CN103215352B - Assay kit for detecting stimulated emission depletion 2 (STED 2) gene mutation - Google Patents
Assay kit for detecting stimulated emission depletion 2 (STED 2) gene mutation Download PDFInfo
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Abstract
The invention relates to an assay kit for detecting stimulated emission depletion 2 (SETD 2) gene mutation. The assay kit comprises a reagent for amplifying an SETD 2 gene and a reagent for detecting the SETD 2 gene mutation. The reagent for amplifying the SETD 2 gene comprises a polymerase chain reaction (PCR) primer for amplifying the SETD 2 gene and a corresponding PCR reaction reagent, wherein the PCR primer for amplifying the SETD 2 gene is a PCR primer which is designed for 21 exons of the SETD 2 gene; and the PCR primer comprises sequences of SEQ ID NO:1-SEQ ID NO:64. The reagent for detecting the SETD 2 gene mutation comprises a sequencing primer for detecting the SETD 2 mutation, wherein the sequencing primer comprises the sequences of SEQ ID NO:65-SEQ ID NO:103. The assay kit can be used for screening and detecting all the known or unknown SETD 2 gene mutations.
Description
Technical field
The present invention relates to a kind of detection kit, particularly a kind of test kit detecting SETD2 transgenation.
Background technology
SETD2 gene is positioned at Human Chromosome 3 the short arm of a chromosome, an important histone modification enzyme of encoding, and can to methylate modification to histone H 3 K36 site.Research shows, H3K36 and another site H3K79 is two crucial histone modification sites, and they play important regulating effect [Berger S., 2007 in genetic transcription extension process; Nature, 447 (7143): 407-412].Carry out genome sequencing discovery to 12 routine acute lymphoblastic system leukemia, the case of 48% contains the sudden change of histone modification enzyme gene, and SETD2 sudden change is one of them [Zhang J., et al, 2012; Nature, 481 (7380): 157-163].Our seminar, to a pair three years old identical twins sister (send out leukemia for, another is then healthy), carries out DNA genome sequencing.Find that leukemia young girl's histone methylated transferase gene M LL (acting on H3K79 site) occurs to reset and form MLL-NRIP3 fusion gene.In addition, also find that SETD2 gene also there occurs two nonsense mutations.Next we analyze 134 routine acute myeloid leukemias (AML) and 107 routine acute lymphoblastic systems leukemia (ALL), find that SETD2 sudden change has generation in AML and ALL, and MLL reset and non-rearrangement case in also all have generation, its Mutation probability accounts for 5.4% of total case (241 routine).Our research finds, the mutation type of SETD2 in leukemia has truncated mutant (truncatingmutation) and missense mutation (missense mutation), the sudden change of these types all makes SETD2 zymoprotein recurring structure destroy, and loses function.Multiple mutation can be there is in addition in SETD2 gene.Because the type of SETD2 transgenation and multiplicity meet tumor suppressor gene, therefore the disappearance of its function plays an important role in the morbidity of acute leukemia.
Therefore the SETD2 transgenation in detection Blood of Patients cell is to leukemic diagnosis, and molecular pathology somatotype, prognostic analysis, medication guide and course of disease monitoring have important clinical meaning.But, up to now, market also there is no detection method and the test kit of a kind of SETD2 of maturation.
Summary of the invention:
The technical problem to be solved in the present invention overcomes the deficiencies in the prior art, provides a kind of test kit detecting SETD2 transgenation.
The technical solution used in the present invention is:
Detect a test kit for SETD2 transgenation, this test kit comprises the reagent of amplification SETD2 gene and detects the reagent of SETD2 transgenation.
The reagent of described amplification SETD2 gene comprises the PCR primer of amplification SETD2 gene and corresponding PCR reaction reagent, wherein
The PCR primer of described amplification SETD2 gene is the PCR primer that pin designs in SETD2 gene 21 exons, and described primer sequence comprises the sequence of SEQ ID NO:1-SEQ ID NO:64.
The reagent of described detection SETD2 transgenation comprises the sequencing primer for detecting SETD2 sudden change, and described sequencing primer sequence comprises the sequence of SEQ ID NO:65-SEQ ID NO:103.
