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CEBPA gene probe design method and application thereof

A design method and DNA probe technology, applied in the field of CEBPA gene probe design, can solve the problems of no CEBPA gene rs762459325 single nucleotide polymorphism detection performance confirmation, etc., to achieve the effect of reducing detection costs

Pending Publication Date: 2020-04-14
苏州元德友勤医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no other literature disclosing the CEBPA gene probe design method and probe sequence, and there is no performance confirmation of the CEBPA gene rs762459325 single nucleotide polymorphism detection using the probe capture method

Method used

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  • CEBPA gene probe design method and application thereof
  • CEBPA gene probe design method and application thereof
  • CEBPA gene probe design method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Detection of target DNA: CEBPA gene contains rs762459325 heterozygous positive DNA samples and rs762459325 negative cell line K562.

[0039] Probe: a combination of probe sequences designed by the present invention as shown in SEQ ID NO.1-SEQ ID NO.55.

[0040] DNA ultrasonic fragmentation: Covaris M220, fragmentation to the range of 200-220bp.

[0041] Library construction kit: kapa hyper Prep kit.

[0042] Experimental steps for library construction: refer to the instructions of the kapa library construction kit.

[0043] Coverage of CEBPA gene sequencing results: 100% (rs762459325 positive samples), 100% (rs762459325 negative samples).

[0044] The average sequencing depth of CEBPA gene sequencing results: 1928 layers (rs762459325 positive samples), 1867 layers (rs762459325 negative samples).

[0045] Site detection results: data analysis results bam files are analyzed using IGV software, such as image 3 as shown, image 3 A is the test result of rs762459325 po...

Embodiment 2

[0047] Detection target DNA: Contains 1% rs762459325 variant DNA (the rs762459325 heterozygous variant sample is mixed with the rs762459325 negative K562 cell line DNA in proportion to obtain a 1% positive control sample).

[0048] Probe: The probe sequence designed by the present invention is combined with the probe sequence of other 85 genes.

[0049] DNA ultrasonic fragmentation: Covaris M220, fragmentation to the range of 200-220bp.

[0050] Library construction kit: kapa hyper Prep kit.

[0051] Experimental steps for library construction: refer to the instructions of the kapa library construction kit.

[0052] CEBPA sequencing result coverage: 100%.

[0053] The average sequencing depth of CEBPA sequencing results: 2319 layers.

[0054] Site detection results: data analysis results bam files are analyzed using IGV software, such as Figure 4 shown.

Embodiment 3

[0056] Detection of target DNA: The CEBPA gene p.Ala44fs and p.Glu316delinsAspGln double mutation-positive samples were detected by first-generation sequencing.

[0057] Probe: The probe sequence designed by the present invention is combined with the probe sequence of other 85 genes.

[0058] DNA ultrasonic fragmentation: Covaris M220, fragmentation to the range of 200-220bp.

[0059] Library construction kit: kapa hyper Prep kit.

[0060] Experimental steps for library construction: refer to the instructions of the kapa library construction kit.

[0061] CEBPA sequencing result coverage: 100%.

[0062] The average sequencing depth of CEBPA sequencing results: 1754 layers.

[0063] Site detection results: data analysis results bam files are analyzed using IGV software p.Ala44fs mutation results are as follows Figure 5 As shown, the result of p.Glu316delinsAspGln mutation is as follows Figure 6 shown.

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Abstract

The invention discloses a CEBPA gene probe design method and an application thereof. For a CEBPA gene coding sequence, according to a sequence reverse complementation principle, a probe sequence witha length of 120bp is tiled and designed every 20bp from an rs762459325 site as a center to two sides; and in combination with a cosmic database, the obtained probe sequence is optimized to obtain an optimal probe sequence. Meanwhile, the invention discloses a DNA probe library constructed by the probe sequence and a detection reagent for CEBPA gene mutation, and discloses a detection method for CEBPA gene mutation. The method has high sensitivity and detection rate for CEBPA gene mutation detection, can reach the international report level, provides a reliable way for disease diagnosis of hematological tumor patients, and has certain market promotion economic value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CEBPA gene probe design method and application thereof. Background technique [0002] CEBPA gene mutation occurs in about 10-15% of acute myeloid leukemia (AML) (PMID: 21177436). European Hematology Network (ELN) in 2017, National Comprehensive Cancer Network (NCCN) in the United States in 2019, Chinese guidelines for diagnosis and treatment of adult acute myeloid leukemia (non-acute promyelocytic leukemia) (2017 edition, Chinese Journal of Hematology, 2017,38 (3): 177-182) AML Prognosis Stratification Guidelines suggest that AML patients with CEBPA double mutations who receive chemotherapy have a better prognosis and belong to the low-risk group. In addition, the World Health Organization (WHO) added a subtype of myeloid tumors with germline susceptibility gene mutations in 2016, and CEBPA germline susceptibility gene mutations are one of them. Therefore, CEBPA doubl...

Claims

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Application Information

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IPC IPC(8): G16B25/20G16B20/00C12Q1/6886C12Q1/6874
CPCG16B25/20G16B20/00C12Q1/6886C12Q1/6874C12Q2600/156Y02A50/30
Inventor 李霆
Owner 苏州元德友勤医学检验所有限公司