WO2008060090A1

WO2008060090A1 - Methods, primers and kits for quantitative detection of jak2 v617f mutants using pyrosequencing

Info

Publication number: WO2008060090A1
Application number: PCT/KR2007/005701
Authority: WO
Inventors: Myunggeun Shin
Original assignee: Industry Foundation of Chonnam National University
Current assignee: Industry Foundation of Chonnam National University
Priority date: 2006-11-15
Filing date: 2007-11-14
Publication date: 2008-05-22
Anticipated expiration: 2009-05-15
Also published as: KR100789731B1

Abstract

Disclosed are a method for quantitative detection of JAK2 V617F mutants using pyrosequencing, and a primer and a kit for the same. According to the method of the invention, JAK2 V617F mutants can be quantitatively detected easily, sensitively and accurately. In addition, the method of the invention has high reproducibility, and time- and cost-effectiveness. Therefore, the method can provide an extremely useful tool for diagnosis, prognosis and prediction of treatment for various myeloid hematological neoplasms including myeloproliferative neoplasms (MPN).

Description

[DESCRI PT ION]

[invention Title]

METHODS, PRIMERS AND KITS FOR QUANTITATIVE DETECTION OF JAK2 V617F MUTANTS USING PYROSEQUENCING

[Technical Field]

The present invention relates to methods, primers and kits for quantitative detection of JAK2 V617F mutants and more specifically, and to methods, primers and kits for quantitative detection of JAK2 V617F mutants which are known to be closely associated with various myeloid hematological neoplasms including myeloproliferative neoplasms (MPN) using pyrosequencing.

[Background Art]

Janus kinase 2 (JAK2) mutation indicates the mutation in exon 12, specifically mutation from 1849G to 1849T, indicating that valine, the 617^th amino acid of the signal transducing molecule JAK2, is substituted with phenylalanine (V617F) . JAK2 mutation is known to be closely associated with the level of gene mutation and the progress of disease, particularly in various myeloid hematological neoplasms including myeloproliferative neoplasms (MPN) . Therefore, quantitative detection of JAK mutants is crucial for the diagnosis, prognosis, and prediction of the treatment.

To detect JAK2 in myeloproliferative neoplasms (MPN) patient, the method to identify/detect a mutant allele in purified granulocyte fraction of bone marrow or peripheral blood using various DNA sequencing platforms has been used. A semi-quantitative detection method has been tried recently, for which specific primers and hybridization probes are designed for real-time polymerase chain reaction (real-time PCR) to distinguish JAK2 mutant allele from wild type by LightCycler (Roche Applied Science) and JAK2 V617F is quantified from the control cell line, unfractionated peripheral blood or bone marrow sample and preserved diagnostic material (Olsen et al., Detection of the JAK2 V617F Mutation in Myeloproliferative Disorders by Melting Curve Analysis Using the LightCycler System, Arch. Pathol. Lab. Med. 2006; 130 : 997-1003) . However, this method enables only semi-quantitative detection, which is not sufficient for the quantitative detection of JAK2 V617F. In the meantime, PCR was performed using the forward primer represented by SEQ. ID. NO: 1 (5'-biotin- GAAGCAGCAAGTATGATGAGCA-3' ) and the reverse primer represented by SEQ. ID. NO: 2 (δ'-TGCTCTGAGAAAGGCATTAGAA- 3') as follows; 50 cycles of denaturation at 94°C for 7 minutes and at 94 ^°C for 30 seconds, annealing at 58 °C for 30 seconds, and extension 72^°C for 30 seconds, final extension at 72°C for 7 minutes and maintenance at 15^°C. And the single-stranded biotinylated PCR product was subjected to pyrosequencing using the sequencing primer represented by

SEQ. ID. NO: 3 (5'-TCTCGTCTCCACAGA-S') to genotyping and quantify allele of JAK2 V617F. As a result, JAK2 mutants were quantitatively detected in 20% of atypical or unclassified myeloproliferative disorder (MPD, UC) , 81% of polycythemia vera (PV) , 41% of essential thrombocythemia

(ET) and 43% of myelofibrosis (MF) (Amy V. Jones et al, Widespread occurrence of the JAK2 V617F mutation in myeloproliferative neoplasms (MPN) , Blood First Edition Paper, prepublished online May 26, 2005) . The disadvantage of this method is that quantitative detection of JAK2 mutation in polycythemia vera is high but low in unclassified myeloproliferative disorder and myelofibrosis.