Described test kit comprises for the step detecting SETD2 transgenation: collect and with selected by flow cytometry apoptosis pathology hemocyte, and with the normal cell compared, then the genomic dna extracting these cells is stand-by as masterplate, utilize the above-mentioned oligonucleotide primer for 21 outer existing sons, polymerase chain reaction (PCR) amplification is carried out to described DNA profiling, then determined dna sequence is carried out to pcr amplification product.The SETD2 sequence of pathology hemocyte and the SETD2 sequence of normal control cells are compared, finds out Candidate Mutant sequence.Then by comparing with existing DNA sequence data storehouse, rejecting crowd's polymorphic sequence, determining SETD2 sudden change result.
The beneficial effect that the present invention has:
Use the method and test kit that the present invention relates to, can all known or unknown sudden changes of SETD2 gene be screened and be detected.Its susceptibility is high, high specificity.It is more comprehensive and accurate method on market so far that the method that the present invention relates to and test kit carry out detection to SETD2 transgenation, and its operation steps is brief and concise, is applicable to very much the requirement of Clinical practice, therefore has significant advantage.
The test kit related in the present invention and detection method are applicable to the SETD2 detection in Gene Mutation that clinical diagnosis is acute leukemic patient.
The determination of acute leukemic patient:
The standard of clinical diagnosis acute leukemia is with reference to WHO2008 " hematopoiesis and lymphsystem tumor are classified " (Swerdlow, Steven H.WhoClassification of Tumours of Haematopoietic and Lymphoid Tissues.4th ed.Lyon, France:InternationalAgency for Research on Cancer, 2008.).
Accompanying drawing explanation
Fig. 1 is the overhaul flow chart of test kit.
Fig. 2 is presented in SETD2 gene can there is various mutations.The kind of sudden change has: truncate mutation (truncating mutation), missense mutation (missense mutation).Sudden change all has generation in AML and ALL; Sudden change can occur in MLL and reset case, also can occur in non-MLL and reset case.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, but do not limit protection scope of the present invention.
Embodiment 1
One, the drawing materials of leukemia cell
Collect the peripheral blood 5ml of acute leukemic patient, extraction mononuclearcell is put in-70 DEG C of Ultralow Temperature Freezers or liquid nitrogen and is preserved.
Two, the drawing materials of normal control tissue
While collecting acute leukemic patient peripheral blood, also need a kind of normal control tissue collecting this patient, as saliva, at least one such as catabasis peripheral blood.The method of collecting normal control tissue is as follows:
A, saliva
1), after acute leukemic patient physiological saline gargles, collutory remaines in container.
2), exhort patient's spitting ptysis, tell several times more as far as possible.Gargle after having told, collutory remaines in container.
3), saliva and collutory respectively with 50ml PBS dilution cleaning, centrifugal 5 minutes of 2000rpm, abandons supernatant.Wash 2 times, after be precipitated.
B, catabasis peripheral blood
Collect acute leukemic patient paracmastic peripheral blood 5ml, and extract mononuclearcell and put
preserve in Ultralow Temperature Freezer or liquid nitrogen.
Three, reagent and key instrument
1. reagent
1) 5 × Tris-boric acid (TBE) damping fluid
2) cell pyrolysis liquid TRIzol Reagent is purchased from Invitrogen
3) monoclonal antibody purchased from American BD company
4) tri methylol amino methane (Tri Base), Silver Nitrate, anhydrous sodium carbonate, glacial acetic acid, urea, ammonium persulphate, formaldehyde, chloroform, Virahol, dehydrated alcohol, sodium ethylene diamine tetracetate (EDTA) are purchased from Tianjin wind ship chemical reagent science and technology limited Company
5) 2 × SSC/ sodium citrate buffer agent powder is purchased from Bo Sheng bio tech ltd, Shanghai
6) archaeal dna polymerase (Taq-DNA polymerase, Cat.#DR011) is purchased from TaKaRa company
2. key instrument
Four. leukemia cell's purifying
In order to ensure the sensitivity of SETD2 abrupt climatic change, the purity requirement of leukemia cell is at least 50%.If the leukemia cell contained in the peripheral blood of leukaemic is less than 50%, need to adopt airflow classification method to improve the purity of leukemia cell.Airflow classification step is as follows:
1) from liquid nitrogen container, take out the peripheral blood freeze pipe of leukaemic, put into rapidly 38 DEG C of water-baths, and frequently shake, in 1 minute, make it melt completely.
2) proceeded to by celliferous nutrient solution in the 12ml PBS solution containing 2% serum, mixing, gets 50 μ l mixed solutions and adds 50 μ l trypan blues, count rapidly under microscope.
