT Activation significantly enhances its resistance to chemotherapeutic drugs. Furthermore, persistent activation of the AKT gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森吴诗扬
Owner SUREXAM BIO TECH
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