CN103045590A - Primer and method for detecting drug-resistant mutation point in BCR/ABL fusion gene ABL kinase zone by using same - Google Patents
Primer and method for detecting drug-resistant mutation point in BCR/ABL fusion gene ABL kinase zone by using same Download PDFInfo
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- CN103045590A CN103045590A CN2012105758172A CN201210575817A CN103045590A CN 103045590 A CN103045590 A CN 103045590A CN 2012105758172 A CN2012105758172 A CN 2012105758172A CN 201210575817 A CN201210575817 A CN 201210575817A CN 103045590 A CN103045590 A CN 103045590A
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Abstract
The invention relates to a primer and a method for detecting a drug-resistant mutation point in a BCR/ABL fusion gene ABL kinase zone by using the same. The method comprises the steps as follows: (1) extracting RNA (Ribose Nucleic Acid); (2) synthetizing cDNA (complementary DNA) through inverse transcription of RNA; (3) taking cDNA as a template, and respectively taking BCR(p210)-F and ABL(p210)-R as an upstream primer and a downstream primer and taking BCR(p190)-F and ABL-(p190)-R as an upstream primer and a downstream primer to carry out the primary PCR (Polymerase Chain Reaction) amplification so as to obtain amplified products; (4) taking ABLKinase-F and ABLKinase-R as an upstream primer and a downstream primer to carry out the secondary PCR amplification; and (5) sequencing the amplified products. The invention further discloses a method for obtaining ABL kinase zone segments in a BCR/ABL fusion gene by nested PCR and sequencing the ABL kinase zone segments by special primers. All common drug-resistant mutation points in the BCR/ABL fusion gene ABL kinase zone can be detected in a reaction system, so that the experimental cost is reduced, and the accuracy and the sensitivity of the sequenced result of the gene is improved greatly.
Description
Technical field
The invention belongs to biological technical field, is the method that primer and this primer detect BCR/ABL fusion gene ABL kinases district resistant mutational site specifically.
Background technology
Chronic granulocytic leukemia (chronic myeloid leukemia, CML) cell has t (9; 22) (q34; Q11) Philadelphia chromosome (Ph chromosome) and the BCR/ABL fusion gene of transposition formation, they occupy critical role in hemopathy research.In more than 30 year after finding Ph karyomit(e), clinical diagnosis and the curative effect monitoring of extensively being CML of cytogenetic methods detection Ph karyomit(e).Especially nearly more than ten years, molecular biology method detected the application of BCR/ABL fusion gene and popularizes gradually.(another name: imatinib mesylate is the highly effective medicine that designs for the BCR/ABL fusion rotein imatinib), in recent years at CML and Ph to imatinib mesylate
+Application gets more and more in acute lymphoblastic leukemia (acute lymphocytic leukemia, the ALL) treatment, but resistance finally appears in some patients were, and the BCR/ABL fusion gene ABL kinases region mutation that resistance is relevant detects and enjoys attention.
The BCR/ABL fusion gene sees 95% above CML patient, also see 20%～25% adult ALL and 2%～4% children ALL patient, about 6% adult acute myelocytic leukemia (acute myeloblastic leukemia, AML), also can detect Ph karyomit(e) and BCR/ABL fusion gene among 1% children AML and 10% acute mixed cell type leukemia (acute mixed lineage leukemia, the AMLL) patient.
Different BCR/ABL fusion gene types is relevant with disease phenotype.In the BCR/ABL fusion gene, mainly carried the main information of malignant change of cell by the ABL section, and the broken site major effect disease phenotype of BCR section.The abl gene broken site is usually located at introne 1, has comprised whole exon 2s and later abl gene sequence.The common breaking point of BCR gene has three: (1) M-BCR district, the zone of 5.8kb between exon b1～b5, be transcribed into b2a2 or b3a2 type mRNA, the BCR/ABL fusion rotein (p210) of coding 210kD sees CML more than 90% and 33% Ph
+Adult BALL patient.(2) m-BCR district, the zone of the 54.4kb between exon e2 ' and e2 is transcribed into e1a2 type mRNA, and the fusion rotein (p190) of coding 190kD sees 2/3 Ph
+Adult BALL, 3% atypia CML and chronic granulocyte monocytic leukemia (chronic myelo-monocytic leukemia, CMML) patient.(3) μ-BCR district is positioned at exons 19 downstreams, is transcribed into e19a2 type mRNA, and the fusion rotein of coding p230kD is mainly seen in Ph
+CNL (chromic neutrophilic leukemia, CNL).
