CN103215352A - Assay kit for detecting stimulated emission depletion 2 (STED 2) gene mutation - Google Patents
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Abstract
The invention relates to an assay kit for detecting stimulated emission depletion 2 (STED 2) gene mutation. The assay kit comprises a reagent for amplifying an STED 2 gene and a reagent for detecting the STED 2 gene mutation. The reagent for amplifying the STED 2 gene comprises a polymerase chain reaction (PCR) primer for amplifying the STED 2 gene and a corresponding PCR reaction reagent, wherein the PCR primer for amplifying the STED 2 gene is a PCR primer which is designed for 21 exons of the STED 2 gene; and the PCR primer comprises sequences of SEQ ID NO:1-SEQ ID NO:64. The reagent for detecting the STED 2 gene mutation comprises a sequencing primer for detecting the STED 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 STED 2 gene mutations.
Description
Technical field
The present invention relates to a kind of detection kit, particularly a kind of test kit that detects the STED2 transgenation.
Background technology
The STED2 gene is positioned at human No. 3 the short arm of a chromosome, and the important histone modification enzyme of encoding can be to the modification that methylates of histone H 3 K36 site.Studies show that H3K36 and another site H3K79 are two crucial histone modification sites, they play important regulatory role [Berger S., 2007 in genetic transcription extension process; Nature, 447 (7143): 407-412].To 12 routine acute lymphoblastics is that leukemia is carried out the genome sequencing discovery, and 48% case contains the sudden change of histone modification enzyme gene, and the SETD2 sudden change is one of them [Zhang J., etal, 2012; Nature, 481 (7380): 157-163].Our seminar carries out the DNA genome sequencing to a pair of three years old identical twins sister (send out leukemia for, another is then healthy).Find that formation MLL-NRIP3 fusion gene takes place to reset leukemia young girl histone methylated transferase gene M LL (acting on the H3K79 site).In addition, find that also two nonsense mutations have also taken place the STED2 gene.Next we are that leukemia (ALL) is analyzed to 134 routine acute myeloid leukemias (AML) and 107 routine acute lymphoblastics, find that the STED2 sudden change all has generation in AML and ALL, and in the case of MLL rearrangement and non-rearrangement generation is arranged all also, its sudden change probability accounts for 5.4% of total case (241 example).We discover, the mutation type of STED2 in leukemia has truncated mutant (truncating mutation) and wrong meaning sudden change (missense mutation), and the sudden change of these types all makes STED2 zymoprotein recurring structure destroy, and loses function.In the STED2 gene multiple mutation can take place in addition.Because the type and the multiplicity of STED2 transgenation meet tumor suppressor gene, so the disappearance of its function plays an important role in the morbidity of acute leukemia.
Therefore detect STED2 transgenation in patient's hemocyte to leukemic diagnosis, the molecular pathology somatotype, prognostic analysis, medication guide and course of disease monitoring all have the important clinical meaning.Yet, up to now, also do not have detection method and the test kit of a kind of sophisticated STED2 on the market.
Summary of the invention:
The technical problem to be solved in the present invention overcomes the deficiencies in the prior art, and a kind of test kit of the STED2 of detection transgenation is provided.
The technical solution used in the present invention is:
A kind of test kit that detects the STED2 transgenation, this test kit comprise the reagent of amplification STED2 gene and detect the reagent of STED2 transgenation.
The reagent of described amplification STED2 gene comprises the PCR primer and the corresponding PCR reaction reagent of amplification STED2 gene, wherein
The PCR primer of described amplification STED2 gene is the PCR primer of pin in 21 exon designs of STED2 gene, and described primer sequence comprises the sequence of SEQID NO:1-SEQ ID NO:64.
The reagent of described detection STED2 transgenation comprises the sequencing primer that is used to detect the STED2 sudden change, and described sequencing primer sequence comprises the sequence of SEQ ID NO:65-SEQ ID NO:103.
