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ApoE kit, primers and use thereof

A kit, the technology of use, applied in the field of biology

Inactive Publication Date: 2016-10-12
XIAMEN RENRUI BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, current methods for determining whether two predetermined loci in a nucleic acid sample have known mutations still need to be improved

Method used

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  • ApoE kit, primers and use thereof
  • ApoE kit, primers and use thereof
  • ApoE kit, primers and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0324] In the example, as figure 1As shown, the primer p112T only matches the nucleic acid sequence of SNP rs429358 being T, the primer p112C only matches the nucleic acid sequence of SNP rs429358 being C, the primer p158T only matches the nucleic acid sequence of SNP rs7412 being T, and the primer p158C only matches the nucleic acid sequence of SNP rs7412 being C nucleic acid sequence match. Therefore, the PCR system containing the primer pair p112T / p158T can only amplify the ApoE allele (ie ε2) in which SNP rs429358 is T and SNP rs7412 is T; the PCR system containing the primer pair p112T / p158C can only amplify SNP rs429358 The ApoE allele with T and SNP rs7412 as C (namely ε3); the PCR system containing the primer pair p112C / p158C can only amplify the ApoE allele with SNP rs429358 as C and SNP rs7412 as C (ie ε4). Therefore, using the above three primer pairs, different alleles of ApoE can be specifically amplified, and the type of human ApoE alleles in the DNA sample can ...

Embodiment 2

[0326] In the example, as figure 2 As shown, the two sections of the DNA molecular probe 3 detached from the fluorophore (F) are coupled with a fluorophore and a quenching group respectively. At this time, since the quenching group can absorb the excitation energy of the fluorophore, therefore, Only background fluorescence signal can be detected in the system. The DNA molecular probe 3 detached from the fluorophore (F) can specifically complement and match the antisense strand 1 of the DNA template of the ApoE gene, and the matching sequence is located between the DNA sequences corresponding to the forward amplification primer 2 and the reverse amplification primer 5 , after the PCR expansion reaction starts, forward amplification primer 2 and the DNA molecule probe 3 that fluorophore (F) breaks off will combine with ApoE gene DNA template antisense strand 1, and reverse amplification primer 5 will combine with ApoE gene The sense strand 4 of the DNA template binds, and unde...

Embodiment 3

[0328] In the example, as image 3 As shown, DNA polymerase 6 starts from the forward amplification primer 2 or the reverse amplification primer 5 during the PCR reaction, using the antisense strand 1 of the ApoE gene DNA template or the sense strand 4 of the ApoE gene DNA template as a template, and starts The extension of DNA, when DNA polymerase 6 encounters the DNA molecular probe 3 that is bound to the fluorescent group (F) on the antisense strand 1 of the ApoE gene DNA template in the process of mediating DNA extension, DNA polymerase 6 will The 5' end of the DNA molecular probe 3, which is detached from the fluorescent group (F) by its exonuclease activity, cuts off the nucleotides on the probe one by one, causing the fluorophore to detach from the probe and combine with the quenching group After separation, the fluorescent signal excited by the fluorophore can be detected at this time, and thus it can be determined that the detection sample contains the ApoE allele typ...

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Abstract

The invention discloses an ApoE kit, primers and use thereof, and a method for determining whether two SNP loci have known mutations. Specifically, the method includes: using a first primer and a second primer to conduct PCR amplification on a nucleic acid sample, and adding a nucleic acid probe into the PCR amplification system, with the nucleic acid probe corresponding to at least a part of corresponding nucleic acid sequence between two predetermined loci; and detecting luminescence of the reaction system so as to determine whether known mutation exists at predetermined loci of the nucleic acid sample. The near 3' end of the first primer contains a basic group corresponding to one of the known mutations of the two predetermined loci, the near 3' end of the second primer contains a basic group corresponding to the other known mutation, and the nucleic acid probe contains a luminescent group formed at the 5' end of the nucleic acid probe and an adjustment group formed at the 3' end of the nucleic acid probe, and the adjustment group can adjust luminescence of the luminescent group. The method can effectively determine whether known mutations exist at the two predetermined loci of the nucleic acid sample.

Description

technical field [0001] The invention relates to an ApoE kit, primers and application thereof, and the determination of whether two SNP sites have known mutations. It specifically relates to a method, kit and primers for determining whether two predetermined positions of a nucleic acid sample have known mutations, and belongs to the field of biology. Background technique [0002] ApoE is an apolipoprotein, and its genetic polymorphism is mainly controlled by two SNP sites rs429358 and rs7412. These two SNP sites determine the three allele types of ApoE through different nucleic acid combinations: ε2( rs429358 is T, rs7412 is T), ε3 (rs429358 is T, rs7412 is C), ε4 (rs429358 is C, rs7412 is C), these three types of alleles encode three ApoE protein subtypes, namely ApoE2 ( The 112th and 158th positions of its protein sequence are both cysteine), ApoE3 (the 112th position of its protein sequence is cysteine, and the 158th position is arginine), ApoE4 (the 112th and 158th posit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/118C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 许华曦卜国军张含
Owner XIAMEN RENRUI BIOMEDICAL TECH CO LTD
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