CN109988832A - A kind of kit and its detection method of the mutation of detection AML patient's prognostic gene - Google Patents

A kind of kit and its detection method of the mutation of detection AML patient's prognostic gene Download PDF

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CN109988832A
CN109988832A CN201711483126.9A CN201711483126A CN109988832A CN 109988832 A CN109988832 A CN 109988832A CN 201711483126 A CN201711483126 A CN 201711483126A CN 109988832 A CN109988832 A CN 109988832A
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pcr amplification
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潘红超
苏燕辉
张瑜
叶茂
蔡宏康
赵琨
程乐华
陈忠
黄士昂
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Shanghai Simplegene Clinical Laboratory Co Ltd
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Abstract

The invention belongs to field of biological technology detection, and in particular to a kind of kit and its detection method of the mutation of detection AML patient's prognostic gene.The kit includes: the PCR amplification primer of NRAS gene, the PCR amplification primer of RUNX1 gene, the PCR amplification primer of TP53 gene, the PCR amplification primer of WT1 gene and universal sequencing primer object connector.NRAS gene, RUNX1 gene, TP53 gene and WT1 gene mutation are detected using sequencing technologies using kit of the present invention.Gene mutation site relevant to AML prognosis can be detected by specific PCR amplimer and with the ddNTP of fluorescent marker, be conducive to the risk degree determination for accurately carrying out AML and layering treatment.

Description

A kind of kit and its detection method of the mutation of detection AML patient's prognostic gene
Technical field
The invention belongs to biotechnology detection and kit fields, and in particular to a kind of detection AML patient's prognostic gene is prominent The kit and its detection method of change.
Background technique
Acute myeloid leukemia (acute myeloid leukemia, AML) is a kind of malignant neoplastic of hemopoietic system Disease, with the proliferation out of control of hematopoietic cell, differentiation is stagnated, and apoptosis, which is obstructed, to be characterized.The biological characteristics that AML is shown be by The genetic alteration of itself is caused with decision, and traditional cytogenetics can be found in the AML patient of more than half All kinds of abnormal karyotypes, these chromosome abnormalities can provide the foundation for judging patient's prognosis for us.But also close to half Patient lacks characteristic chromosome abnormalities, thus is difficult to judge this kind of patient.And Protocols in Molecular Biology is then us It was found that the genetic alteration of microcosmic point provides the broader visual field, there is also very in leukaemia for the exception of molecules label Big heterogeneous, usually there is very big difference in different type leukemia gene exception site, as the research of AML is gradually deeply changed, more Carry out more evidences to show, gene mutation not only plays extremely important effect in the generation of AML, while also controlling disease Progress and final result.Thus, it is found that and verifying some new biological markers for improving its prognosis with extremely important meaning Justice, the discovery that these molecular levels change provide not only the New Century Planned Textbook for explaining AML pathogenesis, and help to establish new AML risk stratification system, for clinic targeted therapy foundation is provided.
Sanger PCR sequencing PCR is generally acknowledged at present, the goldstandard of most of clinical molecular diagnosis projects, specificity even energy Reach 100%, is the classical technology platform in terms of molecular diagnosis.
In order to provide reliable template to Sanger sequencing it may first have to which the characteristics of being directed to the project establishes a stabilization Reliable PCR system.In sport technique segment, there are following two difficult points for the part project PCR: 1. projects need to extract blood-based Because of a group DNA, due to there are numerous uncontrollable factors, often cannot get quality in clinical examination in clinical samples logistics transportation Excellent whole blood sample, therefore the DNA mass obtained can also have biggish inter-sample difference.2. due to the exon that need to be detected Number is more, i.e. PCR target fragment is excessive, leads to PCR condition disunity.
Summary of the invention
For the problems of in the prior art, the purpose of the present invention is to provide a kind of detection AML patient prognosis bases Because of the kit and its detection method of mutation, which can be used for detecting NRAS gene, RUNX1 gene, TP53 gene and WT1 The mutational site of gene order, specific good, high sensitivity.
To achieve the goals above and other related purposes, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of kit of detection AML patient's prognostic gene mutation, the kit packet Include: the PCR amplification primer of NRAS gene, the PCR amplification primer of RUNX1 gene, TP53 gene PCR amplification primer, WT1 gene PCR amplification primer and universal sequencing primer object connector.
Preferably, the PCR amplification primer of the NRAS gene includes at least the nucleotide sequence such as institute of SEQ ID NO.1~4 The primer shown.
Preferably, the PCR amplification primer of the RUNX1 gene includes at least nucleotide sequence such as NO.5~22 SEQ ID Shown in primer.
Preferably, the PCR amplification primer of the TP53 gene includes at least nucleotide sequence such as NO.23~40 SEQ ID Shown in primer.