The step that described test kit is used to detect the STED2 transgenation comprises: collect and with selected by flow cytometry apoptosis pathology hemocyte, and with the normal cell that compares, the genomic dna that extracts these cells then is stand-by as masterplate, utilize above-mentioned oligonucleotide primer at 21 outer existing sons, described dna profiling is carried out polymerase chain reaction (PCR) amplification, then pcr amplification product is carried out determined dna sequence.The STED2 sequence of pathology hemocyte and the STED2 sequence of normal control cell are compared, find out candidate's mutant nucleotide sequence.By comparing with existing dna sequence data storehouse, the polymorphic sequence of rejecting crowd is determined STED2 sudden change result then.The beneficial effect that the present invention had:
Method that use the present invention relates to and test kit can screen and detect all the known or unknown sudden changes of STED2 gene.Its susceptibility height, high specificity.Method that the present invention relates to and test kit to STED2 transgenation to detect be more comprehensive and accurate method on the market so far, its operation steps is brief and concise, is fit to very much the requirement of clinical use, therefore has significant advantage.
Test kit that relates among the present invention and detection method are applicable to that clinical diagnosis is the SETD2 detection in Gene Mutation of acute leukemic patient.
Determining of acute leukemic patient:
The standard reference WHO2008 of clinical diagnosis acute leukemia " hematopoiesis and lymphsystem tumor classification " (Swerdlow, Steven H.Who Classification of Tumours of Haematopoietic and Lymphoid Tissues.4th ed.Lyon, France:International Agency for Research on Cancer, 2008.).
Description of drawings
Fig. 1 is the testing process figure of test kit.
Fig. 2 is presented in the STED2 gene, and various mutations can take place.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
The invention will be further described below in conjunction with specific embodiment, but do not limit protection scope of the present invention.
Embodiment 1
One, the leukemia cell's draws materials
Collect the peripheral blood 5ml of acute leukemic patient, the extraction mononuclearcell is put in-70 ℃ of Ultralow Temperature Freezers or the liquid nitrogen and is preserved.
Two, normal control tissue draws materials
When collecting the acute leukemic patient peripheral blood, also need to collect a kind of normal control tissue of this patient, as saliva, catabasis peripheral blood etc. are at least a.The method of collecting normal control tissue is as follows:
A, saliva
1), after acute leukemic patient physiological saline gargles, collutory remaines in the container.
2), exhort patient's spitting ptysis, tell several times as much as possible.Gargle after having told, collutory remaines in the container.
3), saliva and collutory clean with 50ml PBS dilution respectively, centrifugal 5 minutes of 2000rpm abandons supernatant.Wash 2 times, after obtain the precipitation.
B, catabasis peripheral blood is collected the paracmastic peripheral blood 5ml of acute leukemic patient, and extracts mononuclearcell and put in-70 ℃ of Ultralow Temperature Freezers or the liquid nitrogen and preserve.
Three, important reagent and key instrument
1. important reagent
1) 5 * Tris-boric acid (TBE) damping fluid
2) cell pyrolysis liquid TRlzol Reagent is available from Invitrogen
3) monoclonal antibody is available from U.S. company BD
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 available from Tianjin wind ship chemical reagent science and technology limited Company
5) 2 * SSC/ sodium citrate buffer agent powder is available from the rich bio tech ltd that rises in Shanghai
6) (Taq-DNA polymerase is Cat.#DR011) available from TaKaRa company for archaeal dna polymerase
2. key instrument
Four. leukemia cell's purifying
For the sensitivity that guarantees that the SETD2 sudden change detects, leukemia cell's purity requirement is at least 50%.If the leukemia cell's less than 50% that contains in leukaemic's the peripheral blood needs to adopt the airflow classification method to improve leukemia cell's purity.The airflow classification step is as follows:
1) taking-up leukaemic's peripheral blood freeze pipe from liquid nitrogen container is put into 38 ℃ of water-baths rapidly, and is shaken frequently, in 1 minute it is melted fully.
2) celliferous nutrient solution is changed in the 12ml PBS solution that contains 2% serum, mixing is got 50 μ l mixed solutions and is added 50 μ l trypan blues, and microscopically is counted rapidly.
