CN108531617A - The detecting system of one 42 SNP site for medical jurisprudence individual ancestors' information inference - Google Patents
The detecting system of one 42 SNP site for medical jurisprudence individual ancestors' information inference Download PDFInfo
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Abstract
The invention belongs to medical jurisprudence technical field of gene detection, and in particular to a kind of detection architecture of 42 SNP sites for medical jurisprudence ancestors' information inference, the system can 42 ancestors' information SNP sites of synchronous amplification：rs1012586、rs10496971、rs10506957、rs1205357、rs1211554、rs1510523、rs1800498、rs1876482、rs4466287、rs4756、rs4918664、rs590086、rs67302、rs7226659、rs723220、rs748144、rs7516782、rs7528721、rs7752055、rs7927633、rs8072587、rs8081883、rs8137373、rs931247、rs2056126、rs12142199、rs12419341、rs1366220、rs1399272、rs1453858、rs1475840、rs1557553、rs16891982、rs2080161、rs2267666、rs2289266、rs3176921、rs3827760、rs4749305、rs728404、rs741272、rs830599.The detection architecture method is suitable for the DNA sample of various tissue samples, can be used not only for ancestors' information of extrapolated sample people from source, and can disclose the genetic structure of different groups, infers its Population Genetics information.
Description
Technical field
The invention belongs to medical jurisprudence technical field of gene detection, and in particular to one kind is pushed away for medical jurisprudence individual ancestors' information
The detecting system of 42 disconnected SNP sites.
Background technology
Single nucleotide polymorphism（Single Nucleotide Polymorphism, SNP）It is DNA sequence polymorphism
One type, it is considered to be after short tandem repeat（Short Tandem Repeat, STR）Third generation heredity mark afterwards
Note, SNP showed powerful superiority in medicolegal practice in recent years.SNP is widely present in human genome, about
Per 1000bp, there are a SNP.Compared with STR, the amplified fragments of SNP locus can be shorter, the amplified fragments of single locus
For length usually in 200bp hereinafter, being more suitable for PCR amplification, the DNA abilities of analysis degradation sample are stronger.In forensic science, except answering
Outside for relationship identification and individual identification, it can also be applied to infer ancestors' information of individual, individual phenotypic characteristic identification etc..
With the continuous development of internationalization and economic globalization, people-to-people contacts are more frequent between various countries so that trans-regional,
Transnational crime quantity increasingly rises.On medical jurisprudence, for what is obtained in spot or the large-scale disaster scene of the accident
It is difficult to recognize the height corruption of looks or the corpse of damage, the biological material of height degradation and human blood, the sperm left,
The hereditary information that scene of a crime DNA sample is contained further is excavated, and obtains ancestors' information of these sample people from source,
It is the particularly important research direction in medicolegal genetics field, is conducive to reduce scope of investigation, directive property is provided for the detection of case
Clue.
First ancestor's informative site (Ancestry Informative Markers, AIMs), refers between different crowd
The very big gene loci of allele frequency differences.STR, SNP, mitochondrial DNA（Mitochondrial DNA, mtDNA）
All it is the AIMs there are potential using value, but since the mutation rate of STR and mtDNA are higher, allele between each group
The reasons such as difference is smaller, STR and mtDNA can not be suitable for carrying out the deduction of first ancestor's information as AIMs well.SNP site
Due to its stability is good, has a very wide distribution, allele frequency differences are larger etc. between each group, become first ancestor's information inference
Preferable tool.
Currently, common SNP detection techniques are the SNaPshot technologies based on Single base extension principle and Capillary Electrophoresis.
However, the technology once at most detects 30 or so locus, if tens SNP sites are placed in a reaction system
The optimization of composite amplification, adjustment and reaction system to design of primers, primer concentration and annealing temperature has very high requirement,
It needing repeatedly to react if detecting more SNP locus, the sample amount needed is big, is unfavorable for the detection of trace material evidence,
Limit application of multiple SNP sites in first ancestor infers.Flight time mass spectrum（MassArray）Technology is to combine multiplex PCR
Technology, MassARRAY iPLEX Single base extensions technologies and matrix solid-dispersion flying time mass spectrum analysis skill
Art carries out parting detection, and compared with other classifying methods, the system is quick with detection, parting is accurate, high sensitivity, does not need
Fluorescent marker is easy to the advantages such as extensive and high-throughput operation, can simultaneously be detected in a system SNP more
Point, has a good application prospect.