[Disclosure! [Technical Problem]

The present inventors have conducted extensive studies to develop a novel method for quantitative detection of

JAK2 V617F mutants, which would be easier, more sensitive and accurate. As a result, the present inventors completed this invention by confirming that the object could be successfully achieved by performing PCR under specific conditions and performing pyrosequencing using a sequencing primer having a specific sequence.

It is an object of the present invention to provide a method for quantitative detection of JAK2 V617F mutants using pyrosequencing. It is another object of the present invention to provide a sequencing primer for use in the method.

It is still another object of the invention to provide a kit for quantitative detection of JAK2 V617F mutants comprising the sequencing primer.

[Technical Solution]

One aspect of the present invention provides a method for quantitative detection of JAK2 V617F mutants using pyrosequencing, which comprises the steps of: 1) performing PCR for an extracted total DNA by using the forward primer represented by SEQ. ID. NO: 1 and the reverse primer represented by SEQ. ID. NO: 4; and

2) performing pyrosequencing for the PCR product obtained in step 1) using the sequencing primer represented by SEQ. ID. NO: 5.

According to this method, PCR is preferably performed as follows; 45 cycles of denaturation at 95^°C for 5 minutes and 95^°C for 15 seconds, annealing at 62°C for 30 seconds, and extension at 72 ^°C for 15 seconds, followed by final extension at 95 °C for 5 minutes. Another aspect of the present invention provides a sequencing primer represented by SEQ. ID. NO: 5 for use in quantitative detection of JAK2 V617F mutants using pyrosequencing. Still another aspect of the present invention provides a kit for quantitative detection of JAK2 V617F mutants using pyrosequencing, which comprises:

1) the forward primer and the reverse primer represented by SEQ. ID. NO: 1 and SEQ. ID. NO: 4 respectively for PCR for pyrosequencing; and

2) the sequencing primer represented by SEQ. ID. NO: 5 for pyrosequencing.

[Description of Drawings] The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which:

Figure 1 is a diagram illustrating the nucleotide sequence of jak2 gene in which wild type 1849G and mutant 1849G →T are shown;

Figure 2 is a diagram illustrating the PCR conditions for pyrosequencing;

Figure 3 is a photograph illustrating the result of the examination m which the PCR product for pyrosequencing was electrophoresed on 1% agarose gel and then stained with EtBr, followed by observation under UV light;

Figure 4 is a graph illustrating the result of pyrosequencing to jak2 mutation using the control (1849G type) ;

Figure 5 is a graph illustrating the result of pyrosequencing to jak2 mutation using the sample of myeloproliferative neoplasms (MPN) patient (1849G→T type).

[Mode for Invention]

In the present invention, mutation rate of JAK2 gene is measured, and clinical features and progress of a patient are studied retrospectively to analyze the relationship thereof, by providing the method, the primer and the kit for quantitative detection of JAK2 V617F mutants using pyrosequencing .

In step 1 of the invention, total DNA was extracted and primers were designed. In step 2 of the invention, PCR was performed with a reaction mixture containing the extracted DNA and the primers above. In step 3 of the invention, pyrosequencing was performed with the PCR product obtained above.

First, primers shown in Table 1 were designed based on the JAK2 sequence represented by SEQ. ID. NO: 6 and shown in Figure 1, considering primer size, PCR product size, GC content and T_m .

[Table l] PCR primers and a sequencing primer for pyrosequencing

The PCR composition and condition for pyrosequencing are as shown in Table 2 and Figure 2.

[Table 2] PCR composition and condition

PCR mixture (120 bp) PCR condition

Fl primer 1 ≠ 95 ^°C, 5 minutes

Rl primer i id lOxbuffer 2 Id 95^°C, 15 45 cycles seconds

10 mM dNTP 0.4 Id 62 "C, 30 seconds

Tag o.i id 72°C, 15 polymerase seconds gDNA (20 i id 95 ^°C, 5 minutes nq/βl)

Distilled 13.5 Id water

Total 20 Id

120 bp PCR product was obtained by PCR with the total DNA and primers according to the composition and condition as shown above (see Figure 3). Pyrosequencing was performed with the PCR product using the reagents described in Table 3 and Table 4 (see Figures 4 and 5) .

