Pyrosequencing primer pair for qualitatively detecting ApoE genetic typing and kit

A technology of pyrosequencing and genotyping, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., and can solve the problems of low sensitivity of ApoE genotyping, long detection cycle, cumbersome operation, etc. problems, to achieve the effects of fast sequencing speed, short reaction time, and simple sample processing

Inactive Publication Date: 2016-12-21
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the technical problems of low sensitivity, long detection period, cumbersome operation and high cost in the detection of ApoE genotyping by the above method, the present invention provides a method with high sensitivity, strong specificity, short detection period, simple operation and Pyrosequencing primer pair and kit for qualitative detection of ApoE genotyping that effectively meet clinical testing requirements

Method used

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  • Pyrosequencing primer pair for qualitatively detecting ApoE genetic typing and kit
  • Pyrosequencing primer pair for qualitatively detecting ApoE genetic typing and kit
  • Pyrosequencing primer pair for qualitatively detecting ApoE genetic typing and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: the preparation of kit

[0073] 1. Design and synthesis of primers and probes

[0074] For the polymorphic sites Cys112Arg and Arg158Cys alleles of the human ApoE gene, select specific mutation sites, and use the PyroMark Assay Design2.0 software to design primers; the amplification primers and sequencing primers are first purified by PAGE, and then HPLC Purified, wherein the 5' ends of SEQ ID NO.2 and SEQ ID NO.5 were biotin-labeled.

[0075] Table 1. Mutation site and type:

[0076] Mutation

[0077] The amplified sequence is shown in Table 2:

[0078] Table 2. Specific amplification primers and primer sequences

[0079]

[0080]

[0081] 2. Selection of reference substance

[0082] A synthetic oligonucleotide chain TAYGGTTTGCA control oligo was used as the quality control product; DNase / RNase-Free water was used as the blank control product.

[0083] 3. Composition of PCR reaction solution

[0084] Table 3. Composition of PCR react...

Embodiment 2

[0089] Embodiment 2: the use of kit

[0090] 1. Sample testing

[0091] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, uracil DNA glycosylase, and Taq DNA polymerase, aliquot the system, add sample DNA, blank control substance or positive control substance as templates to form PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0092] The main components of the ApoE Cys112Arg and ApoE Arg158Cys systems are as follows:

[0093] Table 5. The main components of the ApoE Cys112Arg system

[0094]

[0095] Table 6. Main components of ApoE Arg158Cys system

[0096]

[0097] The system reaction procedure is as follows:

[0098] Table 7. PCR reaction program

[0099]

[0100]

[0101] After the amplification is completed, check the PCR results on agarose gel to...

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Abstract

The invention relates to a pyrosequencing primer pair for qualitatively detecting ApoE genetic typing. The primer pair comprises a forward amplification primer, a reverse amplification primer and a sequencing primer, wherein the 5' end of the reverse amplification primer respectively performs biotin labeling. The invention relates to a pyrosequencing kit for qualitatively detecting the ApoE genetic typing. The kit comprises an amplification primer, PCR reaction liquid 1, PCR reaction liquid 2, a sequencing primer, uracil DNA glycosylase and Taq polymerase. The kit has the advantages of accurate detection results, high specificity, short detection period, high simplicity in operation, and capability of effectively meeting clinical examination requirements; in addition, the kit further has the advantages that the reaction process can be monitored in real time, the reaction time is short, pyrosequencing tester sequencing and high-flux sample detection can be performed by only simply treating PCR products, and the sensitivity is higher than that of a gold standard method namely a capillary electrophoresis sequencing method.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a pair of pyrosequencing primers and a kit for qualitative detection of ApoE genotyping. Background technique [0002] Apolipoprotein E (ApolipoproteinE, ApoE) is one of the important apolipoproteins in plasma. Its gene is located on the long arm of the 19th pair of chromosomes. It is 37kb long and contains 4 exons and 3 introns. ApoE has three isoforms, namely ε2, ε3 and ε4. The exchange of two amino acid residues, arginine (Arg) and cysteine ​​(Cys) at positions 112 and 158 of the amino acid sequence of ApoE determines the type of isomer. ApoEε4 is Arg at these two positions; ε2 is Cys; the one with Cys at position 112 and Arg at position 158 is the isomer of ApoEε3. In the natural population, the distribution of gene frequency ε3 is the highest, and the phenotype distribution of ApoEε3 / 3 is about 70%. There can be six different phenotypes in the popu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/156C12Q2565/301C12Q2535/122
Inventor 滕祥云胡玉婷
Owner CHANGSHA 3G BIOTECH
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