3) under 1200r/min speed centrifugal 5 minutes, abandon supernatant liquor, add 50 μ l and contain the PBS of 2% serum, change the streaming pipe over to, add the representative molecule marker protein antibodies of antibody CD45+PerCP-Cy5, leukemia tumor cell surface (by 10 μ l/10 according to cell quantity
6Cell adds).
4) 4 ℃ of lucifuges are hatched 40min.
5) add the PBS2ml contain 2% serum, unnecessary antibody is removed in washing, and under the 1200r/min speed centrifugal 5 minutes, abandon supernatant liquor, add the PBS (1ml/10 that contains 2% serum according to cell quantity
7Cell).
6) establish door with CD45PerCP-Cy5/SSC, the representative molecule marker sorting leukemia cell according to the leukemia tumor cell surface deposits in the mixed system of 10%DMSO, 90% serum, places-70 ℃ of Ultralow Temperature Freezers to preserve.
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 in the test kit
Specification sheets
1) adds 500 μ l BufferAP1 in the 1.5ml centrifuge tube.
2) add 200-250 μ l anticoagulated whole blood in Buffer AP1, with pipettor back and forth suction remain in blood on the suction nozzle with thorough dissolving several times.Cover tight centrifuge tube lid, vortex vibration 10s.
3) add 100 μ l BufferAP2, vortex vibration 10s, the centrifugal 10min of 12,000 * g.
4) will prepare pipe 2ml centrifuge tube (providing in the test kit) will be provided, the filtrate in the step 3) be joined in the preparation pipe the centrifugal 1min of 12,000 * g.
5) abandon filtrate, will prepare pipe and put and get back in the former 2ml centrifuge tube, add 700 μ l BufferW1A, room temperature is placed 2min.The centrifugal 30s of 12,000 * g.
6) abandon filtrate, will prepare pipe and put and get back in the former 2ml centrifuge tube, add the Buffer W2 that 800 μ l have added dehydrated alcohol, the centrifugal 1min of 12,000 * g.
7) will prepare pipe and put and get back in the former 2ml centrifuge tube, add 500 μ l BufferW2 in the preparation pipe, the centrifugal 1min of 12,000 * g.
8) abandon filtrate, will prepare pipe and put back former 2ml centrifuge tube, the centrifugal 1min of 12,000 * g.
9) will prepare pipe another clean 1.5ml centrifuge tube (providing in the test kit) is provided, and prepare the Buffer TE that periosteum central authorities add 80-200 μ l preheating, room temperature leaves standstill 1min.The centrifugal 1min wash-out of 12,000 * g genomic dna.
(2), saliva extracting genome DNA
1), the saliva of acute leukemic patient and collutory clean with 50ml PBS dilution respectively, centrifugal 5 minutes of 2000rpm abandons supernatant.Wash 2 times, after obtain the precipitation.
2), the precipitation that obtains extracts DNA with the hemocyte genome DNA extracting reagent kit of QIAGEN, step is with the specification sheets in the test kit.The difference of DNA extraction amount saliva and collutory can arrive about 7ug.
Six .SETD2 primers are synthetic
For 21 exons of pcr amplification SETD2 gene, need synthetic following amplimer and sequencing primer.
Seven .PCR amplified reactions
1) pcr amplification reaction dosing
Get the PCR pipe of 0.2ml, with the marking pen numbering, pipe is inserted in the particle ice, according to the form below adds reagent:
Total reaction volume 100 μ l do not add light mineral oil or paraffin oil, cover tight PCR pipe, and are with finger bomb tube mixing, centrifugal slightly.