The use of imatinib mesylate makes the CML patient of some hematopoietic stem cell transplantation indication abandon transplanting, but imatinib mesylate can not effect a radical cure CML.Because it plays a role by the kinase activity that suppresses the BCR/ABL fusion rotein, itself can not suppress the formation of fusion rotein, so easily recurrence after the drug withdrawal.The resistance phenomenon is just found in the imatinib mesylate clinical application soon, can be divided into primary drug resistance and Secondary cases resistance.The mechanism of resistance comprises: the ABL kinases district of (1) BCR/ABL fusion rotein undergos mutation, and the space conformation of imatinib mesylate binding site tyrosine kinase domain is changed, and causes the imatinib mesylate can not the competitive binding fusion rotein; (2) BCR/ABL fusion rotein overexpression has surpassed the competition binding ability of imatinib mesylate; (3) amplification of BCR/ABL fusion gene genome copy number often also can cause the overexpression of BCR/ABL fusion rotein; (4) CML clone's hyperplasia not exclusively relies on BCR/ABL, also has other gene unconventionalities, unusual etc. such as multidrug resistance gene.May above-mentioned one or more mechanism cause together resistance, but approximately 80% resistance is that the sudden change of having reported is above 50 kinds because the ABL kinases region mutation of BCR/ABL fusion rotein causes.Approximately 10% resistance is because the overexpression of BCR/ABL fusion gene causes, and may be because amplification or the chromosomal appearance of other atypia Ph of BCR/ABL fusion gene in the genome increases in this case drug dose and usually can reach certain effect.Some patients were just carries the clone of sudden change before using the imatinib mesylate treatment, be the major cause of primary drug resistance.
The amplification of mutant clon may be a progressive process, and initial stage mutant clon ratio is lower, in case but the progress of disease is increased and causes in the appearance meeting very soon.In the situation that stable disease or BCR/ABL fusion gene quantitatively reduce gradually, seldom can detect the situation of sudden change.Patient that imatinib mesylate is just controlled with to have obtained the situation of suddenling change among the patient that complete cytobiology alleviates also more rare.The sudden change of resistance often comes across before the CML acute transformation phase or follows acute transformation phase to occur, so periodic monitoring sudden change situation is necessary, can find early sudden change and corresponding adjustment treatment plan.For the sudden change of some prognosis mala, it is very significant just detecting earlier when low-level.The ABL kinases region mutation of finding at present mainly concentrates on ATP-binding domain (P ring, the 244th～255 amino acid), T315 and region of activation (A ring), and the drug-resistant intensity that different mutational sites cause is different.The patient that partly suddenlys change can have therapeutic response to novel kinase inhibitor Dasatinib (Dasatinib, BMS-354825) or nilotinib (Nilotinib, AMN 107), can be used as the alternative medication of imatinib mesylate.The sudden change resistance that betides the P ring is strong, and is all very high such as sudden change drug-resistant intensities such as G250E, Y253H and E255K.In the sudden change of report, especially the highest with the T315I drug-resistant intensity at present, be that only report is to all sudden changes of resistance of all kinase inhibitor.The sudden change poor prognosis of these height resistances need be used the other treatment scheme as early as possible instead.And the drug-resistant intensities such as M351T, E355G, M244V are not high, can resist resistance by improving dosage.Therefore, BCR/ABL fusion gene ABL kinases district to the patient before with the imatinib mesylate treatment checks order, can effectively do an assessment for situations such as comprehensive and quantitative monitoring and imatinib mesylate medicament-resistant mutations, according to every patient's the state of an illness and advancing of disease, carry out accurate individualized treatment.
Because the BCR/ABL fusion gene is crossed over the sheet segment length, nearly fifties kinds and also have the unknown mutation type of ABL kinases region mutation kinds have been reported, at present for detecting BCR/ABL gene medicament-resistant mutation, only detect with traditional RQ-PCR method for some or certain several known mutations site, can't satisfy the clinician makes correct diagnosis and implements individuation to patient's full appreciation treatment plan far away.This detection adopts nest-type PRC and Sanger sequence measurement to detect all sudden changes of ABL kinases district, and has accuracy and the sensitivity of height.