Preferably, the PCR amplification primer of the WT1 gene includes at least the nucleotide sequence such as institute of SEQ ID NO.41~44 The primer shown.
Preferably, the universal sequencing primer object connector includes at least nucleotide sequence as shown in NO.45~46 SEQ ID Primer connector.
Kit of the invention carries out NRAS gene, RUNX1 gene, TP53 gene, WT1 gene using PCR detection technique Mutational site detection, the PCR amplification primer of NRAS gene, the PCR amplification primer of RUNX1 gene, TP53 gene pcr amplification primer The design of object, the PCR amplification primer of WT1 gene and universal sequencing primer object connector is the key that kit of the present invention.
It is detected based on kit of the present invention using PCR detection technique, so can be in kit Reagent, pcr amplification reaction reagent, sequencing are extracted including conventional reagent required for some other PCR, such as sample genomic dna Use reagent.
Generally, pcr amplification reaction reagent is selected from premix Taq, ddH2O。
Since such PCR common agents can individually be bought through market approach or voluntarily be configured, specifically need by Which reagent is fitted into kit, can be actually needed and be configured according to client, for convenience, can also all be fitted into reagent Box.
Kit of the invention can be the primer pair containing independent packaging, be also possible to containing configured containing drawing The pcr amplification reaction reagent of object pair.
The pcr amplification reaction reagent can be configured voluntarily, can also directly with commercially available without primer pair general PCR reaction Reagent is added primer pair and obtains.For example, premix Taq, ddH can also be contained in the kit2O.Of the invention draw is added Object can be obtained PCR reaction system to, sample to be examined extracting genome DNA object.
Preferably, positive control can also be contained in the kit.The positive control is clinically to be diagnosed as AML The genomic DNA of patient.
Preferably, negative control can also be contained in the kit.The negative control is the sample genome of normal person DNA。
The second aspect of the present invention provides the application method of aforementioned detection kit, includes the following steps:
(1) sample genomic dna is extracted；
(2) PCR of the PCR amplification primer of aforementioned NRAS gene, the PCR amplification primer of RUNX1 gene, TP53 gene is used Amplimer, WT1 gene PCR amplification primer pair sample genomic dna expanded；
(3) it is sequenced after adding aforementioned universal sequencing primer object connector to amplified production；
(4) sequencing result is analyzed.
Preferably, the inspection method is the method for non-disease diagnostic purpose.
In step (1), extraction sample gene group DNA is the prior art.
The third aspect of the present invention provides aforementioned agents box in preparation AML patient's prognostic gene abrupt climatic change product Purposes.
Compared with prior art, the invention has the following beneficial effects:
Using kit of the present invention, using sequencing technologies, to NRAS gene, RUNX1 gene, TP53 gene and WT1 gene Mutation is detected.It can be detected and AML prognosis phase by specific PCR amplimer and with the ddNTP of fluorescent marker The gene mutation site of pass is conducive to the risk degree determination for accurately carrying out AML and layering treatment.
Detailed description of the invention
The sequencing peak figure of Figure 1A: RUNX1 genic mutation type.
The sequencing peak figure of Figure 1B: TP53 genic mutation type.
The sequencing peak figure of Fig. 1 C:WTI gene saltant type.
The sequencing peak figure of Fig. 1 D:NRAS gene saltant type.
Specific embodiment
NRAS gene
RAS is intracellular important proto-oncogene, and Ras gene is the key that many tumour cell signal transduction paths, perhaps More tumour cells have Ras gene mutation.The Ras of Ras gene mutation rate about 25%, the ALL about 6%--20%, CMML of AML are prominent Variability is also very high.The Ras gene family member similar there are three structure, i.e. H-Ras, K-Ras and N-Ras, neutralize AML Relevant mainly NRas gene, full length gene 85kb, cDNA are about 2.1kb, and coding contains the albumen of 495 amino acid, claims It is a kind of film combination G-protein for p21Ras, p21, participates in signal transduction path.The mode of Ras gene activation has 3 kinds: gene point Mutation, the insertion of gene great expression, gene and indexing.It is the most common mode is exactly point mutation that wherein Ras gene, which is activated, more Occur in N-terminal the 12nd, 13 and 61 codons, wherein it is again most common with the 12nd codon mutation, and mostly GGT is mutated into GTT. Different mutational sites are different to the activation mechanism of P21, the 12nd codon mutation can weaken in P21 GTP enzymatic activity, and make Apoptosis is reduced, cell contact inhibits to weaken；61st codon mutation can weaken GAP to the inherent GTP enzymatic activity of P21, And stability of the GAP in conjunction with P21 can be weakened.