Described forward primer and reverse primer are required forward primer and the reverse primer of above-mentioned PCR reaction; The genomic dna template is the poba gene group DNA that obtains in (1) step of the 5th part; 2) pcr amplification reaction condition: the PCR pipe placed on the 9600 or 2400 type PCR instrument increase.95 ℃ of laggard performing PCR circulations of sex change 5min, the PCR loop parameter is 94 ℃ of 25s, 60 ℃ of 45s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 5min; Amplification is provided with 4 ℃ of insulations after finishing.3) PCR product purification and purity are identified: (1) is centrifugal with mixture, and amplified production is transferred in the 1.5ml EP pipe.(2) add 25 μ l sodium-acetate/alcohol mixeding liquids, fully vibration is put on ice 10min with deposit D NA.12000r/min carefully abandons supernatant in 4 ℃ of centrifugal 30min.(3) add the ethanol 50 μ l washing precipitations 2 times of 70% (V/V).12000r/min carefully abandons the liquid pearl of cleer and peaceful tube wall in 4 ℃ of centrifugal 5min, vacuum-drying precipitation 10～15min.Making concentration with deionized water dissolving PCR product D NA is 100ng/ μ l.Detecting DNA purity with spectrophotometer is 1.8-2.0 at A260nm/A280nm generally.
Eight .PCR order-checking
1) PCR sequencing reaction
(1) PCR that gets 0.2ml manages, and with the marking pen numbering, pipe is inserted in the particle ice, and according to the form below adds reagent:
Total reaction volume 5 μ l do not add light mineral oil or paraffin oil, cover tight PCR pipe, and are with finger bomb tube mixing, centrifugal slightly.
(2) the PCR pipe is placed on the 9600 or 2400 type PCR instrument and increase.98 ℃ of laggard performing PCR circulations of sex change 2min, the PCR loop parameter is 96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 4min, 25 circulations, amplification is provided with 4 ℃ of insulations after finishing.
2) sodium-acetate/Ethanol Method purified pcr product
(1) mixture is centrifugal, amplified production is transferred in the 1.5ml EP pipe.
(2) add 25 μ l sodium-acetate/alcohol mixeding liquids, fully vibration is put on ice 10min with deposit D NA.12000r/min carefully abandons supernatant in 4 ℃ of centrifugal 30min.
(3) add the ethanol 50 μ l washing precipitations 2 times of 70% (V/V).12000r/min carefully abandons the liquid pearl of cleer and peaceful tube wall in 4 ℃ of centrifugal 5min, vacuum-drying precipitation 10～15min.
3) processing of order-checking PCR product before the electrophoresis
(1) TSR that adds 12 μ l is in centrifuge tube, and thermal agitation allows its abundant dissolving DNA precipitate, and is centrifugal slightly.
(2) solution is transferred in the isolating 0.2ml PCR of the lid pipe, centrifugal slightly.
(3) carry out thermally denature (95 ℃ of 2min) on the PCR instrument, machine is treated in quenching in the ice.
4) operate the computer
Press the instrumentation specification sheets kapillary is installed, carry out the correction of capillary position, manually the order-checking sequential file of encapsulating and foundation operation.Instrument with automatic encapsulating to kapillary, 1.2kV prerunning 5min, by the program order automatic sampling, again prerunning (1.2kV, 20min), electrophoresis 2h under 7.5kV.Electrophoresis finishes the back instrument and can clean automatically, and encapsulating advances next sample, prerunning and electrophoresis.Each sample electrophoresis total time is 2.5h.Electrophoresis finishes the back instrument can analyze or print the color sequencer collection of illustrative plates automatically.
5) instrument will carry out sequential analysis automatically, and can carry out sequence relatively according to customer requirements.Known as sequencing sequence, can mark difference base place with asterisk by the sequence comparison.
6) the .PCR sequencing data is analyzed and checking
(1) if deliver to Bo Ao biotech firm order-checking, company can return candidate's sudden change that order-checking peak figure and analyzing and testing arrive.The peak map file that will check order imports SMD software (Sequence Mutation Detector, by Boao Biological Co., Ltd's exploitation), use software identification sequencing result (comprising the heterozygosis site), remove the inferior quality sequence at figure two ends, peak, and the reference sequences of high quality sequence and SETD2 gene compared, find out the candidate mutational site.