Summary of the invention
The purpose of this invention is to provide a kind of method that can detect multiple BCR/ABL fusion gene ABL kinases district resistant mutational site.
For achieving the above object, the technical solution used in the present invention is: primer, comprise amplimer and sequencing primer,
Described amplimer sequence is as follows:
BCR_p210-F:5’-TGACCAACTCGTGTGTGAAACTC-3’、
ABL_p210-R：5’-TCCACTTCGTCTGAGATACTGGATT-3’、
BCR_p190-F:5’-ACCGCATGTTCCGGGACAAAAG-3’、
ABL_p190-R：5’-ACAGCATTCCGCTGACCATCAATAAG-3’、
Described sequencing primer sequence is as follows:
ABL?Kinase-F：5’-TGGTTCATCATCATTCAACGGTGG-3’、
ABL?Kinase-R：5’-AGACGTCGGACTTGATGGAGAACT-3’。
Further: described primer sequence is or/and the sequence that 3 ' end prolongs to 5 ' end of amplimer or sequencing primer.
Further: described primer sequence is greater than 85% sequence with amplimer or sequencing primer homology.
Further: described primer sequence is the sequence with amplimer or the complementation of sequencing primer base sequence.
Further: described primer sequence is by any one or more than one the composite sequence in amplimer or the sequencing primer sequence.
A kind of method of utilizing above-mentioned primer to detect BCR/ABL fusion gene ABL kinases district resistant mutational site may further comprise the steps:
(1), extracts RNA;
(2), carry out reverse transcription reaction, acquisition reverse transcription product cDNA;
(3), take the reverse transcription product of step (2) as the PCR reaction template, take BCR_p210-F and ABL_p210-R as the upstream and downstream primer, BCR_p190-F and ABL_p190-R are that the upstream and downstream primer carries out first round pcr amplification, obtain amplified production respectively;
(4), get the amplified production of step (3), take turns pcr amplification take ABL Kinase-F and ABL Kinase-R as the upstream and downstream primer carries out second, obtain amplified production;
(5), get the amplified production of step (4), take ABL Kinase-F and ABL Kinase-R as sequencing primer, the detection of checking order.
Useful technique effect of the present invention is: the BCR/ABL fusion gene has P210 (b2a2, b3a2), P190 (e1a2) hypotype, the present invention has designed respectively two pairs of primers in the first round of nest-type PRC reaction, guaranteed the amplification efficiency to BCR/ABL fusion gene P210 type and P190 type, designed second for abl gene again and taken turns primer, improved sensitivity and the specificity of reaction; Take turns primer as sequencing primer with second, can detect all common BCR/ABL medicament-resistant mutations in a reaction system, reduced the cost of experiment, the accuracy and the sensitivity that have improved greatly gene sequencing result have the prospect of large-scale promotion application.
Description of drawings
Fig. 1 is the structural representation of BCR/ABL fusion gene P210 type;
Fig. 2 is the structural representation of BCR/ABL fusion gene P190 type;
Fig. 3 is the gel electrophoresis figure of primer extension product of the present invention;
Fig. 4 is the gel electrophoresis figure that the method for the invention detects 10 CML sample amplified productions;
Fig. 5 is that the method for the invention detects the normal sequencing result figure of CML sample;
Fig. 6 is the forward sequencer map that the method for the invention detects ABL kinases district F359V sudden change.
Embodiment
Below in conjunction with embodiment technical solution of the present invention is clearly and completely described, obviously, described embodiment only is the present invention's part embodiment, rather than whole embodiment.Based on the embodiment among the present invention, those of ordinary skills belong to the scope of protection of the invention not making the every other embodiment that obtains under the creative work prerequisite.
