Method for amplifying GGC repetitive sequence of NOTCH2NLC gene

A repetitive sequence and gene technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as low efficiency, inability to meet large-scale sample amplification, long amplification time, etc., to achieve convenient use and economical Reagent cost, high reproducibility of effect

Pending Publication Date: 2021-03-26
PEKING UNIV FIRST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Therefore, the technical problem to be solved in the present invention is to overcome the defects that the GGC repeat sequence amplification time of the NOTCH2NLC gene in the prior art is long, the efficiency is low, the amplification of large-scale samples cannot be satisfied, and it is difficult to be widely used in the clinical gene diagnosis of NIID disease , thereby providing a method, a kit, and an application for amplifying the GGC repeat sequence of the NOTCH2NLC gene, the GGC repeat sequence amplification time of the NOTCH2NLC gene is short, and the efficiency is high, which can satisfy the amplification of large-scale samples

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  • Method for amplifying GGC repetitive sequence of NOTCH2NLC gene
  • Method for amplifying GGC repetitive sequence of NOTCH2NLC gene
  • Method for amplifying GGC repetitive sequence of NOTCH2NLC gene

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1 NOTCH2NLC gene GGC repeat sequence amplification method

[0039] (1) Obtain human genomic DNA;

[0040] (2) Take 100ng of the above-mentioned genomic DNA and add it to the PCR amplification reaction system of the GGC repeat sequence of the NOTCH2NLC gene, as shown in Table 1 below;

[0041] Table 1: PCR amplification reaction system

[0042]

[0043]

[0044] (3) Put the PCR reaction solution in step (2) into a PCR instrument for PCR (BioRad MyCycler ThermalCycler) reaction. The temperature change rate of all cycle steps is 1°C / s. The PCR reaction procedure is as follows:

[0045]

[0046] (4) The PCR amplification product obtained in step (3) is subjected to capillary fluorescence electrophoresis detection.

Embodiment 2

[0047] Example 2 NOTCH2NLC Gene GGC Repeat Sequence Amplification Kit

[0048] The kit described in this embodiment includes a PCR amplification reaction system for the GGC repeat sequence of the NOTCH2NLC gene, as shown in Table 2 below.

[0049] Table 2: PCR amplification reaction system

[0050]

[0051]

[0052] Further, the nucleotide sequence of the primer NOTCH2NLC-F is shown in SEQ ID NO.1, the nucleotide sequence of the primer M13-LINKER-R is shown in SEQ ID NO.2, and the primer M13- The nucleotide sequence of (GGC)4(GGA)2-R is shown in SEQ ID NO.3. The 5' end of the above primer NOTCH2NLC-F has a fluorescent group FAM, that is, NOTCH2NLC-F is: 5'-FAM-GGCATTTGCGCCTGTGCTTCGGACCGT-3'.

Embodiment 3

[0053] Example 3 Detection of the repeat sequence GGC in the NOTCH2NLC gene

[0054] The genomic DNA of the NIID patient and the genomic DNA of the normal person were respectively taken to amplify the GGC repeat sequence of the NOTCH2 NLC gene according to the method in Example 1. The obtained PCR reaction product was detected by capillary electrophoresis fluorescence detection method (Denget al., Journal of medical genetics. 2019) to identify whether the GGC repeat number of NOTCH2NLC gene reached the pathogenic abnormal repeat number.

[0055] Identification results such as Figure 1-2 As shown, on the one hand, the above results show that the NOTCH2NLC gene GGC repeat sequence amplification method of the present embodiment can specifically amplify the NOTCH2NLC gene GGC repeat sequence, which greatly shortens the amplification time and effectively reduces the cost. On the other hand, it shows that The NOTCH2NLC gene of NIID patients has an abnormal GGC repeat sequence, and...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for amplifying a GGC repetitive sequence of an NOTCH2NLC gene. The PCR reaction procedure for amplifying the GGC repetitive sequence of the NOTCH2NLC gene comprises the following steps: reacting at 98 DEG C for 10 minutes; then carrying out 9 cycles: reacting at 98 DEG C for 30 seconds, reacting at 66 DEGC for 15 seconds, lowering the temperature by 1 DEG C in each cycle, and reacting at 72 DEG C for 4 minutes; carrying out 30 cycles: reacting at 98 DEG C for 30 seconds, reacting at 58 DEG C for 15 seconds, and reacting at 72 DEG C for 4 minutes; and finally, conducting the extension step at 72 DEG C for 10 minutes. According to the PCR reaction procedure, the PCR time is greatly shortened, the total time is shortened from 10 hours to 4.5 hours, the repeatability is high, the use is convenient, the cost is slightly low, the applicability of the RP-PCR technology in clinical gene diagnosis ofNIID (neuronal intranuclear inclusion disease) is greatly improved, and the demand for the amplification of large-scale samples can be met.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method, a kit and an application for amplifying the GGC repeat sequence of the NOTCH2NLC gene. Background technique [0002] Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disease characterized by eosinophilic hyaline inclusions in the central and peripheral nervous system, as well as in multiple organ tissues. Inclusion bodies are mainly distributed in the central nervous system, peripheral nervous system, and non-nervous tissues. In the central nervous system, they are widely found in neurons and astrocytes in the cerebral cortex, basal ganglia, brainstem, cerebellum, and spinal cord. In the peripheral nervous system, there are sympathetic ganglia, dorsal root ganglia, myenteric plexus ganglia, and Schwann cells, and in non-nervous tissues, there are many somatic cells except skeletal muscle and liver cells. Because NIID is a heterogeneous...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6883
CPCC12Q1/6806C12Q1/6883C12Q2600/156C12Q2525/151C12Q2531/113
Inventor 邓健文王朝霞
Owner PEKING UNIV FIRST HOSPITAL
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