3) under 1200r/min speed centrifugal 5 minutes, abandon supernatant liquor, add 50 μ l containing the PBS of 2% serum, proceed to streaming pipe, according to cell quantity add antibody CD45+PerCP-Cy5, leukemia tumor cells surface representative biomarker proteins proteins antibody (by 10 μ l/10
6cell adds).
4) 4 DEG C of lucifuges hatch 40min.
5) add the PBS2ml containing 2% serum, Excess antibody is removed in washing, under 1200r/min speed centrifugal 5 minutes, abandons supernatant liquor, adds the PBS (1ml/10 containing 2% serum according to cell quantity
7cell).
6) establish door with CD45PerCP-Cy5/SSC, according to the representative molecule marker sorting leukemia cell on leukemia tumor cells surface, deposit in the mixed system of 10%DMSO, 90% serum, be placed in
ultralow Temperature Freezer is preserved.
Five, poba gene group DNA extraction and saliva extracting genome DNA
(1). extract poba gene group DNA with the Blood & cell culture DNA kits of QIAGEN, step is with the specification sheets in test kit
1) add in 500 μ l Buffer AP1 to 1.5ml centrifuge tubes.
2) add 200-250 μ l anticoagulated whole blood in Buffer AP1, with pipettor back and forth suction several times, thoroughly to dissolve the blood remained on suction nozzle.Cover tightly centrifuge tube lid, vortex vibration 10s.
3) 100 μ l Buffer AP2 are added, vortex vibration 10s, 12,000 × g centrifugal 10min.
4) be placed in 2ml centrifuge tube (providing in test kit), by step 3 by preparing pipe) in filtrate joining prepare in pipe, 12,000 × g centrifugal 1min.
5) abandon filtrate, put get back to preparing pipe in former 2ml centrifuge tube, add 700 μ l Buffer W1A, room temperature places 2min.12,000 × g centrifugal 30s.
6) abandon filtrate, put get back to preparing pipe in former 2ml centrifuge tube, add the Buffer W2 that 800 μ l have added dehydrated alcohol, 12,000 × g centrifugal 1min.
7) putting get back to preparing pipe in former 2ml centrifuge tube, adding 500 μ l Buffer W2 to preparing in pipe, 12,000 × g centrifugal 1min.
8) abandoning filtrate, putting back former 2ml centrifuge tube, 12,000 × g centrifugal 1min by preparing pipe.
9) will prepare pipe and be placed in the 1.5ml centrifuge tube (providing in test kit) of another cleaning, preparing periosteum central authorities and add the Buffer TE of 80-200 μ l preheating, room temperature leaves standstill 1min.12,000 × g centrifugal 1min wash-out genomic dnas.
(2), saliva extracting genome DNA
1), the saliva of acute leukemic patient and collutory respectively with 50ml PBS dilution cleaning, centrifugal 5 minutes of 2000rpm, abandons supernatant.Wash 2 times, after be precipitated.
2), the hemocyte genome DNA extracting reagent kit of precipitation QIAGEN that obtains extracts DNA, and step is with the specification sheets in test kit.The difference of DNA extraction amount saliva and collutory can to about 7ug.
Six .SETD2 primer synthesis
In order to 21 exons of pcr amplification SETD2 gene, need to synthesize following amplimer and sequencing primer.
Seven .PCR amplified reactions
1) pcr amplification reaction dosing
Get the PCR pipe of 0.2ml, by marking pen numbering, pipe be inserted in granular ice, according to the form below reagent adding:
Total reaction volume 100 μ l, does not add light mineral oil or paraffin oil, covers tightly PCR pipe, with the mixing of finger bomb tube, slightly centrifugal.
Forward primer needed for above-mentioned PCR reaction of described forward primer and reverse primer and reverse primer; Genomic DNA template is the poba gene group DNA that Part V (1) step obtains;
2) pcr amplification reaction condition: PCR pipe is placed in 9600 or 2400 type PCR instrument and increases.95 DEG C of laggard performing PCR circulations of sex change 5min, PCR loop parameter is 94 DEG C of 25s, 60 DEG C of 45s, 72 DEG C of 1min, 35 circulations; 72 DEG C of 5min; After amplification terminates, 4 DEG C of insulations are set.
3) PCR primer purifying and Purity: (1) is centrifugal by mixture, transfers to amplified production in 1.5ml EP pipe.(2) add 25 μ l sodium-acetate/alcohol mixeding liquids, fully vibrate, put on ice 10min to precipitate DNA.12000r/min, in 4 DEG C of centrifugal 30min, carefully abandons supernatant.
