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Primer group for SBDS gene mutation detection and gene amplification method

A gene amplification and primer set technology, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., can solve the problems of reducing the detection rate of SBDS gene mutation, inability to distinguish effectively, and achieve low cost. , Improve the accuracy, the effect of high detection accuracy

Pending Publication Date: 2021-11-02
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both Sanger sequencing and high-throughput sequencing of commercial capture probes cannot effectively distinguish the sequences of SBDS and SBDSP, thus reducing the detection rate of SBDS gene mutations, and there are certain false positives

Method used

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  • Primer group for SBDS gene mutation detection and gene amplification method
  • Primer group for SBDS gene mutation detection and gene amplification method
  • Primer group for SBDS gene mutation detection and gene amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Using MolPure ® Blood DNA Kit (yasen 18730) extracts human blood genomic DNA and removes RNA. The extracted DNA OD260 / 280 is between 1.8-2.0.

[0036] KOD FX Neo DNA polymerase (TOYOBO KFX-201) was selected as the long fragment PCR amplification reagent. Nest's PCR amplification reagent is 2×Hieff ® PCR Master Mix (With Dye) (yeasen 10102).

[0037] Table 1 Long fragment PCR reaction system:

[0038] components Volume μL 2×PCR buffer for KOD FX Neo 12.5 2mM dNTPs 5 SBDS-LF (10μM) 0.35 SBDS-LR (10μM) 0.35 KOD FX Neo (1U / μl) 0.5 Genome 25ng wxya 2 o

Up to 25μL

[0039] The long-segment PCR amplification program was pre-denaturation at 94°C for 2 minutes, followed by 30 cycles, each cycle at 94°C for 15 seconds, 68°C for 13 minutes, and finally kept at 10°C until use.

[0040] Take 1 μL of the long-fragment PCR amplification product and run electrophoresis. The agarose gel concentration is 0.7%, the elec...

Embodiment 2

[0048] Using the present invention to detect a blood sample of a suspected case of SBDS. The blood DNA of this case was analyzed by high-throughput sequencing, and there were two mutation sites in SBDS or its homologous region. Suspected mutation site 1: Chr7:66994210 (GRCh38 / hg38), T is mutated to C, which is located at the last two positions of intron 2 of SBDS, and is an alternative splicing site; suspected mutation site 2: Chr7:66994286 -66994287 (GRCh38 / hg38), TA is mutated to CT, which is located in exon 2 of SBDS. Due to the short read length of high-throughput sequencing reads (double-ended 150bp), the homologous regions cannot be accurately distinguished, and the detected mutations still need to be determined by another scheme.

[0049] After the DNA of the sample was subjected to long-fragment PCR by SBDS-L, SBDS-2 nested primers were used for the second round of amplification. The PCR product was electrophoresed to confirm that the size of the amplified product was...

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Abstract

The invention provides a primer group for SBDS gene mutation detection, which comprises a pair of long fragment PCR primers and five pairs of nested PCR primers. The sequences of the primers are shown as SEQ ID No.1-12. The invention also discloses a gene amplification method for SBDS gene mutation detection. The invention provides the long-fragment PCR primer for amplifying the full-length gene of the SBDS, and the amplification product is as long as about 12Kb. And five pairs of nested PCR primers are designed for each exon, so that SBDS and SBDSP sequences can be effectively distinguished, and the accuracy of SBDS gene mutation detection is greatly improved. The sequence and the method provided by the invention can effectively distinguish SBDS and pseudogene SBDSP sequences thereof, and have the advantages of high detection accuracy, convenience and rapidness in operation and low cost.

Description

technical field [0001] The patent of the present invention relates to a primer set and gene amplification method for SBDS gene mutation detection, belonging to the field of biotechnology. Background technique [0002] Shwachman-Diamond syndrome (SDS, OMIM260400), also known as Shwachman-Bodian-Diamond syndrome (SBDS), is a rare autosomal recessive genetic disorder first reported by Shwachman in 1964. The main clinical manifestations of SDS are bone marrow failure and pancreatic exocrine dysfunction. Most patients are accompanied by short stature, bone deformity, liver insufficiency, and severe infection. Although the incidence of SDS is only 1:77000, it is one of the common causes of extrapancreatic insufficiency and skeletal dysfunction, and 90% of SDS patients have SBDS gene mutations. [0003] The SBDS gene is located on chromosome 7q11, with a total length of 7907bp, including 5 exons. SBDSP is a pseudogene of SBDS, and the DNA sequences between the two have 97% homolo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2549/119
Inventor 沈小波彭瑜孙晓亮宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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