Ras albumen plays an important role in the starting and development process of leukaemia.Mature Ras albumen closes in endochylema At being positioned at the inner surface of plasma membrane.Then it is transported and modifies.Its modification betides the CAAX functional domain with C-terminal, The assistance of film is needed simultaneously.The first stage of modification is to remove AAX residue first, is contacted with a hydroxylate of cysteine.The latter Envelope transmethylase methyl vinyl.Then, the sulfydryl of Cys-186 is by farnesylation.Second stage is to be located at albumen (HRAS Cys-181 and Cys-184；The Cys-181 of NRAS) hypervariable region cysteine palmitoylation.Then albumen be targeted and It is incorporated into plasma membrane, and is activated there.Mainly by stimulating 1. after Ras albumen receives signal stimulus or itself mutation activation Proliferating way Raf/Mek/Erk 2. protein phosphorylation in two approach of anti-apoptotic approach P13K/Akt, they are played in vivo A series of important biological effects, once this two approach show as albumen because certain reason is in sustained activation state Phosphorylation, will lead to the generation of a variety of malignant diseases (AML).
RUNX1 gene
RUNX1 (runt-related transcription factor 1) is used as important hematopoiesis associated transcription factor, Multiple stages of hematopoietic development play a significant role.Runx1 is Runx family a member, during mammalian development, Runx Family is made of Runx1, Runx2, Runx3, their conserved sequences all containing 128 amino acid compositions constitute DNA and combine knot Structure domain, different members combine the identical site DNA, but different effects is played in growth course.
The main pathogenesis for being thought as RUNX1 genes leukemia in the past is the formation of fusion, such as t (8；21)- AMLl(RUNX1)/ETO(MTG8),t(3；21)-RUNXl/EVll and t (12；21)-TEL(ETV6)/RUNXl.It has now been found that More than 10 are related to fusion caused by the chromosome translocation of RUNX1, although the leukemogenic mechanism of every kind of fusion is different, But its related co-morbid mechanism is all then the activity for inhibiting wild type RUNXl gene.However recent studies have shown that RUNXl gene mutation also assists in the generation of leukaemia, and the mutation of monoallelic can cause familial blood platelet disorders (FPD), and then make the neurological susceptibility enhancing that acute myeloid leukemia (AML) occurs, referred to as FPD/AML.In M0Point mutation is main Occur in the area Runt, and mutation occurs mainly in the end C- in MDS-AML.
Transcription regulatory factor RUNX1 mutation is higher in AML and MDS patient's incidence, and respectively 13.1% and 17.5%. RUNX1 gene encodes RUNX1 albumen, combines to form core-binding factor transcription complex with CBF β, regulates and controls multiple hematopoiesis phases Correlation gene expression.Involving RUNX1 mutation at present is mainly 2 classes: 1. being occurred in the end RUNX1N- (common mutations RUNX1D171N), RUNX1 mainly is influenced in conjunction with DNA in RHD structural domain；2. occurring in the end RUNX1C- (common mutations RUNX1s291fs), RUNX1 can be enhanced in conjunction with DNA, but influence its transcriptional activity mll gene be extremely another MDS and MDS/AML common science of heredity changes.It dashes forward studies have shown that MDS patient disease progress exists simultaneously MLL-PTD and RUNX1 often Become, and in primary and secondary AML, the ratio for existing simultaneously mutation is higher.The detection in Gene Mutation combination genetic chip Detection, can better auxiliary diagnosis AML.
TP53 gene/
TP53 gene is important one of tumor suppressor gene, coding albumen -- tumor suppressor protein P53 can make many Gene keeps its stability, and adjusts the growth, differentiation, aging of cell, and disease is avoided to generate, referred to as " gene defender ". When TP53 gene delection, mutation and adjusting disorder, the function and activity of P53 albumen will be directly affected, so as to cause many swollen The generation of tumor；In addition to this gene appearance of TP53 also becomes the important indicator for judging lymthoma and blood disease prognosis.TP53 base Because two kinds of approach of activity (TIA) that non-transcribed relies in the activity (TA) and cytoplasm by transcribing dependence in nucleus play Press down the effect of cancer.The transcription for the function point analysis gene that TP53 is relied on by transcription, influences cell cycle, DNA reparation, apoptosis, letter Number conduction, transcription and metabolism；By non-transcribed rely on function is apoptosis-induced and autophagy.The mutation and adjusting of TP53 gene are different It often will lead to the generation of many tumours.Although the frequency of TP53 missing and mutation does not have other swollen in blood disease and lymthoma Tumor is high, but the expression of the missing of the gene coding region TP53, rearrangement and mutation P53 albumen that it will be made to encode, stability and Active disorder, the state of TP53 gene have become the important indicator for judging blood disease and lymthoma prognosis.On the other hand, overcome The abnormal novel targets for also becoming disease treatment of TP53.