(2) for the candidate mutational site, at first filter out dbSNP database (build131), 1000genomes Pilot Projects (1,2,3), YanHuang Project, Watson, Venter, Korean, the known group pleomorphism site among the NA18507 is selected nonsynonymous mutation (the non-synonymous mutation in SETD2 gene C DS zone then, cause the sudden change of amino acid change) and be positioned at the variable shearing site sudden change (splice-site mutation) of exon-intron intersection (exon-intron junction), and further distinguish true sudden change and false positive by backward sequencing.The method of backward sequencing is, selects suitable primer backward sequencing (if there is not suitable primer, can with designing under the Primer5.0 software default parameter) according to the position, mutational site.
Whether (3) do the true sudden change of PCR order-checking detection with the primer of this site correspondence exists in the genomic dna of paired normal control tissue (saliva or catabasis peripheral blood).The leukemia cell has but normal control tissue does not have detected sudden change to be considered to somatic mutation (somatic mutation).
7). mono-clonal checks order (choosing is done) if detected somatic mutation is to insert disappearance (indel), can consider the further concrete insertion deletion sequence of affirmation of mono-clonal order-checking.Used instrument: Bio-rad Peltier Thermal Cycler (PCR instrument)
Experimental procedure:
(1), the amplification target fragment, condition is as follows:
(2), get the PCR product, carry out ligation according to pGEM-T Easy vector (Promega) specification sheets, 4 ℃ of connections are spent the night.
(4), get 5 μ l and connect product, join 100 μ l DH5 α competent cells, leave standstill 30min on ice.
(5), the competent cell that connects product be will be added with and 42 ℃ of metal baths, heat shock 1min put into.
(6), after the heat shock, be put into rapidly on ice, leave standstill 2min.
(7), in competent cell, add the LB substratum of 700 μ l37 ℃ preheatings, the 160rpm 1h that vibrates.
(8), get 100 μ l bacterium liquid, be evenly coated in (contain Amp100 μ g/ml, the surface also scribbles X-gal and IPTG) on the LB flat board with spreader, be upside down in 37 ℃ and cultivate 16h.(blue hickie screening)
(9), to single white colony, adopt bacterium colony PCR to carry out positive identification.Male list bacterium colony chosen in an amount of LB liquid nutrient medium (contain Amp100 μ g/ml), 37 ℃ of 200rpm shaken overnight.
(10), extract plasmid, check order with the universal support primer.
(11), sequence that mono-clonal is checked order out, with clustalW and the comparison of SETD2 gene order, be confirmed to be and insert or base sequence that deletion is regional.
Found that, as shown in Figure 2, in the STED2 gene, various mutations can take place.The kind of sudden change has: truncated mutant (truncating mutation), missense mutation (missense mutation).In the STED2 gene multiple mutation can take place in addition.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 STED2 in leukemia has truncated mutant (truncating mutation) and missense mutation (missense mutation), and the sudden change of these types all makes STED2 zymoprotein recurring structure destroy, and loses function.In the STED2 gene multiple mutation can take place in addition.Because the type and the multiplicity of STED2 transgenation meet tumor suppressor gene, so the disappearance of its function plays an important role in the morbidity of acute leukemia.
Embodiment 2
A kind of test kit that detects the STED2 sudden change, test kit comprises:
1)10X PCR buffer 100μl
2) Taq DNA polymerase (2.5u/ μ l) 50 μ l
3) each 4mmol/L 50 μ l of dNTP Mix
4)Mg
2+ 150mmol/L 50μl
5) sterilization deionized water 1000 μ l
6) the PCR primer of described amplification STED2 gene is PCR primer (with deionized water be made into 50pmol/ μ l) the 100 μ ls of pin in 21 exon designs of STED2 gene
Primer sequence is as follows:
7) sequencing primer of STED2 transgenation (being made into 3.2pmol/ μ l) 100 μ l with deionized water
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 (4)
1. test kit that detects the STED2 transgenation is characterized in that: this test kit comprises the reagent of amplification STED2 gene and detects the reagent of STED2 transgenation.
