Patent Application
20130095491
This application claims the priority of Chinese Patent Application No. 201010113309.3, filed on Feb. 24, 2010, the disclosure of which is incorporated herein by reference.
BRAF gene belongs to the RAF gene family. It is an oncogene encoding a serine/threonine kinase, an important member of the RAS-RAF-MEK-ERK signal transduction pathway, which plays important roles in regulating cell proliferation, differentiation and apoptosis (Ikenoue T., Cancer Res., 2003, 63(23):8123-37). Therefore, BRAF gene has been implicated in tumorigenesis and tumor development, and it can serve as potential diagnosis marker and therapy target.
BRAF gene locates at the 7q34 site, encoding a protein of 783 amino acids. Many studies have shown that different mutations can occur in the BRAF gene with various ratios in malignant melanoma, colon cancer, lung cancer, thyroid carcinoma, hepatocarcinoma and pancreatic cancers (Davies H. et al., Nature, 2002, 417(6892): 949-54), wherein about 90% BRAF mutations are located at nucleotide No 1799, in which T is replaced with A, so that the encoded amino acid at position 600 changes from glutamine to valine (Wang L. et al., Cancer Res., 2003, 63(17): 5209-12). The present invention uses real-time quantitative PCR to detect the mutation at the codon encoding amino acid at position 600 in BRAF, a tumor-related gene, so as to predict drug resistance to chemotherapy (Di Nicolantonio F. et al., J. Clin. Oncol., 2008, 26(35): 5705-12).
The detecting method of the present invention has the following advantages: easy manipulation, and easy standardization. Other methods, such as allele specific oligonucleotide probe hybridization method, are very much dependent on hybridization conditions, and therefore require strict control of the experimental conditions. The restriction fragment length polymorphism method, on the other hand, needs a lot of human labor, and can not generate quantitative results. The method of the present invention has short experimental cycle, and can be completed within 2 hours. It doesn't need verification of the results by sequencing, whereas the direct sequencing and high resolution melting analysis need 4 days to 2 weeks. Sensitivity of the method of the present invention is high, which, after optimizing experimental conditions, can reach 1% for detecting mutations, whereas sensitivity of direct sequencing is 20-50%. Specificity of the method of the present invention is also high. Immunohistochemistry (IHC) method can easily get pseudo-positive and pseudo-negative results, and can not determine the position and types of point mutations. The unique advantage of the present invention is accurate quantification. By using absolute quantification method to analyze data, draw standard curve, and accurately determine the content of wild-type gene and mutant gene in the samples, one can obtain ratio of the mutant gene in the samples, which will be helpful for clinical diagnosis and therapeutic selection. Furthermore, the present invention is safe and non-toxic, other methods such as chemical breaking method of mismatched base pairs need isotope and toxic chemical agents.
The question that the present invention addresses is to provide an assay kit for quantitatively detecting an BRAF gene mutation, which can quantitatively detect the following mutations: GTG, the codon encoding amino acid at position 600 in BRAF gene, is replaced with GAG.
To address the above question, the present invention provides quantitative detection kit containing a mixture comprising Taq enzyme, 10× Taq buffer, MgCl2, dNTP mixture, PCR primers which can specifically amplify the sequences at BRAF gene mutation positions, and probes which can specifically identify wild-type sequences and mutant sequences, together with method of the detection, as follows:
(1) Separately design upstream and downstream primers around the mutation positions of Codon 600 of BRAF gene; and design specific probes according to each mutant site. Said probes can specifically bind wild-type sequences or the mutant sequences to be detected at specific BRAF sites, so as to determine whether the tested mutations occur at said sites.
(2) To accurately and quantitatively determine the ratio of the BRAF mutations, standards were designed in the present invention.
(3) Use fluorescent quantitative PCR to detect the samples and standards.
(4) Obtain standard curves for quantitative detection from the detection results of the standards, and calculate the ratios of BRAF gene mutations to the total wild type BRAF gene in the samples to be tested.
Prior to said step (1) it further includes: extracting nucleic acid from the samples, purifying it and determining the content of it.
The probes for fluorescent quantitative PCR specifically bind the sequences at BRAF gene mutation sites under suitable PCR conditions. Preferably, said probes link a fluorescence emitting group at their 5′ end, and link a fluorescence quenching group at their 3′ end. Said fluorescence emitting group is selected from FAM, TET, HEX and ROX. Said fluorescence quenching group is selected from BHQ, TAMARA. Preferably, said emitting group is FAM, and said quencher group is BHQ. Preferably, the sequences of said probes are selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
Said standards include at least one of plasmids, genome DNA or chemically synthesized sequences. Preferably, said standards comprises a wild-type plasmid, a mutant plasmid, or both a wild-type plasmid and a mutant plasmid, wherein said wild-type plasmids include wild-type sequences of BRAF gene, and said mutant plasmids include mutant sequences of BRAF gene. More preferably, said standards are consisted of a wild-type plasmid, a mutant plasmid, or both a wild-type plasmid and a mutant plasmid.
