Method and kit for detecting fragile X syndrome mutation

A kit and reagent technology, applied in the field of kits suitable for this method, can solve the problems of omission, inability to directly and accurately determine the number of CGG repeats, inability to detect mutation types at the same time, etc., and achieve detection false detection and missed detection rate. Low, eliminates the influence of noise, and has a wide detection range

Active Publication Date: 2022-07-29
BERRYGENOMICS CO LTD
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 1. It is impossible to simultaneously detect all mutation types in the same system;
[0009] 2. Since there may be a certain proportion of chimeras in the CGG repeat expansion of FXS, CE and Southern blot may miss some low proportion chimeras;
[0010] 3. CE and Southern blot can only detect CGG repeats greater than 200, and cannot determine a higher number of CGG repeats;
[0011] 4. The judgment of CE on the number of CGG repeats less than 200 depends on the standard with known results, and it is impossible to directly and accurately judge the number of CGG repeats;
[0012] 5. Unable to accurately determine the type of AGG insertion in CGG repeats;
[0013] 6. MLPA cannot determine the exact location of the large fragment deletion;
[0014] 7. Current methods cannot directly detect microdeletions near unknown CGG repeat regions

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method and kit for detecting fragile X syndrome mutation
  • Method and kit for detecting fragile X syndrome mutation
  • Method and kit for detecting fragile X syndrome mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1: Amplify and construct PacBio sequencing library using the PCR method involved in the present invention

[0126] Step 1: Long Fragment and High GC PCR Amplification

[0127] Prepare the reaction system according to Table 1 (long fragment PCR) and Table 2 (high GC PCR) below to amplify peripheral blood, dried blood spot and genomic DNA samples:

[0128]

[0129]

[0130] On the PCR machine, perform pre-amplification according to the conditions shown in Table 3 below:

[0131]

[0132] After the amplification was completed, 5 ul of each sample was taken and tested on a 1% DNA gel. The results were as follows Figure 2A and Figure 2B As shown, using different samples as templates, different fragments of the FMR1 gene can be effectively amplified; at the same time, the amplified products were put into a centrifuge, 10,000 rpm, and centrifuged for 20 min. After centrifugation, it was placed horizontally, and 4 μL of supernatant was added to a new tube ...

Embodiment 2

[0139] Example 2: Construction of PacBio sequencing library using the PCR method involved in the present invention

[0140] Step 1: Long Fragment and High GC PCR Amplification

[0141] Prepare the reaction system according to Table 5 (long fragment PCR) and Table 6 (high GC PCR) to amplify peripheral blood samples with different types of FXS-related gene mutations:

[0142]

[0143]

[0144] On the PCR machine, perform pre-amplification according to the conditions shown in Table 7 below:

[0145]

[0146] After the amplification was completed, the amplified product was placed in a centrifuge at 10,000 rpm for 20 min. After centrifugation, it was placed horizontally, and 4 μL of supernatant was added to a new tube.

[0147] Step 2: Construct PacBio Sequencing Libraries

[0148] The reaction system was prepared according to Table 8 below:

[0149]

[0150] On a PCR machine, perform the reaction as follows: 37ºC for 20 min; 25ºC for 15 min; 65ºC for 10 min. After...

Embodiment 3

[0153]Example 3: Construction of PacBio sequencing library using the PCR method involved in the present invention

[0154] Step 1: Long Fragment and High GC PCR Amplification

[0155] Prepare the reaction system according to Table 9 (long fragment PCR) and Table 10 (high GC PCR) to amplify peripheral blood samples with different types of FXS-related gene mutations:

[0156]

[0157]

[0158] On the PCR machine, perform pre-amplification according to the conditions shown in Table 11 below:

[0159]

[0160] After the amplification was completed, the amplified product was placed in a centrifuge at 10,000 rpm for 20 min. After centrifugation, it was placed horizontally, and 4 μL of supernatant was added to a new tube.

[0161] Step 2: Construct PacBio Sequencing Libraries

[0162] The reaction system was prepared according to Table 12 below:

[0163]

[0164] On a PCR machine, perform the reaction as follows: 37ºC for 20 min; 25ºC for 15 min; 65ºC for 10 min. Aft...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a method and a kit for detecting fragile X syndrome mutation. The kit comprises the following reagents: (1) a reagent for long fragment PCR amplification; (2) a reagent for PCR amplification of a high GC region; and (3) a reagent for constructing a third-generation sequencing library. The method comprises the following steps: (1) obtaining a sample of a subject; (2) carrying out long fragment PCR (Polymerase Chain Reaction) amplification and high GC PCR amplification on the sample; (3) constructing a third-generation sequencing library; and (4) sequencing and analyzing the FMR1 gene mutation type.

Description

technical field [0001] The invention relates to a primer and a method for detecting multiple mutations of FXS by using a three-generation long-read sequencing platform, and a kit suitable for the method. Background technique [0002] Fragile X syndrome (FXS) is an X-linked incomplete penetrance genetic disease, and it is also one of the common chromosomal diseases. It is named after the brittle part that shows like a fracture. [0003] FXS is the most common cause of inherited intellectual disability, with an incidence of 1 / 4000 in males and a carrier rate of 1 / 8000-1 / 4000 in females. In most cases, FXS is caused by expansion of trinucleotide (CGG) repeats in the 5'-UTR region of the FMR1 gene on the X chromosome (Crawford DC et al. Genetics In Medicine, 2001, 3(5): 359-371) . Trinucleotide (CGG) can be divided into pre-mutation (55-200) and complete mutation (>200) according to the number of repeats; pre-mutation is unmethylated and can produce FMR1 protein with sligh...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/686C12Q1/6869C12Q2600/156C12Q2535/122
Inventor 孟万利詹嘉晗毛爱平李佳琪卢玉林张丽任志林
Owner BERRYGENOMICS CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap