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Method for detection of BRAF and PI 3K mutations

A test sample, V600K technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of low abundance, inability to accurately detect low abundance mutations, failure to pass, etc.

Active Publication Date: 2015-06-03
GENOMICA SAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, state-of-the-art methods for the detection of BRAF and PI3K mutations have the disadvantage that any amplification products obtained cannot be detected by combining the former with mutation-specific probes, and especially with microbes containing such probes. array hybridization
[0013] Another related issue is that BRAF and PI3K mutations, which can be present as prognostic factors for tumor stage, metastasis, development, cellular heterogeneity, or allelic heterogeneity, are often found in low abundance in samples with respect to wild-type be found
Also, although many diagnostic methods are available for mutation detection, most cannot accurately detect low-abundance mutations

Method used

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  • Method for detection of BRAF and PI 3K mutations
  • Method for detection of BRAF and PI 3K mutations
  • Method for detection of BRAF and PI 3K mutations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Prepare the following reagent mix for multiplex ARMS amplification of BRAF mutations V600E and V600K in samples / cell lines / clones:

[0187]

[0188] Next, 30 μl of 5 μl of total eluate obtained from paraffin-embedded tissue sections (samples), or from different cell lines or clones, was added to a final reaction volume of 50 μl.

[0189] The thermocycling conditions for PCR are:

[0190]

[0191]

[0192] The results of the BRAF ARMS multiplex amplification are shown in figure 2 and image 3 middle.

[0193] Will figure 2 The different ARMS products of the samples / clones or cell lines indicated in Table 3 were visualized in a 2% agarose gel in which the products of the different amplification reactions were analyzed.

[0194] The lengths of the different amplification products are as follows:

[0195] - Length of the mutation-specific bands corresponding to mutations V600E and V600K: 144bp;

[0196] -The length of the IC band: 101bp (amplified product o...

Embodiment 2

[0204] The compositions of amplification mixes 2 and 3 corresponding to PI3K are shown below. The remaining reaction conditions were the same as those shown in Example 1 for BRAF.

[0205]

[0206]

[0207] Corresponding to 5 μl of the cell line HTC 116 (0.2 ng / μl) containing the PI3K mutation H1047R after their respective amplifications with amplification mix 2 reagents, and subsequent hybridization of the products obtained to the probe microarray, and subsequent visualization (a), or 5 μl of clinical samples containing DNA at a concentration of 20 ng / μl (b) are shown in Figure 4 a) and 4b).

Embodiment 3

[0209] The contents of the amplification mix, forward and reverse primers necessary for internal control and extraction control amplification, and plasmids necessary for internal control amplification such as pBSK with insert, were obtained at the concentrations indicated below, unchanged the result of:

[0210] ARMS Multiplex Reagent

[0211] IC: internal control; EC: extraction control.

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Abstract

The present invention is based on a detection method of the BRAF mutations V600E and V600K, and of the PI3K mutations E542K, E545D, E545K and H1047R, in a sample susceptible of containing one or more of such mutations, based on amplification of the sample with the primers of the present invention. Further, the present invention relates to (i) a kit which comprises, amongst its components, amplification reagents including one or more of the primers of the present invention; (ii) the primers themselves; and (iii) use of the method, kit and primers of above, for the diagnosis / prognosis of a pathological condition in a patient, particularly, of cancer.

Description

Background of the invention [0001] B-raf (or BRAF) is part of the Ras / Raf / MEK / MAP signaling pathway and plays a role in regulating the MAP kinase / ERK signaling pathway. Mutations in this gene are associated with various cancers, such as colorectal cancer (CRC), non-small cell lung cancer (NSCLC), malignant melanoma and adenocarcinoma. [0002] In colon cancer, oncogenic mutations in BRAF have been reported, almost all of which are V600E mutations (Davies H, et al., 2002, Nature 417:949-54; Rajagopalan H, et al., 2002, Nature 418:934) . The V600E mutation is located on exon 15 of the BRAF gene: at position 1799 of the BRAF coding sequence, a T is changed to an A, which results in a change from the valine (V) present in the wild-type BRAF protein to that corresponding to the mutant gene. Glutamine in protein (E). The V600K mutation (1798-1799 GT>AA) was also detected, but not in as large a number, and constitutes the second most abundant mutation in pathologies such as mel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/156C12Q2600/16
Inventor 玛丽亚·路易莎·比利亚埃尔莫萨胡安·莫斯科索德尔普拉多
Owner GENOMICA SAU
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