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 direction of recombinant DNA technology, DNA / RNA fragments, etc., to achieve high sensitivity, simple sample processing, and intuitive results

Active Publication Date: 2014-06-04
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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: the preparation of 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 gene polymorphisms, UGT1A1*6 gene mutations mainly occur in the 211th nucleotide of the first exon G mutation to A, The polymorphism of UGT1A1*28, which causes the encoded glycine to be changed to arginine (G71R), is the (TA) n repeat sequence promoter, using PyroMark Assay Design2.0 software, according to these two mutation sites (Table 1) The PCR amplification primers and pyrosequencing primers were designed and determined (Table 2). The most important factor affecting 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 were compared (see Figure 9 ); the amplification primers and sequencing primers were purified by PAGE first, and then purified by HPLC, among which UGT1A1*6...

Embodiment 2

[0058] Embodiment 2: the use of kit

[0059] 1. sample amplification

[0060] Take the 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 a PCR reaction solution. reaction system for PCR amplification.

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

[0062]

[0063] The main components of the 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 instrument (ABI7500) to start amplification, the reaction program is as follows:

[0067] Table 3. PCR reaction program

[0068]

[0069]

[0070] The annealing temperature of detection site 1 (UGT1A1*6) was set at 61°C, and the annealing temperature of detection site 2 (UGT1A1*28) was set at 59°C.

...

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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 invention relates to the field of in vitro nucleic acid detection, in particular to a pair of sequencing primers for genotyping uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) in clinical samples and a kit thereof. Background technique [0002] Uridine diphosphate glucuronosyltransferase (uridinediphosphoglucuronate glucucronosyltransferase, UGT) is one of the most important enzymes in the second phase of biotransformation in organisms, belonging to the glycosyltransferase superfamily. There are two subfamilies of UGT, UGT1A and UGT2 families. UGT1A1 is an important enzyme that detoxifies many endogenous and exogenous substances and enhances their excretion. The genetic polymorphism of UGT1A1 gene has important clinical significance for reducing the toxic and side effects of drugs. The polymorphisms found so far mainly include the polymorphism of (TA)n repeat promoter (UGT1A1*28) and the polymorphism of UGT1A1*6 gene sequence. In Chinese...

Claims

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Application Information

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