TP53 is located at the 17q13.1 of chromosome, long 19144 nucleotide, wherein 2586 nucleotide are transcribed into TP53's MRNA, including the 1st and exon 25, nontranscribed domain (UTR), the 3 ' nontranscribed domains (UTR) and 2-11 of the 11st exon The code area (~20KB) of exon encodes the P53 albumen being made of 393aa.The combined area DNA (DBD) of P53 albumen is TP53 Gene plays transcription dependence activity and non-transcribed relies on the most important region of activity, so the mutation of TP53 much all occurs In the 4th~8 exon region of encoding D BD.The forfeiture of TP53 normal function mainly passes through gene mutation.Mutation can cause two sides The change in face: (1) mutant loses the binding ability with specific site.Wild type P53 is with tetramer and specific site knot It closes, the expression of trans-activation downstream growth suppressor gene.In some tumours, the forfeiture in one or two single sites P53 makes to be formed The ratio of the tetramer declines, and nonsense mutation causes P53 translation to interrupt, and the missing of the end c acid domain influences the tetramer and formed；Most Commonly wild type and saltant type form the more stable tetramer after missense mutation, weaken the negative regulation of endogenous wild type P53 Effect, so as to cause malignant change of cell.(2) mutation changes the spherical conformation of P53.For example, some mutant can be with heat shock protein White combination, some to be mutated the exposure for causing 213~217 peptide fragments, other then causes the change of acid activatable structural domain, these Mutation prompts the minor alteration of P53 that can cause the change far from the even entire protein conformation of mutational site section.
Gene unconventionality is not only one of pathogenesis of AML, while being also the important Prognostic Factors of AML patient.TP53 base The change of cause is related with age, the complexity of gene unconventionality, special chromosome exception, single caryogram, and indicates that prognosis is poor. There is document report P53 afunction related to chemotherapy such as cytarabine resistance.In recent years again discovery TP53 change with clinically Induction chemother is resisted closely related.There is recurrence after CK-AML patient's treatment of 51% carrying P53 gene mutation, and does not have TP53 base Because of abnormal CK-AML Patients on Recurrence rate only 35%.Carry the median survival time of the CK-AML patient of TP53 gene mutation only Have 4.14 months, and the median survival time of the CK-AML patient without TP53 gene mutation is 10.97 months.Therefore TP53's is prominent Allergic effect this as it is routine clinical detection to instruct clinical application and prompt prognosis.
WT1 gene
WT1 gene, which is located at, is positioned at 11P13, and coding contains the protein of 429 amino acid.The albumen is by the combined area DNA The amino terminal of c-terminus and transcriptional activity regulatory region is constituted.King-Underwood L reports AML for the first time, and there are WT1 genes Mutation, and think that the mutation is related with AML generation.Subsequent research confirms that there are WT1 there are about the AML patient of 10-15% at present Gene mutation.
In recent years WT1 (Wilms ' tumor susceptibility gene) gene tumour generation in effect by Pay attention to, but its definite effect and mechanism of action are still unclear.WT1 gene is initially taken as tumor suppressor gene discovery in Wilms tumor, position It is a kind of transcription factor of regulation kidney development in 11p13.The gene is equal in normal cerebral tissue, placenta, spleen and hematopoietic tissue There is expression.Some growth factors related with Growth and Differentiation are adjusted in WT1 gene: WT1 gene is not a simple tumor suppressor gene, Has the function of oncogene simultaneously.The report WT1 gene-correlation function such as Hosen in 2004 be more likely to a kind of transcriptional activation because Son, can specific recognition and with target gene DNA ining conjunction with, play adjusting transcription.Hu Shaoyan etc. utilizes real-time quantitative Reverse Transcription Polymerase (RQ-RT-PCR) WT1 gene expression dose is bright compared with normal person in reaction technology detection Bone Marrow Cells from Acute Myeloid Leukemia Patients Aobvious to increase, long existence and disease-free survival of the WT1 gene with acute myeloid leukemia are related, prompt WT1 gene that may take part in urgency Property marrow series leukemia pathogenic process, consider using WT1 gene as the index that judges acute myeloid leukemia prognosis and detect micro- The label of small Residual Disease.
WT1 gene is about 50Kb, contains 10 exons, code area about 1300bp, and 5 ' ends encode other transcription factors Regulatory region, 3 ' ends are zinc finger conserved sequence very high homology area.More important in structure is montages that 2 exons are random combine Exon: splice variant I and splice variant II.Splice variant I is located at exon 5, encodes 17 amino acid insertion zinc fingers And control region, referred to as WT1+17AA/WT1-17AA；Splice variant II is between exon 9 and exons 10, by coding 3 Amino acid (lysine-threonine-serine) is inserted between the 3rd and the 4th zinc finger area, referred to as WT1+KTS/WT1-KTS.