[Table 3] Streptavidin bead mixture and sequencing primer mixture

[Table 4]

Purification solution for PCR product immobilized onto streptavidin beads and Pyro Gold reagent

In the present invention, the DNA template amplified by PCR is converted to single-strand, which is bound with the sequencing primer. Then, a substrate, adenosine

5'phosphosulfate (APS) and luciferin are added thereto together with 4 enzymes (DNA polymerase, ATP sulfurylase, luciferase and apyrase) to proceed with reaction. 4 dNTPs are added to the reaction mixture stepwise. Among them, a specific dNTP is used for the synthesis with the template strand using DNA polymerase. When a complementary dNTP is introduced into the template, hybridization occurs and pyrophosphate (PP₁) is released therefrom, and the amount of released PP₁ is the same as the number of hybridized nucleotide. ATP sulfurylase catalyzes ATP production from APS and PP₁ in the reaction solution. The produced ATP activates luciferase to convert luciferin into oxyluciferin with emitting light. The emitted light is in proportion to the amount of ATP which means it is in proportion to the amount of dNTP used for hybridization. The emitted light can be detected by CCD camera and programmed by software. The peak of the light signal is in proportion to the number of hybridized nucleotide, and therefore the types and numbers of the hybridized nucleotide can be detected. Apyrase, the enzyme degrading nucleotide, degrades unreacted residual dNTP and ATP. Once a dNTP is decomposed, another dNTP is added to the reaction solution. One dNTP is added per time. In the meantime, deoxyadenosine alpha-thio triphosphate (dATPαS) can be used instead of dATP, which is to be used only for DNA polymerization but not for the activation of luciferase. As reaction progresses, a DNA strand complementary to the template DNA strand is synthesized, which is represented by a signal peak of pyrogram enabling sequencing. Pyrosequencing enables simultaneous sequencing of samples within 15 minutes, and so it can be used for rapid quantitative detection of genetic variation. The present invention thus provides a method for quantitative detection of JAK2 V617F mutants using pyrosequencing, which not only provides an extremely important tool in diagnosis of myeloid hematological neoplasms including myeloproliferative neoplasms (MPN) but also are important for prognosis and prediction of treatment therefor.

Hereinafter, the present invention will be specifically explained with reference to examples, which are provided only for better understanding of the invention, but should not be construed to limit the scope of the invention in any manner.

In the following examples, 78 myeloproliferative neoplasms (MPN) patients and 20 healthy normal individuals participated as volunteers for pyrosequencing to detect quantitatively JAK2 V617F mutants.

Example 1: DNA extraction and PCR for pyrosequencing

To detect JAK2 V617F mutants quantitatively, DNA was extracted from the peripheral blood of each myeloproliferative neoplasms (MPN) patient (QIAamp DNA Blood mini kit) . DNA extraction was performed according to the manufacturer's instructions. The extracted total DNA was diluted to the concentration of 20 nq/β&, and subjected to

PCR using the forward primer represented by SEQ. ID. NO: 1

(5'-GAAGCAGCTIAGTATGATGAGCA-S') and the reverse primer represented by SEQ. ID. NO: 4 (5'-TGCTCTGAGAAAGGCATTAGAAA- 3')- PCR was performed with a total volume of 20 βi consisting of 5 βl of 10* PCR buffer [20 mM Tris-HCl (pH 8.0),

100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5%

Nonidet P-AO, 50% glycerol], 400 M of dNTP, 2.5 U of Taq polymerase (Solgent F-Taq kit) , 10 pmoles of primers, triple distilled water, and 20 ng of genomic DNA as follows; 45 cycles of denaturation at 95^°Cfor 5 minutes and at 95^°Cfor 15 seconds, annealing at 62 °C for 30 seconds, and extension at 72^°Cfor 15 seconds, and final extension at 95^°Cfor 5 minutes. The PCR product was subjected to electrophoresis on 1% agarose gel and was stained with EtBr₇ followed by observation under UV light. As a result, 120 bp-sized product was obtained. The results are shown in Figure 3.