Invention content
It is an object of the invention to develop a kind of detection for medical jurisprudence individual ancestors 42 SNP sites of information inference
System, the system can 42 SNP sites of synchronous amplification：rs1012586、rs10496971、rs10506957、rs1205357、
rs1211554、rs1510523、rs1800498、rs1876482、rs4466287、rs4756、rs4918664、rs590086、
rs67302、rs7226659、rs723220、rs748144、rs7516782、rs7528721、rs7752055、rs7927633、
rs8072587、rs8081883、rs8137373、rs931247、rs2056126、rs12142199、rs12419341、
rs1366220、rs1399272、rs1453858、rs1475840、rs1557553、rs16891982、rs2080161、
Rs2267666, rs2289266, rs3176921, rs3827760, rs4749305, rs728404, rs741272 and
rs830599。
In embodiments of the invention, the amplification system of the PCR of the system includes Water（HPLC grade）、PCR
Buffer、25mM MgCl2, 25mM dNTP Mix, PCR primer Mix and PCR Enzyme.The SAP reaction systems of the system
Including Water（HPLC grade）, SAP Buffer and SAP Enzyme, the IPLEX reaction systems of the system include Water
（HPLC grade）, iPLEX GOLD Buffer, iPLEX Termination mix, iPLEX extension primers mixture and
iPLEX Enzyme。
Detecting system of the present invention can detect the sample DNA of various tissues, for example, human blood, saliva, hair,
Nail, vaginal secretion or seminal stain equal samples DNA.
When detecting DNA sample, the specific amplification system of kit of the present invention is as follows：
| Reaction system | Concentration | The volume of single hole system each component（µl） |
| Water（HPLC grade） | - | 1.8 |
| PCR Buffer | 10× | 0.5 |
| MgCl2 | 25mM | 0.4 |
| dNTP Mix | 25µM | 0.1 |
| PCR primer MIX | 0.5µU | 1.0 |
| PCR Enzyme | 5U/ml | 0.2 |
| DNA | 10ng/µl | 1.0 |
| Total volume | - | 5.0 |
It is a further object to provide the detection method of the system, the detection method can be used not only for extrapolated sample
First ancestor's information of people from source, and the genetic structure of different groups can be disclosed, the Population Genetics relationship between apparent different groups；Tool
Steps are as follows for body：
1. the preparation of sample：The absorbance of sample DNA requires OD260/OD280 in 1.7-1.9, DNA concentration>10ng/µl.
2. the dilution of primer：By primer dry powder being ranked sequentially according to synthesis of synthesis, 12000rpm is centrifuged 1 minute；Add
Add 1 × TE buffer a concentration of 100 μM to PCR primer, extension primer is 500 μM；Vortex oscillation, centrifugation, stands overnight to it
Preparation primer Mix, spare after dissolving.
3. the preparation of primer Mix
3.1 PCR primer diluted concentrations are 100 μM, prepare final concentration of 0.5 μM of PCR primers Mix；
The preparation of 3.2 extension primer Mix；
The preparation of 3.3 PCR systems.1.8 μ l Water are taken respectively（HPLC grade）, 1 0.5 μM of μ l PCR primers Mix, 0.5
μ l 10 x PCR Buffer, 0.4 μ l 25mM MgCl2, 0.1 μ l 25mM dNTP Mix, 0.2 μ l 5U/ μ l PCR
Enzyme to 1.5m3l pipes preparation PCR Mix.1 μ l DNA are added, be vortexed concussion and centrifugation；
3.4 PCR response procedures：95 DEG C of 2min of pre-degeneration, then 95 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 60sec totally 45
A cycle, 72 DEG C of 5min, last 4 DEG C of preservations.
4.SAP mixes the preparation of liquid
4.1 take 1.53 μ l ultra-pure waters respectively, and 0.17 μ l SAP Buffer, 0.30 μ l SAP Enzyme (1.7U/ μ l) match
SAP processed mixes liquid（The system is single hole）；
4.2 each hole are added the mixed liquid of 2 μ l SAP and plate are sealed with film after the completion, and be vortexed concussion and centrifugation；
The plate sealed is put PCR instrument by 4.3 carries out following procedure：37 DEG C of 40 min, 85 DEG C of 5 min, 4 DEG C of preservations.