The forfeiture of WT1 gene function can lead to cellular overgrowth increment, tumour occurs.The main machine that gene function changes System is point mutation or excalation.Point mutation is the most common reason that WT1 function is lost, mainly prominent including frameshift mutation, nonsense Become, missense mutation.Excalation is another mechanism of WT1 inactivation.There is research to confirm that hematopoiesis can be promoted by destroying WT1 protein function Cell Proliferation and inhibition cell differentiation, also having the presence researches show that WT1 mutation is the shadow of normal karyotype AML patient's prognosis mala The factor of sound, existing research discovery exon 7,8,9,10 are the hot spot being mutated, the further report WT1 gene such as Hollink IH Mutation is concentrated mainly on exon.However, some WT1 of the latest reports such as Damm F are mutated, if SNP rs16754 may be one The advantageous label of independent judging prognosis.WT1 gene expression dose increases or the clinical prognosis of WT1 gene mutation and AML patient It is closely related with therapeutic effect.
Therefore, the present invention provides a kind of kit of detection AML patient's prognostic gene mutation, and the kit includes NRAS The PCR amplification primer of gene, the PCR amplification primer of RUNX1 gene, TP53 gene PCR amplification primer and universal sequencing primer Object connector.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than limiting the scope of protection of the present invention.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment, Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real The existing present invention.
Embodiment 1 detects the preparation of the kit of AML patient's prognostic gene mutation
Kit of the invention includes the PCR amplification primer of NRAS gene, the PCR amplification primer of RUNX1 gene, TP53 base The PCR amplification primer of cause, the PCR amplification primer of WT1 gene and universal sequencing primer object connector, nucleotide sequence are detailed in following table Shown in 1:
Table 1
Embodiment 2 detects the application of the kit of AML patient's prognostic gene mutation
1. sample extracts
Operating procedure:
(1) acquire blood sample to be measured, be placed in 1.5ml centrifuge tube, add 500 μ LBuffer AP1 to the 1.5ml from In heart pipe；
(2) plus 200-250 μ L anticoagulated whole blood is into BufferAP1, covers tightly centrifuge tube lid, and vortex vibrates 10s；
(3) plus 100 μ LBuffer AP2, vortex vibrate 10s, and it is brown that white precipitate and peony lysate mix well into ash It is red；
(4) 12,000 × g are centrifuged 10min；
(5) pipe will be prepared to be placed in 2ml centrifuge tube, the supernatant in step (4) is added to and is prepared in pipe, 12,000 × G is centrifuged 1min；
(6) filtrate is abandoned, pipe will be prepared and put back into former 2ml centrifuge tube, add 700 μ LBuffer W1A, be placed at room temperature for 2min
12,000 × g is centrifuged 30s；
(7) filtrate is abandoned, pipe will be prepared and put back into former 2ml centrifuge tube, 800 μ L is added to add the Buffer W2 of dehydrated alcohol, 12,000 × g is centrifuged 1min；
(8) (optional step) will prepare pipe and put back into former 2mL centrifuge tube, add 500 μ LBuffer W2 to preparing in pipe, 12,000 × g is centrifuged 1min；
(9) filtrate is abandoned, pipe will be prepared and put back into former 2mL centrifuge tube, 12,000 × g is centrifuged 2min；
(10) pipe will be prepared to be placed in the 1.5mL centrifuge tube of another cleaning, uncaps and be placed at room temperature for 1min, in prepares periosteum Centre plus 80-200 μ LBufferTE, are stored at room temperature 1min.12,000 × g is centrifuged 1min and elutes genomic DNA.
Thus it extracts and obtains sample genomic dna.
2. experimentation
(1) using the PCR amplification primer of the NRAS gene in 1 kit of embodiment, RUNX1 gene PCR amplification primer, The PCR amplification primer of TP53 gene, WT1 gene PCR amplification primer totally 22 pairs of amplimers to preparing PCR reaction reagent respectively Totally 22 kinds, for each pair of amplimer to a kind of PCR reaction reagent is corresponded to, the amplifing reagent is as shown in table 2 below:
Table 2
Reagent name Dosage (uL)
premix Taq 10
Upstream primer (5uM) in primer pair 1
Downstream primer (5uM) in primer pair 1
ddH2O 6
Specifically, PCR reaction reagent corresponding to NRAS-2-F and NRAS-2-R is as shown in table 3:
Table 3
Reagent name Dosage (uL)
premix Taq 10
NRAS-2-F(5uM) 1
NRAS-2-R(5uM) 1
ddH2O 6
And so on, in other each PCR reaction reagents, other than difference in primer pair and table 3, other components are homogeneous Together.
