Compositions and methods for multiplex analysis of nras and braf nucleic acids
AssigneeQiagen Mansfield, Inc.
InventorsLilly I. KONG, Kiran Madanahally DIVAKAR
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Abstract
Described herein are methods and assays relating to the detection of NRAS and/or BRAF alterations (e.g. variations in copy number and expression level, and/or the presence of mutations, including point mutations). Existing methods are limited in their clinical usefulness by, e.g., limited sensitivity, inter-lab discordance, or inability to provide the necessary multiplex ability. The methods and assays provided herein permit multimodal, multiplex assaying for faster, more cost-effective testing and screening of patients, permitting improved healthcare.
Claims24
An assay for detecting mutations of NRAS and/or BRAF, the assay comprising
- contacting a portion of a nucleic acid sample with a set of primers,
- wherein the set of primers comprises subsets of primer pairs, wherein each primer pair amplifies a NRAS or BRAF sequence comprising a sequence variation;
- performing a PCR amplification regimen comprising cycles of strand separation, primer annealing, and primer extension on a reaction mixture comprising the portion of the sample and the one or more sets of primers;
- detecting the presence or absence of the amplicon for each primer pair;
- wherein the presence of an amplicon indicates the presence of the sequence variation for which that primer pair is specific;
- and wherein one or more of the primers are selected from the group consisting of SEQ ID NOs:
- 1-39.
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A composition comprising one or more primer pairs selected from the group consisting of molecules having the sequences of:
- SEQ ID NO:s 1 and 8;
- SEQ ID NO:s 2 and 8;
- SEQ ID NO:s 3 and 8;
- SEQ ID NO:s 4 and 8;
- SEQ ID NO:s 5 and 8;
- SEQ ID NO:s 6 and 8;
- SEQ ID NO:s 7 and 8;
- SEQ ID NO:s 9 and 14;
- SEQ ID NO:s 10 and 14;
- SEQ ID NO:s 11 and 14;
- SEQ ID NO:s 12 and 14;
- SEQ ID NO:s 13 and 14;
- SEQ ID NO:s 15 and 18;
- SEQ ID NO:s 16 and 18;
- SEQ ID NO:s 17 and 18;
- SEQ ID NO:s 19 and 33;
- SEQ ID NO:s 20 and 33;
- SEQ ID NO:s 21 and 33;
- SEQ ID NO:s 22 and 33;
- SEQ ID NO:s 23 and 33;
- SEQ ID NO:s 24 and 33;
- SEQ ID NO:s 25 and 33;
- SEQ ID NO:s 26 and 32;
- SEQ ID NO:s 27 and 32;
- SEQ ID NO:s 28 and 32;
- SEQ ID NO:s 29 and 32;
- SEQ ID NO:s 30 and 32;
- SEQ ID NO:s 31 and 32;
- SEQ ID NO:s 34 and 37;
- SEQ ID NO:s 35 and 38;
- and SEQ ID NO:s 36 and 39.
A composition comprising fluorescently-labelled amplification products resulting from the amplification of target polynucleotides with one or more primer pairs selected from the group consisting of molecules having the sequences of:
- SEQ ID NO:s 1 and 8;
- SEQ ID NO:s 2 and 8;
- SEQ ID NO:s 3 and 8;
- SEQ ID NO:s 4 and 8;
- SEQ ID NO:s 5 and 8;
- SEQ ID NO:s 6 and 8;
- SEQ ID NO:s 7 and 8;
- SEQ ID NO:s 9 and 14;
- SEQ ID NO:s 10 and 14;
- SEQ ID NO:s 11 and 14;
- SEQ ID NO:s 12 and 14;
- SEQ ID NO:s 13 and 14;
- SEQ ID NO:s 15 and 18;
- SEQ ID NO:s 16 and 18;
- SEQ ID NO:s 17 and 18;
- SEQ ID NO:s 19 and 33;
- SEQ ID NO:s 20 and 33;
- SEQ ID NO:s 21 and 33;
- SEQ ID NO:s 22 and 33;
- SEQ ID NO:s 23 and 33;
- SEQ ID NO:s 24 and 33;
- SEQ ID NO:s 25 and 33;
- SEQ ID NO:s 26 and 32;
- SEQ ID NO:s 27 and 32;
- SEQ ID NO:s 28 and 32;
- SEQ ID NO:s 29 and 32;
- SEQ ID NO:s 30 and 32;
- SEQ ID NO:s 31 and 32;
- SEQ ID NO:s 34 and 37;
- SEQ ID NO:s 35 and 38;
- and SEQ ID NO:s 36 and 39.
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Dependencies are inferred from the claim text.