5. extension
5.1 take 0.619 μ l sterile deionized waters, 0.2 μ l iPLEX GOLD Buffer, 0.2 μ l iPLEX respectively
Termination mix, 0.94 μ l iPLEX extension primer mixtures, 0.041 μ l iPLEX Enzyme（The system is single
Hole）It prepares iPLEX and extends mixed liquid；
The 5.2 above-mentioned iPLEX of 2 μ l of addition, which extend, to be mixed liquid and mixes, and be vortexed concussion and centrifugation；
5.3 carry out thermal cycle according to following procedure：step 1：94℃ 30s；step 2: 95℃ 5s, 52℃ 5 s, 80
DEG C 5s, 52 DEG C of 5s, 80 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s,
Repeat 40 cycles；step 3: 72℃ 3min；step 4:4 DEG C of long-term preservations.
6. desalting processing
6.1 clean resins（Resin）It paves on 384/6mg dimple plate, air-dries minimum 10 minutes；
6.2 6.2 are added 16 μ l water in each hole for having sample of sample plane.After the completion, plate is sealed with film, be vortexed shake
15min is swung, then 2000rpm centrifuges 5min；
6.3 are added 6 mg cleaning resins（Resin）：Gently by sample plane reversion high up in the air, it is placed on the dimple for having put resin
On plate, then dimple plate are inverted together with sample plane（Two allegros are not horizontally moveable in the process）, resin is allowed to fall
To in hole.Plate is sealed with film, is placed on circulator reverse shake up 15 minutes.Plate is centrifuged 5 minutes with 3200g；
7. starting MassARRAY Nanodispenser RS1000 point sample instruments, the extension products after purifying resin are moved into 384 holes
On SpectroCHIP (Sequenom) chip.
8. the SpectroCHIP chips after point sample are used matrix solid-dispersion flight time mass spectrum point
Analysis, testing result understand the molecular weight peaks of Mass Spectrometer Method using 4.0 softwares of TYPER automatically, and it is right that conversion is shown as SNP site institute
The molecular weight mass spectrum peak figure answered.
The present invention provides a kind of detecting system of 42 SNP sites for medical jurisprudence individual ancestors' information inference, including
Amplification system, the statistics and sequencing result that the reading of sequencing is long are analyzed, and the genotyping result of each SNP site is finally provided.This hair
The bright MassARRAY sequencing technologies that a new generation is utilized carry out parting detection to ancestors' information SNP site, compared to traditional hair
Cons electrophoresis, this technology are capable of providing deeper covering, and an advantage in addition is can different primers to be carried out mixing expansion
Increase, disposably obtain the amplified production of multiple SNP, then carries out parting detection.
Description of the drawings
Fig. 1：Embodiment 1 detects the result in a site DNA sample rs67302.
Fig. 2：Embodiment 1 detects the result in the sites rs67302 in a well.
Fig. 3：Embodiment 1 detects the analysis testing result in the sites rs67302 of 100 parts of different sample DNAs.
Specific implementation mode
It is illustrated the present invention below in conjunction with attached drawing and further detailed description.It should be pointed out that following
Illustrate to be only to claimed technical solution for example, not to any restrictions of these technical solutions.
Protection scope of the present invention be subject to the appended claims record content.
Embodiment 1
When detecting 42 SNP sites of DNA sample, the specific amplification system of system of the present invention is as follows：
| Reaction system | Concentration | The volume of single hole system each component（µl） |
| Water（HPLC grade） | - | 1.8 |
| PCR Buffer | 10× | 0.5 |
| MgCl2 | 25mM | 0.4 |
| dNTP Mix | 25µM | 0.1 |
| PCR primer MIX | 0.5µU | 1.0 |
| PCR Enzyme | 5U/ml | 0.2 |
| DNA | 10ng/µl | 1.0 |
| Total volume | - | 5.0 |
It is a further object to provide the detection method of the system, the detection method can be used not only for extrapolated sample
Ancestors' information of people from source, and the genetic structure of different groups can be disclosed, infer the Population Genetics relationship between different groups；Tool
Steps are as follows for body：
1. the preparation of sample：People's whole blood sample is taken, the DNA of whole blood sample is extracted using paramagnetic particle method and carries out concentration mensuration, is extracted
DNA absorbances require OD260/OD280 between 1.7-1.9, DNA concentration>10ng/µl.
