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Method and primer for detecting mutation of ORF15 exon of RPGR gene

A technology of exons and sequencing primers, applied in the direction of DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low accuracy of sequencing results, mixed peaks of sequencing results, and difficult sequences, so as to enhance integrity and accuracy, The effect of high sensitivity and strong specificity

Inactive Publication Date: 2018-01-23
杭州科宁生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the presence of large purine nucleotide repeats in exon ORF15, it is difficult to accurately detect the sequence of ORF15
[0003] At present, the accuracy of sequencing results is generally low when using next-generation sequencing-based gene detection panels or whole exome sequencing (WES) technology to detect ORF15 sequences
On the other hand, the detection of ORF15 sequences by next-generation sequencing technology requires specific reagents, specific PCR reaction conditions, and multiple pairs of overlapping primers, and the sequencing results usually have heterogeneous peaks

Method used

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  • Method and primer for detecting mutation of ORF15 exon of RPGR gene
  • Method and primer for detecting mutation of ORF15 exon of RPGR gene
  • Method and primer for detecting mutation of ORF15 exon of RPGR gene

Examples

Experimental program
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Effect test

Embodiment 1

[0037] The inventor designed a large number of primers for exon ORF15 of the RPGR gene, and screened primers with good specificity through optimization and comparison of primer reaction conditions. The primers are shown in Table 1.

[0038] Table 1 The present invention provides amplification primers and sequencing primer sequences and their positions on the chromosome

[0039]

[0040] The seven sequencing primers of the present invention were blasted in UCSC, and the amplified fragment of the RPGR gene ORF15 exon primer was located between chrX: 38144608-38146560, with a length of 1953 bp. No other homologous genes, the results are as follows figure 1 It is consistent with the reference sequence of RPGR gene.

[0041] The detection method of RPGR gene ORF15 exon mutation comprises the following steps

[0042] (1) Genomic DNA was extracted using a blood DNA extraction kit (Biteco, DP1801) according to the instructions of the kit.

[0043] The brief operation of extracti...

Embodiment 2

[0071] Example 2 Detection of RPGR gene ORF15 exon mutations in clinical samples

[0072] Three clinical family samples were operated as described in Example 3, DNA extraction, amplification, electrophoresis detection, product purification, sequencing reaction, and result analysis were performed to obtain sequencing results. Such as Figure 10 As shown, the patient has RPGR:c.3031G>T:(p.G1011X) mutation, the patient's father is normal at this site, and the patient's mother is a carrier.

Embodiment 3

[0073] Example 3 compares the accuracy of high-throughput sequencing

[0074] (1) According to the operation described in Example 3, operations such as DNA extraction, amplification and electrophoresis detection were performed.

[0075] (2) Purification of PCR products: DNA purification kit (QIAGEN, Cat. No. 28004) was used to purify PCR products according to the operating instructions. The brief steps are as follows:

[0076] 1) Add 250 μl of Buffer PB to 50 μl of PCR product and mix well.

[0077] 2) Add the mixed solution to the MinElute elution column, centrifuge at 17900×g (13000 rpm) for 1 min at room temperature, and discard the effluent.

[0078] 3) Add 750 μl of Buffer PE to the MinElute elution column, centrifuge at 17900×g (13000 rpm) for 1 min at room temperature, and discard the effluent.

[0079] 4) Centrifuge the MinElute elution column again at 17900×g (13000 rpm) at room temperature for 1 min.

[0080] 5) Put the MinElute elution column into a new centrifug...

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Abstract

The invention discloses a method and a primer for detecting mutation of an ORF15 exon of an RPGR gene. The primer for detecting the mutation of the ORF15 exon of the RPGR gene comprises a positive primer and a reverse primer for amplifying the ORF15 exon of the RPGR gene. The method for detecting the mutation of the ORF15 exon of the RPGR gene comprises the following steps of (1) extracting DNA (deoxyribonucleic acid) of a sample; (2) utilizing a pair of amplification primers RPGR_ORF15_F3 and RPGR_ORF15_R6 to amplify the DNA in step (1), so as to obtain an amplification product; (3) at leastusing one type of sequencing primer to sequence the amplification product, so as to obtain a gene sequence of the amplification product; (4) comparing the sequence of the gene in step (3) and the wildtype reference sequence RPGR-ref of the RPGR gene, so as to determine whether the mutation site exists or not. The primer has the advantages that the specificity is strong, and the sensitivity is high. The detection method has the advantages that the mutation of mononucleotide can be accurately detected, and the frame-shift variation of homozygosis or heterozygosis can be effectively detected.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a method and primers for detecting the mutation of exon of RPGR gene ORF15. Background technique [0002] Retinitis pigmentosa (RP) is a group of the most common hereditary and blinding fundus diseases in which the degeneration of retinal photoreceptor cells and pigment epithelial cells leads to night blindness and progressive visual field defects. The clinical phenotype of male XLRP patients is more severe than that of both autosomal dominant and recessive RP patients, and the rate of XLRP patients is 10-20% of total RP patients. Among the genes related to XLRP, RPGR gene mutations cause the most serious symptoms, and more than 70% of XLRP are caused by RPGR gene mutations. Exon ORF15 on the RPGR gene is a mutation hotspot of XLRP, about 55% of XLRP is caused by ORF15 mutation. However, it is difficult to accurately detect the sequence of ORF15 due to the presence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11
Inventor 王伦钧杨金龙王云霞沈君霞
Owner 杭州科宁生物科技有限公司
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