By above-mentioned 22 kinds of PCR reaction reagents respectively as in corresponding PCR reaction tube, one kind is placed in each reaction tube PCR reaction reagent 20uL obtains 22 kinds of PCR reaction tubes altogether.
(2) each sample genomic dna obtained in above-mentioned steps 1 being all divided into 22 parts, every part of volume is 2 μ L, It is then respectively adding into 22 kinds of PCR reaction tubes, each sample is corresponding to obtain 22 sample reaction tubes；
By same positive control (positive control is the genomic DNA for being clinically diagnosed as AML patient) point It is 22 parts, every part of volume is 2 μ L, is then respectively adding into 22 kinds of PCR reaction tubes, and same positive control is corresponding to obtain 22 A positive control reaction tube；
Same negative control (sample genomic dna that the negative control is normal person) is divided into 22 parts, every part Volume is 2 μ L, is then respectively adding into 22 kinds of PCR reaction tubes, and same negative control 22 negative controls of corresponding acquisition are anti- Ying Guan；
(3) by each sample reaction tube, positive control reaction tube, negative control reaction tube, respectively mix respectively, low speed from The calculation second carries out PCR amplification:
The reaction tube of PCR amplification primer containing NRAS gene and the reaction tube of the amplimer containing RUNX1 gene, When carrying out PCR amplification, PCR reaction condition see the table below 4:
Table 4
The reaction tube of PCR amplification primer containing TP53 gene and the reaction tube of the amplimer containing WT1 gene, into When row PCR amplification, PCR reaction condition see the table below 5:
Table 5
Specific step is as follows for 3.Sanger sequencing:
3.1 sequencing PCR:
3.1.1 preparation of reagents: according to the form below 6 carries out, and operates on ice, -20 DEG C of refrigerators are stored in after packing.
BigDye system:
Table 6
Table 7
Enzyme purification system:
Raw material 1X Need volume (ul/ pipe)
CIP 0.1ul -
Exo I 0.5u l -
Deionized water 1.4ul -
3.2PCR product purification
3.2.1 the purifying reaction solution dispensed is taken out, in the good corresponding number of tube wall subscript.
3.2.2 it takes 9ulPCR product to be added in the pipe accordingly numbered, mixes (attention cannot generate bubble).
3.2.3 sample will be mixed according to PCR instrument on following reaction condition, carry out enzyme purification amplification.
3.2.4 purified sample is used for next step sequencing reaction, as the same day does not have to put freezen protective.
3.3 sequencing reaction
3.3.1 each sample does positive and negative two reactions, and label is finished writing on corresponding thin-wall tube.
3.3.2 every reaction system 5ul, BigDye mixed liquor and 5P primer are fixed plus 1ul, template generally add 2ul, maximum It (is adjusted according to PCR product electrophoresis result) for 3ul, supplies 5ul with water.
3.3.3 the water for first adding corresponding amount, is then added corresponding 1ul sequencing primer connector (M13-F and M13-R) and is added to water In, then in the different places of tube wall add the BigDye mixed liquor of 1ul, it is eventually adding template.
3.3.4 the of short duration centrifugation of lid upper tube cap low speed, vortice mix, the of short duration centrifugation of low speed again.
3.3.5 according to PCR instrument on following reaction condition, sequencing reaction is carried out.
3.4. ethanol purification after sequencing reaction, upper machine.
3.4.1 the PCR product expanded is subjected to of short duration centrifugation.Every pipe side it is adherent be added 2ul 125mM EDTA, Then it in the adherent addition 15ul dehydrated alcohol in the other side of pipe, is vortexed and mixes.
3.4.2 with board-like centrifuge, 4000rpm, centrifugation 30min, then 300rpm is inverted brief centrifugation, removes supernatant.
3.4.3 20 DEG C of 75 ethyl alcohol of pre-cooling of 50ul are added, is vortexed and mixes, 4000rpm, 4 DEG C of centrifugation 15min.
3.4.4 300rpm is inverted brief centrifugation, removes supernatant, is then placed into room temperature, 15min is evaporated completely ethyl alcohol.
3.4.5 after ethyl alcohol volatilizees completely, every pipe is added water and each 5ul of Hi-DiTM, and sufficient vortex vibrates 1min., it is of short duration from 15min. is stood after the heart is completely dissolved sequencing product.
3.4.6 Hi-DiTM lysate is transferred in the corresponding position of 96 orifice plates.It puts to PCR amplification instrument, 95 DEG C pre- It is denaturalized 5min., is transferred quickly to 4 DEG C of cooling 5min on ice chest.Machine is sequenced and analyzes on to 3500 sequenators.
