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Reagent and method for detecting ninth exon mutation of CALR gene

A technology of exons and reagents, applied in the fields of life sciences and biology, can solve the problems of high cost of reagents, inapplicability of daily screening, inapplicability of deletion and insertion mutation detection, etc.

Inactive Publication Date: 2015-04-29
南京艾迪康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

Although the next-generation sequencing technology has high sensitivity and high throughput, the cost of reagents is also high, which is suitable for scientific research services and not suitable for daily clinical screening.
[0006] Other commonly used methodologies for gene mutation detection, such as: HRM, ARMS, etc., because the mutation types of exon 9 of CALR are insertions and deletions, the above two methods cannot distinguish the mutation types, and the ARMS method mainly targets Mutations, less suitable for deletion and insertion mutation detection

Method used

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  • Reagent and method for detecting ninth exon mutation of CALR gene
  • Reagent and method for detecting ninth exon mutation of CALR gene
  • Reagent and method for detecting ninth exon mutation of CALR gene

Examples

Experimental program
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Effect test

Embodiment 1

[0032] The present invention is used to detect the amplification primer of the 9th exon mutation of CALR gene in the sample as shown in the following table:

[0033]

[0034]

Embodiment 2

[0036] Blood DNA extraction: TIANamp Genomic DNA Kit Blood / Cell / Tissue Genomic DNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used. Take 300μl of the whole blood sample to be tested and add it to a 1.5ml centrifuge tube, add 900ul red blood cell lysate and mix it upside down, centrifuge at 10000rpm for 1min, discard the supernatant; add 200uL buffer GA, shake until thoroughly mixed; add 20μl proteinase K solution, mix Mix well; add 200μl buffer GB, mix thoroughly by inversion, and place at 70°C for 10min. Add 200 μl of absolute ethanol, mix well, add the solution to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000rpm for 30s, discard the waste liquid; add 500μl buffer GD to CB3, centrifuge at 12000rpm for 30s, discard Waste liquid; add 600μl rinse solution PW to CB3, centrifuge at 12000rpm for 30s, discard waste liquid; repeat once. Put CB3 back into the collection tube and centrifuge at 12000rpm...

Embodiment 3

[0039] PCR amplification: Configure the PCR amplification system according to the following reagents and reagent volumes, including 0.3ul each of primers CALR-F (10μM) and CALR-R (10μM); 2×KOD Buffer 10ul, KOD enzyme (1U / ul) 0.3ul , d NTP (2mM) 2ul (Toyobo (Shanghai) Biotechnology Co., Ltd.); add 6.1ul of deionized water; finally add 1ul of DNA template. The amplification program is as follows: 95°C for 10 minutes; 98°C for 10s, 62°C for 30s (1°C drop per cycle), 68°C for 30s, 10 cycles; 98°C for 10s, 54°C for 30s, 68°C for 30s, 20 cycles; 68°C ℃ 2min.

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Abstract

The invention provides a reagent and a method for detecting the ninth exon mutation of a CALR gene by using a Sanger sequencing technology. The reagent and the method are high in detection specificity, and the mutation types of insertion and deficiency of the ninth exon of CALR can be specially analyzed, so that the false positive problem is eliminated, and missing inspection of rare mutation can also be avoided. The reagent and the method can be used for clinically screening genes of patients suffering from primary thrombocythemia and primary myelofibrosis.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to a method and primers for identifying and identifying the mutation status of exon 9 of CALR gene by using Sanger sequencing technology and sequencing map analysis. Sanger sequencing technology is still the gold standard and is suitable for the screening of clinical samples. technical background [0002] Myeloproliferative neoplasms (MPNs) are chronic myeloid neoplasms characterized by an overproduction of mature blood cells with possible progression to acute myeloid leukemia (AML). In addition to Ph+ chronic myeloid leukemia (CML), the other three most common MPNs are polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). [0003] Many Ph- MPN patients carry the JAK2V617F mutation, especially PV patients, accounting for about 90%. The mutation rates of JAK2V617F in both ET and PMF patients were 50-60%. JAK2V617F mutation...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6869C12Q2600/156
Inventor 邹媛董越王瑜陈红梅夏成青
Owner 南京艾迪康医学检验所有限公司
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