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One-tube kit for detecting KRAS and BRAF gene mutations in plasma free DNA

A kit and gene technology, applied in the field of molecular biology, to achieve the effects of saving material and labor costs, improving throughput, and quickly and clearly distinguishing results

Active Publication Date: 2020-04-07
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mutated DNA of cancer cells in peripheral blood is an extremely rare mutated gene that exists in the background of a large number of wild-type genes, and its existence rate is generally about 1%. challenge

Method used

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  • One-tube kit for detecting KRAS and BRAF gene mutations in plasma free DNA
  • One-tube kit for detecting KRAS and BRAF gene mutations in plasma free DNA
  • One-tube kit for detecting KRAS and BRAF gene mutations in plasma free DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] 1. KRAS gene mutation detection

[0096] Take the detection of KRAS gene GGT>GCT (G12A) mutation in clinically collected plasma samples as an example.

[0097] KRAS-G12S-F:-ATATAAACTTGTGGTAGTTGGAGCAA- (SEQ ID NO: 1)

[0098] KRAS-G12R-F:-AATATAAACTTGTGGTAGTTGGAACTC- (SEQ ID NO: 2)

[0099] KRAS-G12C-F:-ATATAAACTTGTGGTAGTTGGTGTTT- (SEQ ID NO: 3)

[0100] KRAS-G12S-R:-ACCTCTATTGTTGGATCATATTCG- (SEQ ID NO:4)

[0101] KRAS-G12D-R:-GCACTCTTGCCTACGCCTT- (SEQ ID NO: 6)

[0102] KRAS-G12A-R:-GGCACTCTTGCCTACGTCAG- (SEQ ID NO: 7)

[0103] KRAS-G12V-R:-GCACTCTTTGCCTACGCGAA- (SEQ ID NO:8)

[0104] KRAS-G12D-F:-CACATTTTCATTATTTTATTATAAGGC- (SEQ ID NO:9)

[0105] KRAS-Probe1:FAM-CCTTGACGATACAGCTAATTCAGAATCATTT-BHQ1 (SEQ ID NO:5)

[0106] KRAS-Probe2:FAM-CACAAGTTTATATTCAGTCATTTTCAGCAGGC-BHQ1 (SEQ ID NO: 10)

[0107] KRAS-Blocker1:GCCTAC G CCACCA G CTCCAACT-Pho (underlined bases are marked for nucleic acid modification, 3' -pho indicates phosphorylation modification) (SEQ ID ...

Embodiment 2

[0132] 1. BRAF gene mutation detection

[0133] Take the detection of BRAF gene V600E mutation in clinically collected plasma samples as an example.

[0134] KRAS-G12S-F:-ATATAAACTTGTGGTAGTTGGAGCAA- (SEQ ID NO: 1)

[0135] KRAS-G12R-F:-AATATAAACTTGTGGTAGTTGGAACTC- (SEQ ID NO: 2)

[0136] KRAS-G12C-F:-ATATAAACTTGTGGTAGTTGGTGTTT- (SEQ ID NO: 3)

[0137] KRAS-G12S-R:-ACCTCTATTGTTGGATCATATTCG- (SEQ ID NO:4)

[0138] KRAS-G12D-R:-GCACTCTTGCCTACGCCTT- (SEQ ID NO: 6)

[0139] KRAS-G12A-R:-GGCACTCTTGCCTACGTCAG- (SEQ ID NO: 7)

[0140] KRAS-G12V-R:-GCACTCTTTGCCTACGCGAA- (SEQ ID NO:8)

[0141] KRAS-G12D-F:-CACATTTTCATTATTTTATTATAAGGC- (SEQ ID NO:9)

[0142] KRAS-Probe1:FAM-CCTTGACGATACAGCTAATTCAGAATCATTT-BHQ1 (SEQ ID NO:5)

[0143] KRAS-Probe2:FAM-CACAAGTTTATATTCAGTCATTTTCAGCAGGC-BHQ1 (SEQ ID NO: 10)

[0144] KRAS-Blocker1:GCCTAC G CCACCA G CTCCAACT-Pho (underlined bases are marked for nucleic acid modification, 3' -pho indicates phosphorylation modification) (SEQ ID NO: 11) ...

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Abstract

The invention relates to a one-tube kit for detecting KRAS and BRAF gene mutations in plasma free DNA, and belongs to the technical field of molecular biology. The kit of the invention comprises: KRAS-G12S-F, KRAS-G12R-F, KRAS-G12C-F, KRAS-G12S-R, KRAS-G12D-R, KRAS-G12A-R, KRAS-G12V-R, KRAS-G12D-F, KRAS-Probe1, KRAS-Probe2, KRAS-Blocker1, BRAF-V600-F, BRAF-V600-R and BRAF-Probe. The kit of the invention can quickly detect mutations of the human KRAS gene and the BRAF gene in the free DNA sample of peripheral blood simultaneously in one tube.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a tube of primers capable of detecting seven hot spot mutations of the KRAS gene and one hot spot mutation of the BRAF gene in human plasma free DNA and a kit thereof. Background technique [0002] There are three genes in the RAS gene family that are closely related to human tumors: HRAS, KRAS and NRAS. The proteins encoded by these three genes have about 90% amino acid sequence homology, and the molecular weight is 21KDa, also known as RASp21 protein. The KRAS signaling pathway is the downstream pathway of EGFR and other signal transduction. When the RAS gene is mutated, the ability of the encoded RAS protein to hydrolyze GTP-RAS is reduced, so that the signal transduction pathway is continuously activated and stimulates cell proliferation and differentiation. , eventually leading to malignant transformation of cells. [0003] KRAS is a proto-oncogene, about 35kb in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/156C12Q2531/113C12Q2547/101C12Q2561/101C12Q2537/163
Inventor 张伟付亚成邬麟
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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