The tested samples include fresh tissue, paraffin embedded tissues, cell lines, blood, pleural effusion, peritoneal effusion, saliva, digestive juice, urine and feces.
Said primers consist of upstream primers and downstream primers. Preferably, said primers are selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
Said quantitative detection kit for BRAF gene mutations includes the agents selected from: the above-mentioned primers, probes and standards. Preferably, said kit further includes Taq enzyme, 10× Taq buffer, MgCl2, and dNTP mixture. Preferably, the ratio of said primer to probe is 2:1-10:1, and said primer comprises a forward primer and reverse primer in a ratio of 1:3-3:1. Said standards include a mixture of said plasmids in a certain ratio, wherein the ratio of the content of wild-type plasmids to mutant plasmids is 0%-100%.
The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the technology and together with the description.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make use of the present invention, and are neither intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. The experimental conditions not indicated in the Examples, are generally conventional, such as those disclosed in “Molecular Cloning, A Laboratory Manual, 3rd ed, (Sambrook J.)”, or those suggested by the manufacturer.
The tumor cell lines we tested included cell lines of: non-small-cell lung carcinoma (NSCLC; A549, H460, H838 and H1703), breast cancer (MCF-7, BT474 and HuL100), malignant mesothelioma (H513, H2052, H290, MS-1 and H28), thyroid carcinoma (KAT10), colon cancer (SW480, S1-M1-80), head and neck cancer (U87), cervical carcinoma (Hela), sarcoma (Mes-SA, Saos-2 and A204).
The fresh human tumor tissues, peripheral blood, paraffin embedded tissues we tested included: NSCLC, mesothelioma, colon cancer, malignant melanoma, renal carcinoma, esophagus cancer, thyroid carcinoma, malignant cancer and ovarian cancer.
Extraction of Sample DNA
DNA extracting kit from Qiagen Inc., Promega Inc., or Roche Inc. can be used to extract genomic DNA from the samples. Content and purity of the extracted DNA can be determined by using Nanodrop ND1000 (Gene Inc.) (OD260/OD280 is about 1.8, OD260/OD230 is more than 2.0). For example, the sample DNA may be extracted using the DNA Extracting Kit (Promega Inc.) as follows:
1. DNA Extraction From Fresh Tissues
(1) cut a bean-sized tissue using scissors, put it into a mortar, cut it into pieces, and ground it into powder by adding liquor nitrogen.
(2) add 600 μl pre-cooled lysate into the mortar, blow it 6 times using 1 ml tip, sufficiently mix the tissue powder and the lysate, transfer the mixture into a 1.5 ml EP tube, then turn it over 6 times, water bath under 65° C. for 20 minutes.
(3) add 3 μl RNase, turn over 6 times to mix homogenously, water bath under 37° C. for 20 minutes.
(4) cool it to room temperature, add 200 μl protein precipitation agent, turn over 6 time to mix homogenously, place it on ice for 5 minutes, 13000×g centrifuge 4 minute at room temperature.
(5) transfer the supernatant into a new EP tube pre-added with 600 μl isopropanol (room temperature), gently mix 6 times, then 13,000×g centrifuge at room temperate for 1 minutes.
(6) discard the supernatant, add 600 μl 70% ethanol (room temperature) into precipitate, 13,000×g centrifuge at room temperate for 1 minutes.
(7) aspirate out ethanol, air dry for 15 minutes.
(8) add 40 μl DNA dissolving solution into the precipitate, incubate at 65° C. for 1 hour or 4° C. overnight. 2. DNA Extraction From Paraffin Embedded Tissues
(1) add 1 mg or less tissues into 1.5 ml centrifuge tube.
(2) add freshly prepared 100 μl incubation buffer/proteinase K solution, and incubate at 56° C. overnight based on the type of the samples.
(3) take out the incubated sample tube, add two times volume of lysate buffer.
(4) vortex-oscillate the resin for 10 seconds until the resin is fully suspended, add 7 μl fully suspended resin, vortex-oscillate the resin for 3 seconds, then incubate at room temperature for 5 minutes.
