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Gene chip for detecting mutation of 18 loci of susceptibility genes of type 2 diabetes

A type 2 diabetes and gene chip technology is applied in the field of gene chips for rapid detection of multiple site mutations in type 2 diabetes susceptibility genes, which can solve the problems of limited detection gene sites, cumbersome operations, and high cost, and achieve consistent fluorescence intensity. The effect of good sex and simple operation

Active Publication Date: 2013-03-06
GUANGZHOU IMPROVE MEDICAL TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The traditional diabetes susceptibility gene detection methods mainly include: PCR-RFLP, AS-PCR and DNA sequencing methods, etc. These methods play an important role in the detection of gene mutations, but there are obvious deficiencies generally. The traditional diabetes susceptibility Gene detection methods mainly include: PCR-RFLP, AS-PCR and DNA sequencing methods, etc. These methods play an important role in the detection of gene mutations, but there are generally obvious deficiencies, such as limited detection of gene sites, long time-consuming, operation Complicated, etc., not suitable for large-scale and systematic testing, which brings difficulties to the clinical diagnosis of diabetes
[0012] The PCR-RFLP method is a classic biological method, but due to the limited restriction endonuclease recognition sites, it is difficult to detect multiple sites at the same time, and it takes a long time; although the AS-PCR method can accurately detect mutations, it requires The amplification conditions are very optimized, and the primers must be highly specific, otherwise false-limited or false-positive results are prone to occur; DNA sequencing is currently recognized as the gold standard for detecting new mutations, but it cannot be sequenced due to its special structural DNA sequence , and can only be detected when the heterogeneity level is > 25%, and the heterogeneity level in peripheral blood leukocytes of commonly used clinical samples usually does not meet this standard, so it is easy to miss detection
[0013] Currently commonly used SNaPshot and Sequenom technologies have high application costs and require the purchase of expensive special equipment

Method used

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  • Gene chip for detecting mutation of 18 loci of susceptibility genes of type 2 diabetes
  • Gene chip for detecting mutation of 18 loci of susceptibility genes of type 2 diabetes
  • Gene chip for detecting mutation of 18 loci of susceptibility genes of type 2 diabetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Probe and Primer Design

[0051] The present invention screens type 2 diabetes susceptibility gene site mutations, designs related probes, and obtains a group of probes with high hybridization specificity and high accuracy after repeated tests, screening and verification. The probe sequences are shown in Table 1. Show.

[0052] Table 1 The wild-type and mutant probe sequences of 18 sites

[0053]

[0054]

[0055] When the probe is synthesized, 15 more poly-Ts are synthesized at the 5' end, and are modified by amino groups (using standard phosphoramidite chemical method, 5' or 3' amino modification with 5'-Amino-Modifier C6, TFA- protected is introduced in the last step after synthesis). The poly-T modified probe required for the chip (the probe was synthesized by InvitrogenG) was resuspended with TE Buffer (100mM Tris-Cl pH 8.0, 10mM EDTA pH 8.0) to a solution with a concentration of 50μM, and the concentration was 1:1 before sample application. The ratio was ...

Embodiment 2

[0061] Glass medium gene chip preparation

[0062] Aldehylation modification of the glass slide; resuspend the pre-synthesized probe (the probe was synthesized by InvitrogenG) with TEBuffer (100mM Tris-Cl pH 8.0, 10mM EDTA pH 8.0) to a solution with a concentration of 50uM, press 1 : 1 ratio mixed with spotting buffer (Micro Spotting Solution Plus 2X, Telechem), the final concentration is 25 μ M; figure 1 ) are arranged in a 96-well plate; PBS solution washes the spotting needle of the spotting instrument (SpotBot3, Telechem), calibrates, and presses figure 1 Matrix spotting; room temperature hydration fixation.

Embodiment 3

[0064] Detection methods of susceptibility loci

[0065] (1) Sample DNA extraction

[0066] Peripheral blood DNA was obtained, and genomic DNA was extracted using a blood genome DNA extraction kit (blood genome extraction kit DP318-02, TIANGEN) according to the manufacturer's instructions.

[0067] (2). Target DNA amplification, hybridization, detection:

[0068] The PCR reaction solution is composed of 10×buffer, 10μmol / L forward primer, 10μmol / L reverse primer, 25mmol / LMgCl 2 , 10mmol / L dNTPs (containing 0.5nM Cy3-dCTP, provided by Amersham Company) and sterile double distilled water. Amplification conditions are pre-denaturation at 95°C for 3 min, 35 cycles (denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s), extension at 72°C for 7 min; denaturation at 100°C, quenching; pre-hybridization at 42°C for 30 min to block non-specificity Binding; preheated denatured Cy3-DNA and hybridization solution (DIG Easy Hyb, Roche) were mixed at a rat...

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Abstract

The invention discloses a gene chip for detecting mutation of 18 loci of susceptibility genes of type 2 diabetes. The chip takes probes shown as SEQ ID No.1-36 which are fixed on a solid phase carrier as a detection system, and further comprises positive control, negative control and parallel control which form a quality control system package. The invention also discloses supporting primers for the detection of the gene chip. The primers can perform polymerase chain reaction (PCR) at the same annealing temperature, amplify and accomplish Cy3 marking at the same time, and perform hybridization reaction at one time to detect the 18 gene loci. The gene chip has the advantages of accurate detection result, fastness and high efficiency, can systematically screen the mutation of the 18 loci of the susceptibility genes of the type 2 diabetes which are reported at home and abroad at present, and is suitable for large-sample low-cost association studies on the susceptibility genes of the type 2 diabetes.

Description

technical field [0001] The invention relates to the field of gene chip detection, in particular to a gene chip for rapidly detecting multiple site mutations of type 2 diabetes susceptibility genes, and the sites cover susceptibility genes clearly related to type 2 diabetes at present. Background technique [0002] Diabetes is a complex metabolic disease caused by defects in insulin secretion and / or insulin action. younger. Chronic complications caused by persistent hyperglycemia have become the main cause of renal failure, blindness and cardiovascular and cerebrovascular diseases, bringing a heavy burden to individual, social and national health care, and becoming a global social health and economic problem. Because T2DM has familial characteristics, and there are obvious differences in the incidence among different races, and the incidence and condition of identical twins and fraternal twins are also different, so genetic components play an important role in the pathogenes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 邓冠华谢焱刘松梅周新马海波郑璇袁媛丁峰王春芳梁纯子
Owner GUANGZHOU IMPROVE MEDICAL TECH CO LTD
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