(3) the ethanol 50 μ l washing precipitation 2 times of 70% (V/V) is added.12000r/min, in 4 DEG C of centrifugal 5min, carefully abandons the liquid pearl of cleer and peaceful tube wall, vacuum-drying precipitation 10 ~ 15min.Concentration is made to be 100ng/ μ l with deionized water dissolving PCR primer DNA.Detecting DNA purity with spectrophotometer is generally 1.8-2.0 at A260nm/A280nm.
Eight .PCR order-checkings
1) PCR sequencing reaction
(1) get the PCR pipe of 0.2ml, by marking pen numbering, pipe be inserted in granular ice, according to the form below reagent adding:
Total reaction volume 5 μ l, does not add light mineral oil or paraffin oil, covers tightly PCR pipe, with the mixing of finger bomb tube, slightly centrifugal.
(2) PCR pipe is placed in 9600 or 2400 type PCR instrument and increases.98 DEG C of laggard performing PCR circulations of sex change 2min, PCR loop parameter is 96 DEG C of 10s, 50 DEG C of 5s, 60 DEG C of 4min, 25 circulations, arranges 4 DEG C of insulations after amplification terminates.
2) sodium-acetate/Ethanol Method purified pcr product
(1) mixture is centrifugal, amplified production is transferred in 1.5ml EP pipe.
(2) add 25 μ l sodium-acetate/alcohol mixeding liquids, fully vibrate, put on ice 10min to precipitate DNA.12000r/min, in 4 DEG C of centrifugal 30min, carefully abandons supernatant.
(3) the ethanol 50 μ l washing precipitation 2 times of 70% (V/V) is added.12000r/min, in 4 DEG C of centrifugal 5min, carefully abandons the liquid pearl of cleer and peaceful tube wall, vacuum-drying precipitation 10 ~ 15min.
3) process of the PCR primer that checks order before electrophoresis
(1) add the TSR of 12 μ l in centrifuge tube, thermal agitation, allow its abundant dissolving DNA precipitate, slightly centrifugal.
(2) solution is transferred in the 0.2ml PCR pipe of lid separation, slightly centrifugal.
(3) in PCR instrument, carry out thermally denature (95 DEG C of 2min), quenching in ice, treats machine.
4) operate the computer
By instrumentation specification sheets, kapillary is installed, carries out the correction of capillary position, the order-checking sequential file that encapsulating and foundation manually runs.Instrument by automatic glue filling to kapillary, 1.2kV prerunning 5min, by program order automatic sampling, then prerunning (1.2kV, 20min), electrophoresis 2h under 7.5kV.Electrophoresis terminates rear instrument and can automatically clean, and encapsulating, enters next sample, prerunning and electrophoresis.Each sample electrophoresis total time is 2.5h.Electrophoresis terminates rear instrument meeting automatic analysis or prints color sequencer collection of illustrative plates.
5) instrument will carry out sequential analysis automatically, and can require to carry out gene comparision according to user.As sequencing sequence is known, mark distinguishing base place by gene comparision with asterisk.
6) .PCR sequencing data analysis & verification
(1) if deliver to the order-checking of Bo Ao biotech firm, company can return order-checking peak figure and the Candidate Mutant that arrives of analyzing and testing.
The peak map file that will check order imports SMD software (Sequence Mutation Detector, developed by Boao Biological Co., Ltd), use software identification sequencing result (comprising heterozygous sites), remove the inferior quality sequence at figure two ends, peak, and the reference sequences of high quality sequence and SETD2 gene is compared, find out Candidate Mutant site.
(2) for Candidate Mutant site, first dbSNP database (build131) is filtered out, 1000genomes Pilot Projects (1, 2, 3), YanHuang Project, Watson, Venter, Korean, known group pleomorphism site in NA18507, then nonsynonymous mutation (the non-synonymous mutation in SETD2 gene C DS region is selected, cause the sudden change of amino acid change) and be positioned at the variable sheer site mutation (splice-site mutation) of exon: intron intersection (exon-intron junction), and distinguish true sudden change and false positive further by backward sequencing.The method of backward sequencing is, selects appropriate primer backward sequencing (if do not have appropriate primer, can design with under Primer5.0 software default parameter) according to position, mutational site.
