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Probes and uses thereof

A technology of probes and fragments, which is applied in the system field of determining the nucleic acid sequence of the FLG gene coding region of the sample to be tested, and can solve the problems of not obtaining specific sequences or sequencing failures, FLG gene detection methods need to be improved, and FLG cannot be fully interpreted, etc. Achieve the effect of small workload, low cost and good specificity

Active Publication Date: 2016-05-25
WUHAN BGI CLINICAL LAB CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this sequence is not only complex in combination, but also has individual specificity. When using this method, specific sequences may not be obtained or sequencing failures may occur. The experimental process is very unstable, resulting in incomplete interpretation of FLG.
In addition, the number of samples sequenced by the first-generation sequencer at the same time is small, and it needs to go through the PCR process first. The workload is large, the throughput is small, and the detection rate is less than 30%.
[0003] Therefore, the current FLG gene detection method still needs to be improved

Method used

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  • Probes and uses thereof

Examples

Experimental program
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Embodiment 1

[0110] According to the method for determining the nucleic acid sequence of the FLG gene coding region of the test sample according to the present invention, refer to Figure 16 , Follow the steps below to perform FLG gene detection on samples to be tested:

[0111] Among them, the sample to be tested is the patient information as follows:

[0112]

[0113] 1. Preparation of probes and chips

[0114] Design a set of capture probes that specifically recognize the coding region of the FLG gene, and use the CustomArrayB3P platform to synthesize probes to prepare multiple chips. The design and synthesis parameters of the probes are as follows:

[0115] (1) The length of the probe is 75bp;

[0116] (2) The probe specifically recognizes the sequence between 10 bp upstream and 10 bp downstream of the FLG gene coding region;

[0117] (3) Probes that specifically recognize regions with GC content higher than 0.6 and lower than 0.3, with a multiplier greater than 2;

[0118] (4) The melting tempera...

Embodiment 2

[0265] According to the method of Example 1, FLG gene detection was performed on 59 patients with suspected ichthyosis vulgaris. Among them, this embodiment uses the first-generation direct sequencing method (that is, PCR is performed first, and then the PCR product is nested and amplified to obtain a specific target sequence, and then combined with sanger sequencing to interpret the FLG sequence) as a control.

[0266] The results are shown in the following table. Using the method of the present invention, 49 patients were detected to have mutations in the FLG gene, and the detection rate was 83%. Compared with the first-generation direct sequencing detection rate (less than 30%), it is greatly improved.

[0267] Patient ID

[0268] P7

[0269] P42

[0270] Note: "Het" means heterozygous, "Hom" means homozygous

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Abstract

The present invention discloses probes and uses thereof. The group of the probes specifically recognize at least one part of a FLG gene coding region, and meet at least one condition selected from the following conditions that (1) the length of the probe is 75 bp; (2) the probe specifically recognizes the sequence between the upstream 10 bp and the downstream 10 bp of the FLG gene coding region; (3) the multiplier of the probe specifically recognizing the region having the GC content of higher than 0.6 and lower than 0.3 is more than 2; (4) the melting temperatures of the probe and the target sequence is 60-10 DEG C, preferably 80 DEG C; (5) the probe does not contain a hairpin structure; (6) the probe is matched with two sites on a reference genome at the most; and (7) the window sliding size is 10 bp during the probe selection. According to the present invention, the group of the probes provides good capture specificity, high sensitivity and high coverage for at least one part of the specifically-recognized FLG gene coding region, and can be effectively used for the capture detection of the FLG gene coding region.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to the technical field of high-throughput sequencing of target genes. Specifically, the present invention relates to probes and their uses. More specifically, the present invention relates to a set of probes, a method for constructing a high-throughput sequencing library, a method for determining the nucleic acid sequence of the FLG gene coding region of a sample to be tested, and a method for constructing a high-throughput A device for sequencing a library and a system for determining the nucleic acid sequence of the FLG gene coding region of the sample to be tested. Background technique [0002] Ichthyosis vulgaris (Ichthyosisvulgaris) is an autosomal dominant genetic disease, the incidence of the disease is 1:250-1:1000. The main clinical symptoms include skin damage in the form of "fish scales" or "snakeskin", keratinized pimples of hair follicles, punctate pores, and many, deep, and di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12M1/34C40B50/06C40B60/14
Inventor 魏晓明杨昀田甜孙岩
Owner WUHAN BGI CLINICAL LAB CO LTD
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