Embodiment 1:
Primer and this primer detect the method for BCR/ABL fusion gene ABL kinases district resistant mutational site;
The primer that BCR/ABL fusion gene ABL kinases district resistant mutational site detects is as follows:
Amplimer:
BCR_p210-F:5’-TGACCAACTCGTGTGTGAAACTC-3’；
ABL_p210-R：5’-TCCACTTCGTCTGAGATACTGGATT-3’；
BCR_p190-F:5’-ACCGCATGTTCCGGGACAAAAG-3’；
ABL_p190-R：5’-ACAGCATTCCGCTGACCATCAATAAG-3’；
Sequencing primer:
ABL?Kinase-F：5’-TGGTTCATCATCATTCAACGGTGG-3’；
ABL?Kinase-R：5’-AGACGTCGGACTTGATGGAGAACT-3’；
1, the extraction of RNA:
(1) in the 15ml centrifuge tube of processing through autoclaving, adds the fresh blood of collection and the erythrocyte cracked liquid of 7ml, the vortex oscillation mixing.
(2) ice bath is after 15 minutes, low-speed centrifugal 3 minutes (1500r/min).
(3) centrifugal complete after, outwell supernatant liquid, add again the erythrocyte cracked liquid of 4ml in the precipitation, behind the vortex oscillation mixing, low-speed centrifugal 3 minutes (1500r/min).
(4) remove supernatant after centrifugal, add again 8ml PBS, vortex oscillation mixing, low-speed centrifugal 3 minutes (1500r/min).
(5) centrifugally remove supernatant after complete.
(6) add the PBS of 1.5ml in the precipitation, will precipitate the piping and druming mixing after, get respectively 1ml and 0.5ml to two through in the clean centrifuge tube of sterilizing.1ml is experiment usefulness, and 0.5ml gives over to backup.
(7) above-mentioned centrifuge tube is carried out low-speed centrifugal 3 minutes (1500r/min).
(8) centrifugally remove supernatant after complete, in two pipes, add the Trizol of 1ml and the write cell lysis buffer of 300ul respectively, and the piping and druming mixing.Adding Trizol is to test usefulness, and what add write cell lysis buffer is backup, puts into-20 ℃ of preservations.
(9) the backup pipe of writing a clean sterilization for each 1ml Trizol pipe, and add the trichloromethane of 200ul in the centrifuge tube that has added 1ml Trizol, behind the thermal agitation, 4 ℃, the centrifugal 10min of 12000r/min.
(10) in centrifugal process, to backing up the Virahol that adds 500ul in the pipe.Etc. centrifugal complete after, supernatant is taken in the corresponding clean centrifuge tube, up and down behind the mixing ,-20 ℃ of refrigeration 20min.
After (11) 20 minutes, centrifuge tube is taken out, 4 ℃, the centrifugal 12min of 15000r/min.
(12) centrifugal complete after, remove supernatant, add again the ethanol of 1ml75%, up and down behind the mixing ,-4 ℃, the centrifugal 8min of 12000r/min.
(13) after centrifugal, outwell supernatant, in stink cupboard that ethanol is air-dry, to sinking to the bottom an amount of ultrapure water of middle adding, obtain total RNA again.
2, reverse transcription step: the M-MLV PCR RT Kit test kit specification sheets with reference to Promega company is reversed to cDNA with RNA, the reverse transcription system:
| Reagent | Volume (uL) |
| |
6 |
| Random primer | 1 |
| M-MLV | 0.25 |
| dNTP | 0.5 |
[0063]?
| The RNA inhibitor | 0.25 |
| 10×M-MLV?buffer | 2 |
| |
10 |
The reverse transcription reaction condition:
| RNA and random primer be the system of 7uL altogether | 70℃5min |
| Add again altogether 10uL system of reverse transcription reaction mixed solution 3uL | 37℃60min |
3, nest-type PRC reaction
Get reverse transcription product 2ul, as the PCR reaction template, take BCR (p210)-F and ABL (p210)-R as the upstream and downstream primer, BCR (p190)-F and ABL (p190)-R are that the upstream and downstream primer carries out first round pcr amplification, and the PCR reaction system is 20ul.Concrete prescription is as follows:
| Composition | Add-on (μ l) |
| LA Taq enzyme | 0.2 |
| 10×LA?PCR?Buffer?II(Mg 2+Plus) | 2 |
| dNTP?Mixture（10mM?each） | 0.8 |
| First round primer (5 μ M each) | 2 |
| |
2 |
| ddH 2O | 13 |
| Cumulative volume | 20 |
The amplification condition of first round PCR is as follows:
Take turns the PCR reaction with getting 2ul after 50 times of dilutions of first round PCR product for second, ABL Kinase-F and ABL Kinase-R are the upstream and downstream primer, and system is prepared the same first run.
