Sequencing primer for qualitative detection of genetic typing of uridinediphosphoglucuronate glucuronosyltransferase 1A1 and kit thereof

A uridine diphosphate glucose, qualitative detection technology, applied in the field of in vitro nucleic acid detection, to achieve good specificity, fast detection speed, and accurate qualitative results

Active Publication Date: 2013-01-30
CHANGSHA 3G BIOTECH
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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 the genetic polymorphism of UGT1A1

Method used

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  • Sequencing primer for qualitative detection of genetic typing of uridinediphosphoglucuronate glucuronosyltransferase 1A1 and kit thereof
  • Sequencing primer for qualitative detection of genetic typing of uridinediphosphoglucuronate glucuronosyltransferase 1A1 and kit thereof
  • Sequencing primer for qualitative detection of genetic typing of uridinediphosphoglucuronate glucuronosyltransferase 1A1 and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of the kit

[0044] 1. Design and synthesis of primers

[0045] Studies have shown that the current UGT1A1 gene polymorphisms are mainly UGT1A1*6 and UGT1A1*28 two gene polymorphisms. UGT1A1*6 gene mutations mainly occur at the 211th nucleotide of exon 1 with a G mutation to A. The glycine encoded by it was changed to arginine (G71R). The polymorphism of UGT1A1*28 is the (TA) n repeat sequence promoter. Using PyroMark Assay Design 2.0 software, according to these two mutation sites (Table 1) Designed and determined PCR amplification primers and pyrosequencing primers (Table 2). The most important thing in the kit that affects the accuracy of the kit is the primers, including amplification primers and sequencing primers. In the initial stage of the design, we designed Compare multiple sets of primers (see Picture 9 ); wherein the amplification primers and sequencing primers are purified by PAGE first, and then purified by HPLC. Among them, UGT1A1*6 fo...

Embodiment 2

[0058] Example 2: Use of the kit

[0059] 1. Sample amplification

[0060] Take blank control, positive control and sample DNA respectively as PCR reaction templates, add UNG enzyme, Taq polymerase, specific PCR reverse amplification primers, and PCR reaction solution containing PCR forward amplification primers to form PCR Reaction system, PCR amplification.

[0061] The main components of UGT1A1*6 system are as follows:

[0062]

[0063] The main components of UGT1A1*28 system are as follows:

[0064]

[0065] 2. PCR reaction program

[0066] Set up the PCR reaction program, put the reaction tube into the fluorescent PCR machine (ABI7500) to start amplification, the reaction program is as follows:

[0067] Table 3. PCR reaction program

[0068]

[0069]

[0070] The annealing temperature for detection site one (UGT1A1*6) is set to 61°C, and the annealing temperature for detection site two (UGT1A1*28) is set to 59°C.

[0071] 1. Pyrosequencing

[0072] ①Pretreatment of PCR products

[007...

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PUM

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Abstract

The invention provides a sequencing primer for qualitative detection of genetic typing of uridinediphosphoglucuronate glucuronosyltransferase 1A1 and a kit thereof, and belongs to the field of in vitro nucleic acid detection. The kit comprises uracil DNA glycosylase, Taq polymerase, a PCR reaction liquid, PCR primers, pyrosequencing primers, and a positive control product. The kit provided by the invention has high sensitivity and good specificity; the PCR products can be simply treated for sequencing on a phosphate sequenator; and the kit has advantages of simple operation, short reaction time, and higher sensitivity than a gold standard-capillary electrophoresis sequencing, and is more suitable for mutation analysis.

Description

Technical field [0001] The present invention relates to the field of in vitro nucleic acid detection, in particular to a sequencing primer pair for genotyping of uridine diphosphate glucuronyltransferase 1A1 (UGT1A1) in clinical samples and a kit thereof. Background technique [0002] Uridinediphosphoglucuronate glucucronosyltransferase (UGT) is one of the most important enzymes for the second phase of biotransformation in organisms, and belongs to the glycosyltransferase superfamily. UGT has 2 subfamilies, UGT1A and UGT2. UGT1A1 is an important enzyme that detoxifies many endogenous and exogenous substances and strengthens their excretion. The genetic polymorphism of UGT1A1 gene has important clinical significance in reducing the toxic and side effects of drugs. The polymorphisms found so far mainly include the polymorphism of the (TA) n repeat promoter (UGT1A1*28) and the polymorphism of the UGT1A1*6 gene sequence. Among Chinese, UGT1A1*28 only finds two types: (TA)6 and (TA)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 周姗
Owner CHANGSHA 3G BIOTECH
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