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Methods for detecting mutations in JAK2 nucleic acid

A nucleic acid and amino acid technology, which can be used in biochemical equipment and methods, microbial determination/inspection, extracellular fluid diseases, etc.

Inactive Publication Date: 2009-06-24
QUEST DIAGNOSTICS INVESTMENTS INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, characterizing the conjugative state of cell populations from samples such as blood cells, bone marrow cells, or buccal cells is difficult using standard assays because the wild-type JAK2 sequence from normal cells is tested along with any mutants and may contain homozygous or A sample of a hemizygous cell population appears to be heterozygous

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  • Methods for detecting mutations in JAK2 nucleic acid
  • Methods for detecting mutations in JAK2 nucleic acid
  • Methods for detecting mutations in JAK2 nucleic acid

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Embodiment

[0113] Sensitivity to detect JAK2 mutations from plasma was determined as follows. The HEL cell line (92.1.7, obtained from American Type Culture Collection, Manassas, VA) - carrying only the JAK2V617F mutation (no wild-type allele) - was maintained in RPMI 1640 with 10% fetal bovine serum. Lysates were prepared and mixed with plasma from normal (JAK2 wild type) individuals at various concentrations.

[0114] Total RNA was extracted from the mixture using the NucliSense Extraction Kit (bioMerieux Inc., Durham, NC) as recommended by the manufacturer. A PCR primer pair was designed to amplify the region including the JAK2 gene encoding amino acid 671. The primer sequences used for PCR and sequencing were as follows: JAK2-F (5'-GAC TAC GGT CAA CTG CAT GAA A-3') SEQ ID NO: 5, and JAK2-R (5'-CCA TGC CAA CTG TTT AGC AA -3') SEQ ID NO:6. One-step RT-PCR was performed in a 25 microliter reaction volume using the SuperScript III One-Step RT-PCR System with Platinum Taq (Invitrogen, ...

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Abstract

The present invention relates to methods for detecting JAK2 nucleic acid in acellular bodily fluid samples from patients with neoplastic disease and determining if the nucleic acid contains one or more mutations or one mutation and one deletion. The methods are useful for diagnosing patients that have cells with mutations in the JAK2 gene that effect kinase activity. The detection of such mutations can be used to determine treatment for patients or stratifying patients for therapy and management.

Description

field of invention [0001] The present invention relates to the field of cancer detection and more particularly to diagnostic methods useful for patients suffering from neoplastic diseases such as myeloproliferative diseases. Background of the invention [0002] The following discussion of the background to the invention is at least provided to assist the reader in understanding the invention and is not considered to describe or constitute prior art to the invention. [0003] Includes non-CML myeloproliferative disorders (MPDs) such as polycythemia vera (PV), essential thrombocythemia (ET), and chronic idiopathic myelofibrosis (IMF) as well as so far unclassified myeloproliferative disorders (MPD-ND) Certain neoplastic disorders are characterized by an abnormal increase in blood cells. See, for example, Vainchenker and Constantinescu, Hematology (American Society of Hematology (American Society of Hematology) Society of Hematology)) , 195-200 (2005). This increase is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12Q1/68
CPCC12Q2600/112C12Q2600/156C12Q1/6883C12Q1/6886C12Q2600/106C12Q2600/118A61P7/02A61P35/00A61P35/02A61P43/00
Inventor M·阿尔比塔W·马
Owner QUEST DIAGNOSTICS INVESTMENTS INC