(3) do the true sudden change of PCR order-checking detection with the primer that this site is corresponding whether to exist in the genomic dna of the normal control tissue (saliva or catabasis peripheral blood) of pairing.Leukemia cell has but the sudden change that normal control tissue does not detect is considered to somatic mutation (somatic mutation).
7). mono-clonal order-checking (choosing is done)
If the somatic mutation detected is insertion and deletion (indel), can consider that mono-clonal order-checking confirms concrete insertion and deletion sequence further.Instrument: Bio-rad Peltier Thermal Cycler (PCR instrument)
Experimental procedure:
(1), increase target fragment, and condition is as follows:
(2), get PCR primer, carry out ligation according to pGEM-T Easy vector (Promega) specification sheets, 4 DEG C of connections are spent the night.
(4), get 5 μ l connection products, join 100 μ l DH5 α competent cells, leave standstill 30min on ice.
(5), 42 DEG C of metal baths are put into, heat shock 1min by being added with the competent cell connecting product.
(6), after heat shock, be put into rapidly on ice, leave standstill 2min.
(7), in competent cell, add the LB substratum of 700 μ l37 DEG C preheatings, 160rpm vibrates 1h.
(8), get 100 μ l bacterium liquid, be evenly coated in (containing Amp100 μ g/ml, surface also scribbles X-gal and IPTG) on LB flat board with spreader, be upside down in 37 DEG C and cultivate 16h.(blue hickie screening)
(9), to single white colony, bacterium colony PCR is adopted to carry out positive identification.Single bacterium colony of the positive is chosen in appropriate LB liquid nutrient medium (containing Amp100 μ g/ml), 37 DEG C of 200rpm shaken overnight.
(10), extract plasmid, check order with universal support primer.
(11) sequence, to mono-clonal checking order out, with the comparison of clustalW and SETD2 gene order, is confirmed to be the base sequence inserting or delete region.
Found that, as shown in Figure 2, various mutations can occur in SETD2 gene.The kind of sudden change has: truncated mutant (truncatingmutation), missense mutation (missense mutation).Multiple mutation can be there is in addition in SETD2 gene.Sudden change all has generation in AML and ALL; Sudden change can occur in MLL and reset case, also can occur in non-MLL and reset case.
The mutation type of SETD2 in leukemia has truncated mutant (truncating mutation) and missense mutation (missense mutation), and the sudden change of these types all makes SETD2 zymoprotein recurring structure destroy, and loses function.Multiple mutation can be there is in addition in SETD2 gene.Because the type of SETD2 transgenation and multiplicity meet tumor suppressor gene, therefore the disappearance of its function plays an important role in the morbidity of acute leukemia.
Embodiment 2
Detect a test kit for SETD2 sudden change, test kit comprises:
6) PCR primer of described amplification SETD2 gene is that pin is in PCR primer (being made into 50pmol/ μ l with deionized water) the 100 μ l of SETD2 gene 21 exon designs
Primer sequence is as follows:
7) sequencing primer (being made into 3.2pmol/ μ l with deionized water) the 100 μ l of SETD2 transgenation
8) sequence of sequencing primer is as follows:
The using method of this test kit is with embodiment 1, and its flow process as shown in Figure 1.
Claims (1)
1. detect a test kit for the SETD2 transgenation relevant to acute leukemia, it is characterized in that: this test kit comprises the reagent of amplification SETD2 gene and detects the reagent of SETD2 transgenation; The reagent of described amplification SETD2 gene comprises the PCR primer of amplification SETD2 gene and corresponding PCR reaction reagent, the PCR primer of wherein said amplification SETD2 gene is the PCR primer for 21 the exon designs of SETD2 gene, and described PCR primer sequence comprises the sequence of SEQ ID NO:1-SEQ ID NO:64; The reagent of described detection SETD2 transgenation comprises the sequencing primer for detecting SETD2 sudden change, and described sequencing primer sequence comprises the sequence of SEQ ID NO:65-SEQ ID NO:103.
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| CN113106157B9 (en) * | 2021-05-24 | 2023-08-25 | 温州医科大学附属第二医院（温州医科大学附属育英儿童医院） | Kit for predicting prognosis survival time of tumor immunotherapy and application thereof |
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| SET/TAF-1β的研究进展;许波群等;《癌变.畸变.突变》;20091130;第21卷(第06期);参见其正文第4节 * |
| 表观遗传修饰与白血病;陈燕等;《中国实验血液学杂志》;20060820;第14卷(第04期);参见全文 * |
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