Mutational analysis of JAK2

A mutant, part-technology application in the field of analysis of genetic mutations in the JAK2 gene to address problems such as overproduction of white blood cells and platelets

Inactive Publication Date: 2014-08-13
EPISTEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It may also lead to an overproduction of white blood cells and platelets

Method used

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  • Mutational analysis of JAK2
  • Mutational analysis of JAK2
  • Mutational analysis of JAK2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: JAK2

[0052] figure 1 The sequence (SEQ ID NO 4) of the wild-type JAK2 mRNA sequence is given. Residue 2343 is highlighted.

[0053] figure 2 Sequences of forward and reverse primers and probe sequence for binding wild-type JAK2 are given.

[0054] The V617F mutation in JAK2 is caused by the G2343T mutation in the nucleic acid sequence. The probe binds to the region spanning the mutation; if the mutation (or indeed any other mutation in the sequence) is present, the Tm of the hybridizing probe is lowered.

[0055] Clearly, the probe sequence is perfectly complementary to the associated wild-type target sequence and has one base mismatch compared to each possible mutant sequence. The mismatch is within the probe, not at either end. The probe is A probe having a pair of fluorophores that alters its emission when the probe adopts a double-stranded duplex compared to the single-stranded form.

[0056] DNA samples containing the JAK2 region were amplifie...

Embodiment 2

[0073] Example 2 - Comparison with other tests

[0074] We performed a comparison of the method described herein with other assays to determine reliability, accuracy and sensitivity. Twenty blinded DNA samples were obtained for testing and assayed using three different methods: 1. Genedrive assay (using the Genedrive thermal cycler from Epistem as described herein); 2. Using the Roche LightCycler 480II 3. JAK2V617F MutaQuant kit (Ipsogen) on Roche LightCycler480II.

[0075] 1. Genedrive assay

[0076] Assays are based on amplicon melting curve analysis of WT and mutant peak heights. Blind test samples on Genedrive TM analyzed on three different Genedrive TM Three independent analytical runs on the part were performed in singular. V617F peak melting temperature (Tm) = 56-58°C. JAK2Wt peak Tm = 61-65°C.

[0077] Image 6 Exemplary melting curves from wild-type samples and mixed wild-type-V617F mutant samples are shown.

[0078] Figure 7 Shows the thermal cycling steps...

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Abstract

An assay for mutations in JAK2 is described. The assay uses selective amplification of mutant alleles with a blocker probe which preferentially hybridises to wild type alleles. The same probe is then used to detect presence or absence of wild type sequences. It is not necessary to know the specific mutant sequence beforehand.

Description

field of invention [0001] The present invention relates to methods for the analysis of genetic mutations and in particular single nucleotide polymorphisms (SNPs) in the JAK2 gene. Aspects of the invention also relate to nucleic acid probes and primers for analyzing such mutations. Background of the invention [0002] Janus kinase 2 (Janus kinase2, commonly referred to as JAK2) is a protein that has been involved in type II cytokine receptor family (such as interferon receptor), GM-CSF receptor family (IL-3R, IL-5R and GM - CSF-R), signaling human proteins of members of the gp130 receptor family (eg IL-6R) and single chain receptors (eg Epo-R, Tpo-R, GH-R, PRL-R). JAK2 signaling is activated downstream of protein receptors. Mutations in JAK2 have been implicated in polycythemia vera, essential thrombocythemia and other myeloproliferative disorders. This mutation, valine to phenylalanine at position 617, appears to make hematopoietic cells more sensitive to growth factors s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2600/156C12Q1/6883C12Q1/6858C12Q2527/107C12Q2537/163C12Q2565/107C12Q2537/159C12Q1/6813C12Q1/6844
Inventor 本·科布
Owner EPISTEM
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