PCR kit used for detecting CGC replication number and AGG insert information of fragile X syndrome

An X chromosome and information insertion technology, applied in the field of PCR kits, can solve the problems of difficult operation, long detection time, and high cost

Inactive Publication Date: 2014-08-13
江苏佰龄全基因生物医学技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Existing detection methods for Fragile X chromosome syndrome use fluorescent probes, MLPA, Southern

Method used

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  • PCR kit used for detecting CGC replication number and AGG insert information of fragile X syndrome
  • PCR kit used for detecting CGC replication number and AGG insert information of fragile X syndrome
  • PCR kit used for detecting CGC replication number and AGG insert information of fragile X syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of PCR kit for CGG repeat number and AGG insertion information of fragile X syndrome

[0036] 1. Primer design

[0037]A pair of primers F and R flanking the CGG repeat region are provided for amplifying the CGG repeat region; two different pairs of primers are provided in addition, including the first primer containing the search probe and the corresponding annealing primer located on the flank of the CGG repeat region; one of One pair is detection primers M1 and R1, and the other pair is verification primers F1 and M2. Two pairs of different primers each contain a first primer M1 and M2 as a search probe, wherein the first primer M1 of the detection primer M1 and R1 is used as an upstream primer, and the first primer M2 of the verification primer M2 and F1 is used as a downstream primer. primers. M1 includes CGG, GCG, GGC repeats and AGG, GAG, GGA repeats, M2 includes GCC, CGC, CCG repeats and TCC, CTC, CCT repeats. M1 and M2 consist of a sequ...

Embodiment 2

[0043] Example 2 CGG repeat number accurate quantification

[0044] 1) Use a commercially available genomic DNA extraction kit to extract human peripheral blood DNA, the concentration of which is 100 ng / μl, the 260 / 230 value is greater than 2.0, and the 260 / 280 value is greater than 1.8.

[0045] 2) Prepare PCR reaction system: prepare 50 μl PCR reaction system in a 200ul thin-walled PCR test tube,

[0046] The PCR amplification system is as follows:

[0047]

[0048] Wherein, the primers are SEQ ID No: 2 and SEQ ID No: 4.

[0049] Composition of PCR enhancer: betaine (2.5mol / L) 5 μl, GCEnhancer 5 μl and DMSO 4 μl.

[0050] PCR reaction program: 95°C pre-denaturation for 8 min, followed by 30 cycles: denaturation at 95°C for 50 s, annealing at 65°C for 45 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min.

[0051] 3) After the reaction, use 2% agarose gel to load the sample, electrophoresis under the electric field condition of 10V / cm for 45min, and ...

Embodiment 3A

[0057] Example 3 AGG Insertion Information Accurate Quantification

[0058] 1) Use a commercially available genomic DNA extraction kit to extract human peripheral blood DNA, the concentration of which is 100 ng / μl, the 260 / 230 value is greater than 2.0, and the 260 / 280 value is greater than 1.8.

[0059] 2) Prepare PCR reaction system: Prepare 50 μl PCR reaction system in a 200ul thin-walled PCR tube, and the PCR amplification system is as follows:

[0060]

[0061]

[0062] Wherein, the primers are SEQ ID No: 5 and SEQ ID No: 11.

[0063] Composition of PCR enhancer: betaine (2.5mol / L) 4 μl and GCEnhancer 4 μl.

[0064] PCR reaction program: 95°C pre-denaturation for 8 min, followed by 30 cycles: denaturation at 95°C for 50 s, annealing at 68°C for 45 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min.

[0065] 3) After the reaction, use 2% agarose gel to load the sample, electrophoresis under the electric field condition of 10V / cm for 45min, and u...

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Abstract

The invention provides a PCR kit used for detecting the CGC replication number and AGG insert information of fragile X syndrome. The PCR kit comprises a DNA polymerase emboitement, a PCR reinforcing agent, a primer group and a correction Marker; and the primer group comprises primers F and R used for detecting the CGG replication number, and detection primers M1 and R1 and verification primers M2 and F1 used for detecting the AGG insert information. The kit can accurately and rapidly analyze the CGC replication number and the AGG insert information of a sample, can breakthrough the problems of high sequencing difficulty, long time and high cost of present fragile X syndrome detection methods adopting a fluorescence probe, MLPA and Southern Blot, and can directly analyze the range of the CGC replication number and the AGG insert information according to a gel electrophoresis result in order to rapidly diagnose the characteristics of the sample; and the product resolution can be obtained by separating products through a high-resolution device, and the CGC replication number and the AGG insert information are obtained through a mathematic model.

Description

technical field [0001] The invention relates to a PCR kit for detecting CGG repeat number and AGG insertion information of fragile X chromosome syndrome, which belongs to the field of molecular biology and clinical diagnosis of genetic diseases, especially relates to fragile X chromosome syndrome and other causes of triplet, The field of clinical diagnosis of genetic diseases caused by highly repeated quadruple nucleotides. Background technique [0002] Fragile X syndrome is a hereditary mental retardation syndrome with an incidence rate second only to congenital stupidity, and it is also one of the most common causes of autism known so far. The incidence rate accounts for 0.05% of all children, showing X-linked inheritance, accounting for 40% of X-linked mental retardation. The molecular genetic basis of more than 95% of patients with fragile X syndrome is caused by the dynamic mutation of the FMR-1 gene (CGG)n structure expansion, and less than 5% are due to missense muta...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858
Inventor 潘海波华琴邢楠楠谢阳
Owner 江苏佰龄全基因生物医学技术有限公司
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