Second to take turns the pcr amplification condition as follows:
The PCR product is got 3ul and is used for the agarose electrophoresis evaluation, and the result as shown in Figure 3.
4, sequencing reaction
(1) PCR product enzymolysis, digestion: configure the enzyme digestion reaction system according to following requirement:
| ddH 2O | 1.2μL |
| 10×SAP?Buffer | 0.3μL |
| SAP enzyme (1U/ μ L) | 1μL |
| Exo I enzyme (5U/ μ L) | 0.5μL |
| The PCR product | 5μL |
| TotaL | 8μL |
After system configurations is complete, cover tightly 8 unions lid, the vibration mixing is also centrifugal rapidly, then places the following temperature bath program of operation on the PCR instrument:
| 37℃ | 30min |
| 95℃ | 15min |
| 12℃ | ∞ |
The enzymolysis after product should be kept at 4 ℃ (continuing downstream process in the 24h) or-20 ℃ (surpassing 24h can not use).
(2) sequencing reaction operation:
Configure the sequencing reaction system according to following requirement: use ABL Kinase-F and ABL Kinase-R to carry out forward and reverse order-checking as sequencing primer.
| ddH 2O | 6μL |
| 5×Sequencing?Buffer | 1.8μL |
| BigDye3.1 | 0.4μL |
| Sequencing primer (5 μ M) | 1μL |
| Template behind the enzymolysis *1 | 0.8μL |
| Total | 10μL |
After system configurations is complete, cover tightly 8 unions lid, the vibration mixing is also centrifugal rapidly, then places the following temperature bath program of operation on the PCR instrument:
The sequencing reaction product should keep in Dark Place at-20 ℃.
(3) ethanol deposition and purification
Every hole adds 1 μ L125mM EDTA solution and 1 μ L3M sodium acetate solution (pH=5.2) in the respective aperture of 96 orifice plates, adds whole sequencing reaction products and 40 μ L dehydrated alcohols again.
After centrifugal the finishing, take off 8 union dished covers, get 3 layers of dust-free paper, pad is placed on 96 orifice plates at least 4 thieving papers together.After the Roberval's balance trim, be inverted 96 orifice plates+dust-free paper+thieving paper, rotating speed 500g, 4 ℃ of temperature, centrifugal 1min dries all supernatants.
Take out 96 orifice plates, discard dust-free paper and thieving paper, every hole adds 100 μ L75% dehydrated alcohols, covers 8 union dished covers, does not want mixing, and after the Roberval's balance trim, forward is placed 96 orifice plates in refrigerated centrifuge, rotating speed 2100g, 4 ℃ of temperature, centrifugal 5min.
After centrifugal the finishing, take off 8 union dished covers, get 3 layers of dust-free paper, pad is at least 4 thieving papers.Be inverted 96 orifice plates (dust-free paper is close to 96 orifice plates) to dust-free paper+thieving paper, most of supernatant liquor is absorbed by dust-free paper.
Again get 3 layers of dust-free paper, pad is placed on 96 orifice plates at least 4 thieving papers together.After the Roberval's balance trim, be inverted 96 orifice plates+dust-free paper+thieving paper, rotating speed 500g, 4 ℃ of temperature, centrifugal 1min dries all supernatants.
Take out 96 orifice plates, discard dust-free paper and thieving paper, 96 orifice plates are placed on 12-15min in the lucifuge box, make the ethanol volatilization clean.
Every hole adds 10 μ L Hi-Di methane amides, covers 8 union dished covers, and of short duration centrifugal 96 orifice plates with 95 ℃ of sex change 2min of 96 orifice plates, are then preserved on ice, machine order-checking in the preparation.
(4) machine testing on the sequenator: use 3500xL genetic analysis instrument to carry out capillary electrophoresis and detect.
The clinical sample checking: the whole blood sample of collecting 10 routine patients CML is tested, and uses detection method of the present invention to test.