Data analysis: sequence alignment analysis is carried out using sequencing analysis software.The sequence that sequencing is obtained It is compared with the standard sequence retrieved in NCBI, the gene order of confirmatory sample.
4. clinical samples compare: preclinical Samples detection has determined that 30, sample of mutational site, kit of the present invention Testing result coincidence rate 90%.
Solution temperature (Tm) is also a critically important parameter in PCR, is necessary, conjunction for setting PCR annealing temperature Suitable annealing temperature can effectively reduce non-specific binding, moreover it is possible to guarantee that aim sequence is effectively annealed.So guaranteeing spy In the case where the opposite sex, the Tm value of design primer is close as far as possible, is consistent the annealing temperature of PCR, to reach PCR item The unification of part.
Therefore, using kit of the present invention, using sequencing technologies, to NRAS gene, RUNX1 gene, TP53 gene and WT1 Gene mutation is detected.It can be detected pre- with AML by specific PCR amplimer and with the ddNTP of fluorescent marker Relevant gene mutation site afterwards is conducive to the risk degree determination for accurately carrying out AML and layering treatment.
Further, since present invention employs special universal sequencing primer object connector, sequencing primer connector cannot be equal to PCR Amplimer, sequencing primer jointing requirements want more tightened up, and sequencing primer requires 3, and end has to match completely, cannot contain Base, and length 20bp or so are mixed, G/C content is between 50%-60%；In addition, due to the exon number that need to detect compared with More, i.e. required primer is more when Sanger is sequenced, and will cause larger workload in sequencing reaction, while also increasing error rate Generation, cause big inconvenience to experiment, the present invention is by the way that in specific PCR amplimer 5, end is logical plus special sequencing With primer connector, by comparison, it was found that, in addition the sequencing is not added in Sanger sequencing when ratio after the sequencing universal primer Primer connector can be saved the time of 15min-30min, while can substantially reduce the error rate as caused by non-universal sequencing primer, To significantly improve working efficiency.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should be covered by the claims of the present invention.
Sequence table
<110>Co., Ltd, brilliant medical test institute is newly trained in Shanghai
<120>a kind of kit and its detection method of the mutation of detection AML patient's prognostic gene
<130> 176816
<160> 46
<170> SIPOSequenceListing 1.0
<210> 2
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtaaaacgac ggccagtggt tttcatttcc attgattata gaa 43
<210> 3
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggaaacagct atgaccatgt cagcgggcta cca 33
<210> 3
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtaaaacgac ggccagtggg cagaaatggg cttga 35
<210> 4
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggaaacagct atgaccatgc tgtagaggtt aatatccgca aatg 44
<210> 5
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gtaaaacgac ggccagtgat gtagggctag aggggtga 38
<210> 6
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ggaaacagct atgaccatga caggcaaagc tgagcaaaa 39
<210> 7
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gtaaaacgac ggccagtgcc acgtgcataa ggaacagt 38
<210> 8
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ggaaacagct atgaccatgt ggaatcagca gaaacagcc 39
<210> 9
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gtaaaacgac ggccagtgaa aggcccctga acgtgtat 38
<210> 10
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ggaaacagct atgaccatga aagctgagac gagtgcctc 39
<210> 11
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
gtaaaacgac ggccagtgtc attgctattc ctctgcaacc 40
<210> 12
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
ggaaacagct atgaccatgt gtgggtttgt tgccatgaa 39
<210> 13
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gtaaaacgac ggccagtggc caaaattccg ggagtgtt 38
<210> 14
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ggaaacagct atgaccatgg gtccctgagt ataccagcc 39
<210> 15
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gtaaaacgac ggccagtgga cagtggcccc aaattcag 38
<210> 16
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
ggaaacagct atgaccatgc tgttggttcg aggcctttc 39
<210> 17
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gtaaaacgac ggccagtgtg aacaagggcc actcatttc 39
<210> 18
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
ggaaacagct atgaccatgc caactccttc atgcacctc 39
<210> 19
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gtaaaacgac ggccagtgct aggcggtatc atcctggg 38
<210> 20
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ggaaacagct atgaccatga tggagaactg gtaggagcc 39
<210> 21
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
gtaaaacgac ggccagtgcc ctcctaccac ctgtactac 39
<210> 22
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
ggaaacagct atgaccatgt tgtcgcgaac aggaggccc 39
<210> 23
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
gtaaaacgac ggccagtgag cagccattct tttcctgct 39
<210> 24
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