(5) vortex-oscillate the resin for 2 seconds, put the tube on a magnetic separation rack (MagneSphere®), immediately conduct magnetic separation.
(6) carefully remove all solution, without touching the resin on the tube wall.
(7) add 100 μl lysate buffer, remove the tube from the magnetic separation rack, vortex-oscillate for 2 seconds.
(8) put the tube back to the magnetic separation rack, remove all the lysate.
(9) add 100 μl 1× washing fluid, remove the tube from the magnetic separation rack, vortex oscillate 2 seconds.
(10) put the tube back to the magnetic separation rack, remove all the lysate.
(11) repeat step (9) and (10) twice, totally wash three times, and remove all the liquid after the last wash.
(12) open the lid, put the tube on the magnetic separation rack, air dry for 5 minutes.
(13) add 25 μl eluate.
(14) close the lid, vortex oscillate for 2 seconds, incubate at 65° C. for 5 minutes.
(15) take out the incubated tube, vortex oscillate for 2 seconds, immediately put it on the magnetic separation rack.
(16) carefully transfer the DNA solution into a selected container.
3. DNA Extraction of Whole Blood
(1) obtain 300 μl anticoagulant whole blood, add 900 μl cell lysate, blow 6 times using 1 ml tip, so that the whole blood and the cell lysate are sufficiently mixed, place it under room temperature for 10 minutes, blow with the tip three times.
(2) 13,000×g centrifuge under room temperature for 20 seconds, discard the supernatant, shake violently, add 300 μl pre-cooling lysate, blow with 1 ml tip until the precipitate are totally dissolved.
(3) add 1.5 μl RNase, turn over 6 times to mix homogenously, water bath under 37° C. for 20 minutes.
(4) cool to room temperature, add 100 μl protein precipitation agent, turn over 6 times to mix homogenously, place it on ice for 5 minutes, 13,000×g centrifuge under room temperature for 4 minutes.
(5) transfer the supernatant to a new EP tube previously added 300 μl isopropanol (room temperature), gently mix 6 times, centrifuge under room temperature for 1 minutes.
(6) discard the supernatant, add 1 ml 70% ethanol (room temperature) into the precipitate, turn over 6 times to mix homogenously, 13,000×g centrifuge under room temperature for 1 minutes.
(7) aspirate out ethanol, air dry for 15 minutes.
(8) add 40 μl DNA dissolving solution, stay at 65° C. for 1 hour or 4° C. overnight.
4. DNA Extraction of Pleural Effusion
(1) obtain 5 ml pleural effusion, 2000 rpm centrifuged at room temperature for 10 minutes, remove the supernatant, add 1 ml cell lysate, turn over 6 times to mix homogenously, stay under room temperature for 10 minutes.
(2) 13,000×g centrifuged under room temperature for 20 seconds, discard the supernatant, shake violently, add 1 ml pre-cooling lysate, mix until the precipitate totally dissolved.
(3) add 3 μl RNase, turn over 6 times to mix homogenously, water bath under 37° C. for 20 minutes.
(4) cool to room temperature, add 200 μl protein precipitation agent, turned over 6 times to mix homogenously, place it on ice for 5 minutes, 13,000×g centrifuged under room temperature for 4 minutes.
(5) transfer the supernatant to a new EP tube previously added 5 ml isopropanol (room temperature), gently mix 6 times, 13,000×g centrifuged under room temperature for 1 minutes.
(6) discard the supernatant, add 1 ml 70% ethanol (room temperature) into the precipitate, turn over 6 times to mix homogenously, 13,000×g centrifuged under room temperature for 1 minutes.
(7) aspirate out ethanol, air dry for 15 minutes.
(8) add 40 μl DNA dissolving solution, stay at 65° C. for 1 hour or 4° C. overnight.
5. DNA Extraction From Cell Lines
(1) obtain at least 1×106 cells, transfer them into a 1.5 ml EP tube, 13,000×g centrifuged at room temperature for 10 seconds. If the cells are adherent cells, they should be digested by trypsin before collecting them.
(2) discard the supernatant, add 200 μl PBS to wash the cells, 13,000×g centrifuged under room temperature for 10 seconds, discard the supernatant, shake violently until the precipitate is suspended.
(3) add 600 μl pre-cooling lysis solution, blow to mix homogenously with 1 ml tip until no visual cell blocks.
(4) add 3 μl RNase, turn over 6 times to mix homogenously, water bath under 37° C. for 20 minutes.
(5) cool to room temperature, add 200 μl protein precipitation agent, turn over 6 times to mix homogenously, place it on ice for 5 minutes, 13,000×g centrifuged under room temperature for 4 minutes.