Used amplimer and sequencing primer that BCR/ABL fusion gene ABL kinases district resistant mutational site detects are as follows:
Amplimer:
BCR(p210)-F:5’-TGACCAACTCGTGTGTGAAACTC-3’；
ABL(p210)-R：5’-TCCACTTCGTCTGAGATACTGGATT-3’；
BCR(p190)-F:5’-ACCGCATGTTCCGGGACAAAAG-3’；
ABL(p190)-R：5’-ACAGCATTCCGCTGACCATCAATAAG-3’；
Sequencing primer:
ABL?Kinase-F：5’-TGGTTCATCATCATTCAACGGTGG-3’；
ABL?Kinase-R：5’-AGACGTCGGACTTGATGGAGAACT-3’
1, the extraction of RNA:
(1) in the 15ml centrifuge tube of processing through autoclaving, adds the fresh blood of collection and the erythrocyte cracked liquid of 7ml, the vortex oscillation mixing.
(2) ice bath is after 15 minutes, low-speed centrifugal 3 minutes (1500r/min).
(3) centrifugal complete after, outwell supernatant liquid, add again the erythrocyte cracked liquid of 4ml in the precipitation, behind the vortex oscillation mixing, low-speed centrifugal 3 minutes (1500r/min).
(4) remove supernatant after centrifugal, add again 8mlPBS, vortex oscillation mixing, low-speed centrifugal 3 minutes (1500r/min).
(5) centrifugally remove supernatant after complete.
(6) add the PBS of 1.5ml in the precipitation, will precipitate the piping and druming mixing after, get respectively 1ml and 0.5ml to two through in the clean centrifuge tube of sterilizing.1ml is experiment usefulness, and 0.5ml gives over to backup.
(7) above-mentioned centrifuge tube is carried out low-speed centrifugal 3 minutes (1500r/min).
(8) centrifugally remove supernatant after complete, in two pipes, add the Trizol of 1ml and the write cell lysis buffer of 300ul respectively, and the piping and druming mixing.Adding Trizol is to test usefulness, and what add write cell lysis buffer is backup, puts into-20 ℃ of preservations.
(9) the backup pipe of writing a clean sterilization for each 1mlTrizol pipe, and add the trichloromethane of 200ul in the centrifuge tube that has added 1ml Trizol, behind the thermal agitation, 4 ℃, the centrifugal 10min of 12000r/min.
(10) in centrifugal process, to backing up the Virahol that adds 500ul in the pipe.Etc. centrifugal complete after, supernatant is taken in the corresponding clean centrifuge tube, up and down behind the mixing ,-20 ℃ of refrigeration 20min.
After (11) 20 minutes, centrifuge tube is taken out, 4 ℃, the centrifugal 12min of 15000r/min.
(12) centrifugal complete after, remove supernatant, add again the ethanol of 1ml75%, up and down behind the mixing ,-4 ℃, the centrifugal 8min of 12000r/min.
(13) after centrifugal, outwell supernatant, in stink cupboard that ethanol is air-dry, to sinking to the bottom an amount of ultrapure water of middle adding, obtain total RNA again.
2, reverse transcription step: the M-MLV PCR RT Kit test kit specification sheets with reference to Promega company is reversed to cDNA with RNA, the reverse system:
| Reagent | Volume (uL) |
| |
6 |
| Random primer | 1 |
| M-MLV | 0.25 |
| dNTP | 0.5 |
| The RNA inhibitor | 0.25 |
| 10×M-MLV?buffer | 2 |
| |
10 |
The reverse transcription reaction condition:
| RNA and random primer be the system of 7uL altogether | 70℃5min |
| Add again altogether 10uL system of reverse transcription reaction mixed solution 3uL | 37℃60min |
3, nest-type PRC reaction
Get reverse transcription product 2ul, as the PCR reaction template.Take BCR (p210)-F and ABL (p210)-R as the upstream and downstream primer, BCR (p190)-F and ABL (p190)-R are that the upstream and downstream primer carries out first round pcr amplification.The PCR reaction system is 20ul.Concrete prescription is as follows:
| Composition | Add-on (μ l) |
| LA Taq enzyme | 0.2 |
| 10×LA?PCR?Buffer?II(Mg 2+Plus) | 2 |
| dNTP?Mixture（10mM?each） | 0.8 |
| First round primer (5 μ M each) | 2 |
| |
2 |
| ddH 2O | 13 |
| Cumulative volume | 20 |
The amplification condition of first round PCR is as follows:
Take turns the PCR reaction with getting 2ul after 50 times of dilutions of first round PCR product for second, ABL Kinase-F and ABL Kinase-R are the upstream and downstream primer, and system is prepared the same first run.