ggaaacagct atgaccatga ttttcgcttc ccacaggtct 40
<210> 25
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
gtaaaacgac ggccagtgcc ccctctgagt caggaaaca 39
<210> 26
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
ggaaacagct atgaccatga ggaagccaaa gggtgaagag 40
<210> 27
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
gtaaaacgac ggccagtgga ggtgtagacg ccaactct 38
<210> 28
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
ggaaacagct atgaccatga tcagtgagga atcagaggc 39
<210> 29
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
gtaaaacgac ggccagtgcc atgagcgctg ctcagatagc g 41
<210> 30
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
ggaaacagct atgaccatgt actgctcacc tggagggcca ctg 43
<210> 31
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gtaaaacgac ggccagtgtt gccacaggtc tccccaag 38
<210> 32
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
ggaaacagct atgaccatgc cggggatgtg atgagaggt 39
<210> 33
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
gtaaaacgac ggccagtgcc agaaaggaca agggtggt 38
<210> 34
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
ggaaacagct atgaccatga tctgaggcat aactgcaccc 40
<210> 35
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
gtaaaacgac ggccagtgcc aagggtgcag ttatgcct 38
<210> 36
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ggaaacagct atgaccatga gctacaacca ggagccatt 39
<210> 37
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
gtaaaacgac ggccagtgta cttacttctc cccctcctct g 41
<210> 38
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
ggaaacagct atgaccatga gatggggtca gctgccttt 39
<210> 39
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
gtaaaacgac ggccagtggg cccttcaaag cattggtc 38
<210> 40
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
ggaaacagct atgaccatgt ctcccaaaca tccctcacag 40
<210> 41
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
gtaaaacgac ggccagtgca tggggatctg gagtgtga 38
<210> 42
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
ggaaacagct atgaccatgt cagaatgcaa aatggcccc 39
<210> 43
<211> 42
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
gtaaaacgac ggccagtgtg cagacattgc aggcatggca gg 42
<210> 44
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
ggaaacagct atgaccatgg cactattcct tctctcaact gag 43
<210> 45
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
gtaaaacgac ggccagtg 18
<210> 46
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
ggaaacagct atgaccatg 19

Claims (10)

1. a kind of kit of detection AML patient's prognostic gene mutation, the kit includes: the pcr amplification primer of NRAS gene Object, the PCR amplification primer of RUNX1 gene, the PCR amplification primer of TP53 gene, the PCR amplification primer of WT1 gene and general Sequencing primer connector.
2. kit according to claim 1, which is characterized in that the PCR amplification primer of the NRAS gene includes at least Nucleotide sequence primer as shown in NO.1~4 SEQ ID.
3. kit according to claim 1, which is characterized in that the PCR amplification primer of the RUNX1 gene includes at least Nucleotide sequence primer as shown in NO.5~22 SEQ ID.
4. kit according to claim 1, which is characterized in that the PCR amplification primer of the TP53 gene includes at least Nucleotide sequence primer as shown in NO.23~40 SEQ ID.
5. kit according to claim 1, which is characterized in that the PCR amplification primer of the WT1 gene includes at least core Nucleotide sequence primer as shown in NO.41~44 SEQ ID.
6. kit according to claim 1, which is characterized in that the universal sequencing primer object connector includes at least nucleotide Sequence primer connector as shown in NO.45~46 SEQ ID.
7. kit according to claim 1, which is characterized in that the kit further includes that sample genomic dna extracts One of reagent, pcr amplification reaction reagent, sequencing reagent are a variety of.
8. kit according to claim 7, which is characterized in that the pcr amplification reaction reagent be selected from premix Taq, ddH2One of O or a variety of.
9. kit according to claim 1, which is characterized in that the kit also contains positive control or negative control One of or it is a variety of.
10. kit is in preparation AML patient's prognostic gene abrupt climatic change product as described in claim 1~9 any claim In purposes.
CN201711483126.9A 2017-12-29 2017-12-29 A kind of kit and its detection method of the mutation of detection AML patient's prognostic gene Pending CN109988832A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105861674A (en) * 2016-04-27 2016-08-17 上海荻硕贝肯生物科技有限公司 Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis
CN106381332A (en) * 2016-08-31 2017-02-08 天津协和华美医学诊断技术有限公司 Detection kit for detecting AML related gene group

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105861674A (en) * 2016-04-27 2016-08-17 上海荻硕贝肯生物科技有限公司 Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis
CN106381332A (en) * 2016-08-31 2017-02-08 天津协和华美医学诊断技术有限公司 Detection kit for detecting AML related gene group

Non-Patent Citations (2)

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Title
DOMINIC ROSE ET AL: "Subtype-specific patterns of molecular mutations in Acute Myeloid Leukemia", 《LEUKEMIA》 *
OCHI: "M13测序引物名称及碱基序列", 《微博》 *

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Application publication date: 20190709