Pyrophosphoric acid sequencing kit for detecting CYP3A4*4 genotyping

A technology of pyrosequencing and genotyping, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effects of high sensitivity, simple steps, and simple sample processing

Active Publication Date: 2012-10-10
韩勇
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no product on the market that uses pyrophosphate technology to detect CYP3A4*4 gene polymorphisms

Method used

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  • Pyrophosphoric acid sequencing kit for detecting CYP3A4*4 genotyping
  • Pyrophosphoric acid sequencing kit for detecting CYP3A4*4 genotyping
  • Pyrophosphoric acid sequencing kit for detecting CYP3A4*4 genotyping

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the preparation of kit

[0032] 1. Design and Synthesis of Primers and Probes

[0033] Studies have shown that the current CYP3A4*4 gene mutation is mainly concentrated in base 13871, select specific mutation sites (Table 1), and use PyroMark Assay Design2.0 software to design and determine primers (Table 2). The most important thing that affects the accuracy of the kit is the primers, including amplification primers and sequencing primers. In the early stage of design, we designed multiple sets of primers for comparison (see Figure 5 ); wherein the amplification primer and the sequencing primer were first purified by PAGE, and then purified by HPLC, wherein the 5' end of the forward amplification primer shown in SEQ ID NO.1 was labeled with biotin.

[0034] Table 1 Mutation site and type

[0035]

[0036] Table 2 Sequences of specific amplification primers and sequencing primers

[0037]

[0038] 2. Control substance selection

[0039] A syn...

Embodiment 2

[0043] Embodiment 2: the use of kit

[0044] 1. sample testing

[0045] 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, UNG enzyme, and Taq DNA polymerase, aliquot the system, add sample DNA, and a blank control as templates to form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0046] The main components of the system are shown in Table 4:

[0047] Table 4 The main components of the system (the amount of UNG enzyme is relatively small and negligible)

[0048]

[0049] The system reaction procedure is as follows:

[0050] Table 5 PCR reaction program

[0051]

[0052] After the amplification is completed, check the PCR results on agarose gel to proceed to the next step.

[0053] 2. Pyrosequencing

[0054] The sequencing operation was performed accordi...

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Abstract

The invention discloses a kit for detecting CYP3A4*4 genetyping, belongs to the testing field of in-vitro nucleic acid, and particularly relates to a pyrophosphoric kit for detecting CYP3A4*4 genetyping. The pyrophosphoric kit for detecting CYP3A4*4 genetyping comprises uracil DNA (deoxyribonucleic acid)glycosylase, Taq polymerase, a PCR (Polymerase Chain Reaction) amplimer and a sequencing primer. The pyrophosphoric kit provided by the invention has high sensitivity and good specificity; and after simple treatment, a PCR product can be subjected to sequencing by utilizing phosphoric acid sequencing instrument, the operation is simple and convenient, the reaction time is short, the sequencing operation by adopting the pyrophosphoric kit provided by the invention has higher sensitivity when being compared with a gold-standard CE (capillary electrophoresis) sequencing operation, thus the pyrophosphoric kit is particularly suitable for mutation analysis.

Description

technical field [0001] The invention relates to the field of in vitro nucleic acid detection, in particular to a pyrosequencing kit for detecting CYP3A4*4 genotyping. Background technique [0002] The full name of CYP3A4 gene is called cytochrome P450 isoenzyme 3A4. Cytochrome P450 (Cytochrome P450, CYP450) is a group of proteases containing heme, also known as mixed function monooxygenase system. It mainly exists in the human liver and plays an important role in the disposal of exogenous substances and the metabolism of endogenous substances. It has been found that the CYP450s closely related to drug metabolism mainly include CYP1-4, among which the CYP3A4 subfamily is the most important, accounting for 30% of the total CYP450 in human liver. host. [0003] There are many types of drug metabolism that CYP3A4 participates in. In clinical practice, more than 50% of drug metabolism is through CYP3A4, mainly including: docetaxel, paclitaxel, etoposide, irinotecan (irino -tec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 韩勇
Owner 韩勇
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