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Methods and kits for detecting chromosomal rearrangements

A chromosome and kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as high technical requirements, complex operation, and difficulty in forming loops, and achieve less DNA consumption, simple operation, and economical effect of time

Active Publication Date: 2020-12-29
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has disadvantages such as complex operation and high technical requirements; and the fragments after restriction enzyme digestion are too long and difficult to form loops, etc.

Method used

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  • Methods and kits for detecting chromosomal rearrangements
  • Methods and kits for detecting chromosomal rearrangements
  • Methods and kits for detecting chromosomal rearrangements

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] LD-PCR combined with qPCR two-step method

[0064] Experimental operation

[0065] The experiment is divided into two steps: the first step is 12 cycles of LD-PCR, the second step is 12 cycles of LD-PCR products and the original DNA that was not subjected to 12 cycles of LD-PCR for M1 and M2 loci qPCR.

[0066] Select samples from hemophiliacs and prepare the system according to the table below.

[0067] Table 1-1

[0068]

[0069] Table 1-2

[0070] Primer and Probe Sequences

[0071]

[0072] Table 1-3

[0073] 12 cycles of LD-PCR reaction solution components

[0074]

[0075] After the configuration of the sample reaction system is completed, the sample is placed in the Bio-Rad S1000 Thermal Cycler PCR instrument for PCR reaction. The program is: 98°C, 1min; 98°C for 10s, 68°C for 10min (+20s / cycle), for 12 cycles; 72°C for 10min; 4°C hold. See Tables 1-4 below.

[0076] Table 1-4

[0077]

[0078]After 12 cycles of LD-PCR reactions, 2 μL of 12 ...

Embodiment 2

[0095] LD-PCR combined with qPCR one-step method

[0096] Experimental operation

[0097] Configure the system for the samples to be tested according to Table 3-1, 3-2 and 3-3. After the reaction system of the sample was prepared, the sample was placed in a Bio-Rad C1000 Touch Thermal Cycler CFX96 Q-PCR instrument. Open the Bio-RadCFX Manager3.1 software, select the Probe program in PrimePCR, set the program and set the plate respectively. Set the program according to Table 3-4. When setting up the plate, select the two fluorescence detection channels "FAM" and "HEX", and select the position of the sample for detection. After all settings are completed, click "Start Run" to start the qPCR reaction.

[0098] Table 3-1 Main Reagents

[0099]

[0100] Table 3-2 Primer and probe sequences

[0101]

[0102] Table 3-3 Reaction solution components

[0103]

[0104] Table 3-4 Reaction program

[0105]

[0106] Analysis of test results

[0107] After the qPCR exper...

Embodiment 3

[0125] One-step detection of intron 22 inversion of F8 gene is not affected by the amount of substrate DNA

[0126] Experimental operation

[0127] Configure the system for the samples to be tested according to Table 3-1, 3-2 and 3-3. After the reaction system of the sample was prepared, the sample was placed in a Bio-Rad C1000 Touch Thermal Cycler CFX96 Q-PCR instrument. Open the Bio-RadCFX Manager3.1 software, select the Probe program in PrimePCR, set the program and set the plate respectively. Set the program according to Table 3-4. When setting up the plate, select the two fluorescence detection channels "FAM" and "HEX", and select the position of the sample for detection. After all the settings are completed, click "Start Run" to start the qPCR reaction.

[0128] Analysis of test results

[0129] After the qPCR experiment, set the threshold value of "FAM" to 97.69, and the threshold value of "HEX" to 98.53. The Ct values ​​of M1 and M2 obtained from the tested sampl...

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Abstract

Provided in the present invention are a method and kit for detecting chromosome rearrangement. Specifically, the method for detecting chromosomal rearrangement of the present invention comprises: obtaining sample DNA from a sample of a subject; performing long-distance PCR (LD-PCR) and quantitative PCR (qPCR) on the sample DNA; comparing a detection result with a result of a normal control sample; and from the result of the comparison, inferring whether a chromosomal rearrangement is present in the sample DNA. The LD-PCR and qPCR may be performed in succession or in the same reaction container. Further provided in the present invention are a kit for performing the method, as well as primers and probes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and a kit for detecting chromosome rearrangement, such as chromosome fragment deletion, duplication, inversion, translocation and the like. The invention also specifically relates to a method and a kit for detecting the inversion of No. 22 intron of the F8 gene. Background technique [0002] A chromosomal rearrangement is a chromosomal abnormality that involves a change in the structure of the natural chromosome. The alterations can involve several different types, such as deletions, duplications, inversions, translocations, and the like. Typically, these changes are due to a break in two different places in the DNA double helix, followed by rejoining of the broken ends to create a new chromosomal arrangement of genes that differs from the order of genes in the chromosomes before the break. Chromosomal rearrangements can cause various diseases. [0003] Hemophilia A is an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6848
CPCC12Q1/6848C12Q1/6883C12Q2600/156C12Q2549/119C12Q2531/113C12Q2545/114C12Q2563/107C12Q2537/143
Inventor 丁春明金胜男尚庆娟
Owner WENZHOU MEDICAL UNIV
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