Second to take turns the pcr amplification condition as follows:
The PCR product is got 3ul and is used for the agarose electrophoresis evaluation, and the result as shown in Figure 4.
4, sequencing reaction
(1) PCR product enzymolysis, digestion: configure the enzyme digestion reaction system according to following requirement:
| ddH 2O | 1.2μL |
| 10×SAP?Buffer | 0.3μL |
| SAP enzyme (1U/ μ L) | 1μL |
| Exo I enzyme (5U/ μ L) | 0.5μL |
| The PCR product | 5μL |
| TotaL | 8μL |
After system configurations is complete, cover tightly 8 unions lid, the vibration mixing is also centrifugal rapidly, then places the following temperature bath program of operation on the PCR instrument:
| 37℃ | 30min |
| 95℃ | 15min |
| 12℃ | ∞ |
The enzymolysis after product should be kept at 4 ℃ (continuing downstream process in the 24h) or-20 ℃ (surpassing 24h can not use).
(2) sequencing reaction operation:
Configure the sequencing reaction system according to following requirement: use ABL Kinase-F and ABL Kinase-R to carry out forward and reverse order-checking as sequencing primer.
| ddH 2O | 6μL |
| 5×Sequencing?Buffer | 1.8μL |
| BigDye3.1 | 0.4μL |
| Sequencing primer (5 μ M) | 1μL |
| Template behind the enzymolysis *1 | 0.8μL |
| Total | 10μL |
After system configurations is complete, cover tightly 8 unions lid, the vibration mixing is also centrifugal rapidly, then places the following temperature bath program of operation on the PCR instrument:
The sequencing reaction product should keep in Dark Place at-20 ℃.
(3) ethanol deposition and purification
Every hole adds 1 μ L125mM EDTA solution and 1 μ L3M sodium acetate solution (pH=5.2) in the respective aperture of 96 orifice plates, adds whole sequencing reaction products and 40 μ L dehydrated alcohols again.
After centrifugal the finishing, take off 8 union dished covers, get 3 layers of dust-free paper, pad is placed on 96 orifice plates at least 4 thieving papers together.After the Roberval's balance trim, be inverted 96 orifice plates+dust-free paper+thieving paper, rotating speed 500g, 4 ℃ of temperature, centrifugal 1min dries all supernatants.
Take out 96 orifice plates, discard dust-free paper and thieving paper, every hole adds 100 μ L75% dehydrated alcohols, covers 8 union dished covers, does not want mixing, and after the Roberval's balance trim, forward is placed 96 orifice plates in refrigerated centrifuge, rotating speed 2100g, 4 ℃ of temperature, centrifugal 5min.
After centrifugal the finishing, take off 8 union dished covers, get 3 layers of dust-free paper, pad is at least 4 thieving papers.Be inverted 96 orifice plates (dust-free paper is close to 96 orifice plates) to dust-free paper+thieving paper, most of supernatant liquor is absorbed by dust-free paper.
Again get 3 layers of dust-free paper, pad is placed on 96 orifice plates at least 4 thieving papers together.After the Roberval's balance trim, be inverted 96 orifice plates+dust-free paper+thieving paper, rotating speed 500g, 4 ℃ of temperature, centrifugal 1min dries all supernatants.
Take out 96 orifice plates, discard dust-free paper and thieving paper, 96 orifice plates are placed on 12-15min in the lucifuge box, make the ethanol volatilization clean.
Every hole adds 10 μ L Hi-Di methane amides, covers 8 union dished covers, of short duration centrifugal 96 orifice plates.
With 95 ℃ of sex change 2min of 96 orifice plates, then preserve machine order-checking in the preparation on ice.
(4) machine testing on the sequenator: use 3500xL genetic analysis instrument to carry out capillary electrophoresis and detect.
