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Method of quantifying mutant allele burden of target gene

An allele and target gene technology, applied in biochemical equipment and methods, microbial assay/inspection, etc., can solve problems such as underestimation of allele burden

Pending Publication Date: 2019-10-01
CHANG GUNG MEDICAL FOUND CHANG GUNG MEMORIAL HOSPITAL CHIAYI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although DNA from JAK2-mutant UKE-1 and HEL cells is commonly used in the establishment of standard curves for the quantification of the JAK2 V617F mutation, neither cell is considered ideal because HEL cells harbor multiple JAK2 complexes. This, as well as UKE-1 cells undergo clonal evolution with increased JAK2 replicas during in vitro culture, which may lead to an underestimation of the JAK2 V617F mutant allelic burden

Method used

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  • Method of quantifying mutant allele burden of target gene
  • Method of quantifying mutant allele burden of target gene
  • Method of quantifying mutant allele burden of target gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1. Construction of a recombinant plasmid as a standard for quantifying JAK2 V617F mutant allele burden (mutant allele burden)

[0084] To minimize copy number variation and normalize the quantification of JAK2 V617F allele burden, the pair recombinant plasmids of the present invention were constructed as follows.

[0085] Since fewer somatic mutations are present in exon 21 of the JAK2 gene, applicants chose this region as an internal control. use TRI Genome DNA extracted from HEL cells was used as a template and PCR was carried out using the forward primer H_JAK2_clon_exon21_F and the reverse primer H_JAK2_clon_exon21_R shown in Table 1, and the control PCR product (264bp) with JAK2 exon 21 partial DNA sequence It was obtained. The resulting control PCR product was purified and then ligated into a cloning vector using the yT&A cloning kit (Yeastern Biotech, Taipei, Taiwan, China) according to the manufacturer's instructions to obtain the plasmid JAK2_Ctrl_yT...

Embodiment 2

[0090] Example 2. Verification of the accuracy of the recombinant plasmid of the present invention as a standard for quantifying JAK2 V617F mutant allele burden

[0091] experimental method:

[0092] The wild-type and mutant target recombinant plasmids JAK2_WT_Ctrl_yT&A and JAK2_V617F_Ctrl_yT&A (representing 0% and 100% JAK2 V617F mutant allele burden, respectively) obtained from Example 1 were obtained in various different ways as shown in Table 2 below. ratio to obtain six recombinant plasmid mixtures (i.e., 100%, 50%, 10%, 1%, 0.1% and 0.01%) each with a different JAK2 V617F mutant allele burden, which Supplied as a standard dilution for the JAK2 V617F mutant allele.

[0093] Table 2

[0094] JAK2 V617F mutant allele burden (%) 100 50 10 1 0.1 0.01 JAK2_WT_Ctrl_yT&A (μL) 0 50 90 99 99.9 99.99 JAK2_V617F_Ctrl_yT&A (μL) 100 50 10 1 0.1 0.01

[0095] The JAK2 V617F mutation in the above-mentioned recombinant plasmid mixture was test...

Embodiment 3

[0099] Example 3. Quantification of JAK2 V617F mutant allele burden in recombinant plasmid mixtures and clinical samples by quantitative duplex PCR assay

[0100] A. Establishment of standard curve for JAK2 V617F mutant allele burden

[0101] 1. Plasmid-based standard curve:

[0102] Seven recombinant plasmid mixtures (with JAK2 V617F mutant allele burdens of 100%, 10%, 1%, 0.1%, 0.01%, 0.001% and 0%, respectively) were prepared according to the method described in Example 2. is prepared. Each of these recombinant plasmid mixtures was used as a DNA template (template) for quantitative double-PCR analysis, which was performed in Rotor-Gene Q (RGQ) 5plexHRM using the PCR reaction mixture (PCR reaction mixture) and reaction conditions shown in Table 3. Platform (Rotor-Gene Q 5plex HRM platform) (Cat No.: 9001580, Qiagen, Germany) was carried out. JAK2_exon 14 mutant allele-specific primer pairs and probes and JAK2_exon21-specific primer pairs and probes as listed in Table 4 we...

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Abstract

The invention relates to a method and kit for quantifying a mutant allele burden of a target gene in a subject. The invention also discloses use of a nucleic acid agent for preparing the kit for quantifying a mutant allele burden of a target gene in a subject. The nucleic acid agent includes a first plasmid, a second plasmid and a reaction mixture, wherein steps for quantifying a mutant allele burden of a target gene by utilizing the kit include providing the first plasmid that includes a mutant allele sequence and an internal control sequence, and the second plasmid that includes a wild-typeallele sequence and the internal control sequence; and subjecting DNA of the subject to quantitative polymerase chain reaction to measure a mutant allele expression level of the target gene, so as todetermine the mutant allele burden of the target gene in the subject based on a standard curve of the mutant allele burden of the target gene created by serial dilution of the first and second plasmids.

Description

【Technical field】 [0001] The present invention relates to a method for quantifying the mutant allele burden of a target gene by using a recombinant plasmid pair as a standard. 【Background technique】 [0002] Classic myeloproliferative neoplasms (MPNs) are multipotent hematopoietic stem cell disorders characterized by an overproduction of various blood cells. MPNs include three main clinical entities, namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (primary myelofibrosis, PMF). A hallmark of the genetic background of MPNs that constitute the diagnostic criteria of the World Health Organization (WHO) classification are MPN-restricted driver mutations, including those in the Janus kinase 2 ( Janus kinase 2, JAK2), calreticulin (CALR), and myeloproliferative leukemia virus (myeloproliferative leukemia virus, MPL) in those. Mutations in any of the mutually exclusive driver genes lead to continuous activation of the downstream signaling...

Claims

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Application Information

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IPC IPC(8): C12Q1/6827
CPCC12Q1/6827C12Q2545/114C12Q2537/163C12Q2545/10C12Q2545/107C12Q1/6851C12Q1/686C12Q2600/16C12Q2600/156C12Q2600/166C12Q1/6876
Inventor 陈志丞许家祯
Owner CHANG GUNG MEDICAL FOUND CHANG GUNG MEMORIAL HOSPITAL CHIAYI
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