2. according to the described a kind of test kit that detects the STED2 transgenation of claim 1, it is characterized in that: the reagent of described amplification STED2 gene comprises the PCR primer and the corresponding PCR reaction reagent of amplification STED2 gene, the PCR primer of wherein said amplification STED2 gene is the PCR primer of pin in 21 exon designs of STED2 gene, and described PCR primer sequence comprises the sequence of SEQ ID NO:1-SEQ ID NO:64.
3. according to claim 1 or 2 described a kind of test kits that detect the STED2 transgenation, it is characterized in that: the reagent of described detection STED2 transgenation comprises the sequencing primer that is used to detect the STED2 sudden change, and described sequencing primer sequence comprises the sequence of SEQ ID NO:65-SEQ ID NO:103.
4. the using method of each described test kit of claim 1-3, comprise the steps: to collect and with selected by flow cytometry apoptosis pathology hemocyte, and with the normal cell that compares, the genomic dna that extracts these cells then is stand-by as masterplate, utilize above-mentioned oligonucleotide primer at 21 outer existing sons, described dna profiling is carried out polymerase chain reaction (PCR) amplification, then pcr amplification product is carried out determined dna sequence.The STED2 sequence of pathology hemocyte and the STED2 sequence of normal control cell are compared, find out candidate's mutant nucleotide sequence.By comparing with existing dna sequence data storehouse, the polymorphic sequence of rejecting crowd is determined STED2 sudden change result then.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103529202A (en) * | 2013-09-13 | 2014-01-22 | 中国科学院合肥物质科学研究院 | Method for reducing spontaneous mutation background of wild type human-rat hybridoma AL cells |
| CN113106157A (en) * | 2021-05-24 | 2021-07-13 | 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 prognosis survival prediction of tumor immunotherapy and application thereof |
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| US7252968B2 (en) * | 1995-05-10 | 2007-08-07 | Boehringer Ingelheim International Gmbh | Chromatin regulator genes |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103529202A (en) * | 2013-09-13 | 2014-01-22 | 中国科学院合肥物质科学研究院 | Method for reducing spontaneous mutation background of wild type human-rat hybridoma AL cells |
| CN103529202B (en) * | 2013-09-13 | 2015-04-15 | 中国科学院合肥物质科学研究院 | Method for reducing spontaneous mutation background of wild type human-rat hybridoma AL cells |
| CN113106157A (en) * | 2021-05-24 | 2021-07-13 | 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 prognosis survival prediction of tumor immunotherapy and application thereof |
| CN113106157B (en) * | 2021-05-24 | 2022-09-20 | 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 prognosis survival prediction of tumor immunotherapy and application thereof |
| 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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Owner name: BEIJING INSTITUTE OF GENE SCIENCE, CHINESE ACADEMY Effective date: 20150615 |
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Effective date of registration: 20150615 Address after: 300020 No. 288, Nanjing Road, Heping District, Tianjin Patentee after: Blood Diseases Hospital (Institute of Hematology), Chinese Academy of Medical Sciences Patentee after: Beijing Institute of Genomics, Chinese Academy of Sciences Address before: 300020 No. 288, Nanjing Road, Heping District, Tianjin Patentee before: Blood Diseases Hospital (Institute of Hematology), Chinese Academy of Medical Sciences |
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Address after: 300020 No. 288, Nanjing Road, Heping District, Tianjin Patentee after: Hematology Hospital of Chinese Academy of Medical Sciences (Institute of Hematology, Chinese Academy of Medical Sciences) Patentee after: BEIJING INSTITUTE OF GENOMICS, CHINESE ACADEMY OF SCIENCES Address before: 300020 No. 288, Nanjing Road, Heping District, Tianjin Patentee before: Hematology Hospital of Chinese Academy of Medical Sciences (Institute of Hematology) Patentee before: BEIJING INSTITUTE OF GENOMICS, CHINESE ACADEMY OF SCIENCES |