5, sequencing result
The result is as shown in the table;
| Sample | Sequencing result | |
| 8 | |
|
| 1 | |
|
| 1 | E255K |
Normal sequencing result is seen Fig. 5, and the F359V sequencing result is seen Fig. 6.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. primer comprises amplimer and sequencing primer, it is characterized in that:
Described amplimer sequence is as follows:
BCR_p210-F:5’-TGACCAACTCGTGTGTGAAACTC-3’、
ABL_p210-R：5’-TCCACTTCGTCTGAGATACTGGATT-3’、
BCR_p190-F:5’-ACCGCATGTTCCGGGACAAAAG-3’、
ABL_p190-R：5’-ACAGCATTCCGCTGACCATCAATAAG-3’、
Described sequencing primer sequence is as follows:
ABL?Kinase-F：5’-TGGTTCATCATCATTCAACGGTGG-3’、
ABL?Kinase-R：5’-AGACGTCGGACTTGATGGAGAACT-3’。
2. described primer according to claim 1, it is characterized in that: described primer sequence is or/and the sequence that 3 ' end prolongs to 5 ' end of amplimer or sequencing primer.
3. described primer according to claim 1 is characterized in that: described primer sequence is greater than 85% sequence with amplimer or sequencing primer homology.
4. described primer according to claim 1 is characterized in that: described primer sequence is the sequence with amplimer or the complementation of sequencing primer base sequence.
5. described primer according to claim 1, it is characterized in that: described primer sequence is by any one or more than one the composite sequence in amplimer or the sequencing primer sequence.
6. described this primer of any one detects the method for BCR/ABL fusion gene ABL kinases district resistant mutational site according to claim 1-5, it is characterized in that may further comprise the steps:
(1), extracts RNA;
(2), carry out reverse transcription reaction, acquisition reverse transcription product cDNA;
(3), take the reverse transcription product of step (2) as the PCR reaction template, take BCR_p210-F and ABL_p210-R as the upstream and downstream primer, BCR_p190-F and ABL_p190-R are that the upstream and downstream primer carries out first round pcr amplification, obtain amplified production respectively;
(4), get the amplified production of step (3), take turns pcr amplification take ABL Kinase-F and ABL Kinase-R as the upstream and downstream primer carries out second, obtain amplified production;
(5), get the amplified production of step (4), take ABL Kinase-F and ABL Kinase-R as sequencing primer, the detection of checking order.
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| CN104232765A (en) * | 2014-09-03 | 2014-12-24 | 南京百捷生物科技有限公司 | Primer pair and kit for detecting BCR-ABL fusion genes by using pyrosequencing method |
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| RU2609641C1 (en) * | 2015-12-07 | 2017-02-02 | Федеральное Государственное Бюджетное Учреждение Науки Институт Молекулярной Биологии Им. В.А. Энгельгардта Российской Академии Наук (Имб Ран) | Method of analysis of somatic mutations in the bcr/abl chimeric gene using rt-pcr and subsequent hybridization with oligonucleotide biological microchip (biochip) |
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| CN102676638A (en) * | 2011-03-08 | 2012-09-19 | 苏州大学附属第一医院 | Method and kit for detecting drug-resistance mutation site of ABL kinase domain of BCR/ABL fusion gene |
| CN102827937A (en) * | 2012-09-06 | 2012-12-19 | 上海源奇生物医药科技有限公司 | Primer and probe for detecting relative fusion genes of leukemia and kit of primer and probe |
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| CN102533740A (en) * | 2011-09-07 | 2012-07-04 | 杭州艾迪康医学检验中心有限公司 | Reverse transcription-polymerase chain reaction (RT-PCR) primer of human leukemia fusion gene BCR-ABL and application method thereof |
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| CN107190091A (en) * | 2017-07-21 | 2017-09-22 | 大连晶泰生物技术有限公司 | Suitable for the real-time quantitative PCR method of the quantitative gene expression of FFPE samples |
| CN107190091B (en) * | 2017-07-21 | 2021-04-20 | 大连晶泰生物技术有限公司 | Real-time quantitative PCR method suitable for gene expression quantification of FFPE sample |
| CN109777864A (en) * | 2018-12-29 | 2019-05-21 | 武汉康圣达医学检验所有限公司 | A method of detection BCR-ABL fusion ABL kinases region mutation |
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