(6) transfer the supernatant to a new EP tube previously added 600 μl isopropanol (room temperature), gently mix 6 times, 13,000×g centrifuged under room temperature for 1 minutes.
(7) discard the supernatant, add 600 μl 70% ethanol (room temperature) into the precipitate, turn over 6 times to mix homogenously, 13,000×g centrifuged under room temperature for 1 minutes.
(8) aspirate out ethanol, air dry for 15 minutes.
(9) add 40 μl DNA dissolving solution, stay at 65° C. for 1 hour or 4° C. overnight.
1. Construction of Wild-Type Plasmids (
1.1 Preparation of the Carrier
TA cloning carrier pMD18-T was purchased from TAKARA Inc.
1.2 Preparation of the Insert
The insert is prepared using PCR. The template of PCR is the sample genome DNA extracted in Step 1. The reaction system and amplification condition are shown in the following tables (Table 1, Table 2 and Table 3):
1.3 After recovering the target fragment using QIAgen Gel Recover Kit, insert said fragment into pMD18-T (purchased from TAKARA Inc.) by TA colonizing.
1.4 Amplify the constructed plasmid in E. coli DH5α strain, and harvest by extraction and purification (the methods are showed in Molecular Cloning, A Laboratory Manual, 3rd ed. pages 96-99 and 103.
1.5 Identify the plasmid by double enzyme digestion of BamHI and HindIII.
1.6 Sequence the strains having positive result, and use the strains with correct sequence as the standard containing wild-type sequence (
2. Construction of mutant plasmids: design mutant primers of mutant sites, obtain the standards containing mutant sequences by DPN1 method.
2.1 Design the mutant primers (
2.2 Use 5 ng wild-type plasmid as template, and use mutant primers and Pfu enzyme to mutate the target sites. The amplification system and condition are shown in Table 1, Table 4 and Table 3.
During the preparation of the plasmid containing BRAF Codon 600 GTG→GAG mutant sequence, BRAF-1-F (SEQ ID NO:7) and BRAF-1-R (SEQ ID NO:8) primers are needed to add into the amplification system.
2.3 treat the product obtained in step 2.2 with DPN1 enzyme, recover the product after incubating at 37° C. for 1 hour, amplify in E. coli DH5α strain, and harvest by extraction and purification.
2.4 Identify the plasmid by double enzyme digestion of BamHI and HindIII.
2.5 Sequence the strains having positive result, and use the strains with correct sequence as the standard containing mutant sequence (
1. The templates for fluorescent quantitative PCR are the genome DNA of thyroid carcinoma and colon cancer samples extracted in Example 1, and the standards prepared in Example 2. Double-distilled water is served as negative control. For drawing the standard curves, the standards are diluted as 1 ng/μl, 0.5 ng/μl, 0.25 ng/μl, 0.125 ng/μl, 0.0625 ng/μl, 0.03125ng/μl.
2. The reaction system and condition are shown in Table 2, Table 5, Table 6 and Table 7, wherein the fluorescent emission group bound to the probe is selected from FAM, TET, HEX or ROX, the quench group is selected from BHQ or TAMARA.
For detecting the mutations in BRAF Codon 600, it needs to prepare two systems, in which all reagents are same except the probes. Specifically, for detecting BRAF Codon 600 wild-type genes, it needs to add BRAF-W-1 (SEQ ID NO: 9) or BRAF-W-2 (SEQ ID NO: 10) probes into the system; for detecting BRAF Codon 600 GTG→GAG mutant gene, it needs to add BRAF-M-1 (SEQ ID NO: 11) or BRAF-M-2 (SEQ ID NO: 12) probes into the system.
3. Drawing the Standard Curve
The standard curve is drawn based on the CT values obtained from the standard in Step 3.
4. Calculation of the Ratio of Specific BRAF Mutation in a Sample
According to the standard curve, the copy numbers of wild-type and mutant genome DNA are calculated from the CT values of the sample. Then we obtain the ratio of mutant BRAF DNA to total BRAF DNA (wild-type plus all mutants at said site). As shown in
5. Result of Detection
In this Example, we detected the BRAF gene mutation in 80 cases of tissues, whole blood and cell line samples of thyroid carcinoma and colon cancer, and found that 8 cases had mutations. The mutation ratios, i.e. the ratio of mutant gene to non-mutant gene in those samples, can be seen in Table 8.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20101013309.9 | Feb 2010 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN2011/000270 | 2/22/2011 | WO | 00 | 8/24/2012 |