Example 2: Pyrosequencing 20 βl of sterilized triple distilled water was added to 20 βl of the PCR product (total volume: 40 βl) , followed by immobilization with 40 βl of streptavidin sepharose beads™ mixture. Sequencing primer mixture was prepared and distributed on plate low by 40 β& per each. 10 minutes later, the plate containing the PCR product and the plate low were placed on worktable. To wash the PCR product immobilized on streptavidin beads, a probe was dipped in the PCR product immobilized on streptavidin beads, 70% ethanol, 0.2 N NaOH, and 1 x PSQ washing buffer, in that order 5 seconds for each and 5 second intermission for drying between dipping was allowed. After a while, PSI was turned off and the probe was placed on the plate low. The product immobilized on streptavidin beads were shaken off, followed by reaction for one minute at 95 ^°C The substrate mixture and nucleotide (dATPαS, dCTP, dGTP, dTTP) (kit Biotage) were distributed in the cartridge of PSI, which was then run according to the program set up. 4 dNTPs were added sequentially, one at a time, for the polymerization. PP₁ (inorganic pyrophosphate) from the polymerized dNTP emitted light by the enzymatic reaction. The emitted light was shown to signal peak at the order of sequentially added dNTP, and each peak was shown high or low, in proportion to the number of reacted dNTP.

The results are shown in Figure 4 and Figure 5. As explained hereinbefore, experiments were performed with 78 myeloproliferative neoplasms (MPN) patients and 20 normal controls. As a result, JAK2 mutants were quantitatively detected in 77% of polycythemia vera, 26% of essential thrombocythemia, 100% of myelofibrosis, 67% of unclassified myeloproliferative disorder and 53% at average. By contrast, JAK2 mutants were not detected at all in normal controls. [industrial Applicability]

According to the present invention, JAK2 V617F mutants can be quantitatively detected by pyrosequencing easily, sensitively and accurately. The method of the invention has high reproducibility, and time- and cost-effectiveness.

Therefore, the method can provide an extremely useful tool for diagnosis, prognosis and prediction of treatment for various myeloid hematological neoplasms including myeloproliferative neoplasms (MPN) , particularly unclassified myeloproliferative disorder and myelofibrosis.

[Sequence Listing Free Text]

SEQ. ID. NO: 1 is a nucleotide sequence of the forward primer for PCR according to the prior art and the present invention;

SEQ. ID. NO: 2 is a nucleotide sequence of the reverse primer for PCR according to the prior art;

SEQ. ID. NO: 3 is a nucleotide sequence of the sequencing primer for pyrosequencing according to the prior art;

SEQ. ID. NO: 4 is a nucleotide sequence of the reverse primer for PCR according to the present invention;

SEQ. ID. NO: 5 is a nucleotide sequence of the sequencing primer for pyrosequencing according to the present invention;

SEQ. ID. NO: 6 is a nucleotide sequence of JAK2 gene

Claims

[CLAIMS!

[Claim l]

A method for quantitative detection of JAK2 V617F mutants using pyrosequencing, which comprises the steps of: 1) performing PCR with an extracted total DNA by using the forward primer represented by SEQ. ID. NO: 1 and the reverse primer represented by SEQ. ID. NO: 4; and

[Claim 2]

The method for quantitative detection of JAK2 V617F mutants according to claim 1, wherein the PCR is performed with 45 cycles of denaturation at 95°Cfor 5 minutes and at 95 ^°C for 15 seconds, annealing at 62 ^°C for 30 seconds, and extension at 72^°Cfor 15 seconds, and final extension at 95 ^°C for 5 minutes.

[Claim 3]

A sequencing primer represented by SEQ. ID. NO: 5, which is used for the quantitative detection of JAK2 V617F mutants using pyrosequencing.

[Claim 4] A kit for quantitative detection of JAK2 V617F mutants using pyrosequencing, which comprises:

2) the sequencing primer represented by SEQ. ID. NO: 5 for pyrosequencing.