2. the dilution of primer：By primer dry powder being ranked sequentially according to synthesis of synthesis, 12000rpm is centrifuged 1 minute；Add
Add 1 × TE buffer a concentration of 100 μM to PCR primer, extension primer is 500 μM；Vortex oscillation, centrifugation, stands overnight to it
Preparation primer Mix, spare after dissolving.
3. the preparation of primer Mix
3.1 PCR primer diluted concentrations are 100 μM, prepare final concentration of 0.5 μM of PCR primers Mix；
The preparation of 3.2 extension primer Mix；
The preparation of 3.3 PCR systems.1.8 μ l Water are taken respectively（HPLC grade）, 1 0.5 μM of μ l PCR primers Mix, 0.5 μ
L 10 x PCR Buffer, 0.4 μ l 25mM MgCl2, 0.1 μ l 25mM dNTP Mix, 0.2 μ l 5U/ μ l PCR Enzyme
PCR Mix are prepared to 1.5ml pipes.1 μ l DNA are added, be vortexed concussion and centrifugation；
3.4 PCR response procedures：95 DEG C of 2min of pre-degeneration；Then 95 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 60sec totally 45
A cycle；72 DEG C of 5min, last 4 DEG C of preservations.
4.SAP mixes the preparation of liquid
4.1 take 1.53 μ l ultra-pure waters respectively, and 0.17 μ l SAP Buffer, 0.30 μ l SAP Enzyme (1.7U/ μ l) match
SAP processed mixes liquid（The system is single hole）；
4.2 each hole are added the mixed liquid of 2 μ l SAP and plate are sealed with film after the completion, and be vortexed concussion and centrifugation；
The plate sealed is put and carries out following procedure in PCR instrument by 4.3：37 DEG C of 40 min, 85 DEG C of 5 min, 4 DEG C of preservations.
5. extension
5.1 take 0.619 μ l sterile deionized waters, 0.2 μ l iPLEX GOLD Buffer, 0.2 μ l iPLEX respectively
Termination mix, 0.94 μ l iPLEX extension primer mixtures, 0.041 μ l iPLEX Enzyme（The system is single
Hole）It prepares iPLEX and extends mixed liquid；
The 5.2 above-mentioned iPLEX of 2 μ l of addition, which extend, to be mixed liquid and mixes, and be vortexed concussion and centrifugation；
5.3 carry out thermal cycle according to following procedure：step 1：94℃ 30s；step 2: 95℃ 5s, 52℃ 5 s, 80
DEG C 5s, 52 DEG C of 5s, 80 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s,
Repeat 40 cycles；step 3: 72℃ 3min；step 4:4 DEG C of long-term preservations.
6. desalting processing
6.1 clean resins（Resin）It paves on 384/6mg dimple plate, air-dries minimum 10 minutes；
6.2 are added 16 μ l water in each hole for having sample of sample plane.After the completion, plate is sealed with film, be vortexed concussion
15min, then 2000rpm centrifuge 5min；
6.3 are added 6 mg cleaning resins（Resin）：Gently by sample plane reversion high up in the air, it is placed on the dimple for having put resin
On plate, then dimple plate are inverted together with sample plane（Two allegros are not horizontally moveable in the process）, resin is allowed to fall
To in hole.Plate is sealed with film, is placed on circulator reverse shake up 15 minutes.Plate is centrifuged 5 minutes with 3200g.
7. starting MassARRAY Nanodispenser RS1000 point sample instruments, the extension products after purifying resin are moved
On 384 hole SpectroCHIP (Sequenom) chips.
8. the SpectroCHIP chips after point sample are used matrix solid-dispersion flight time mass spectrum point
Analysis, testing result understand the molecular weight peaks of Mass Spectrometer Method using 4.0 softwares of TYPER automatically, and it is right that conversion is shown as SNP site institute
The molecular weight mass spectrum peak figure answered.
Claims (5)
1. a kind of detecting system of 42 first ancestor's SNP sites for medical jurisprudence ancestors' information inference, the detection architecture can synchronize
42 first ancestor's SNP sites of augmentation detection are：rs1012586、rs10496971、rs10506957、rs1205357、
rs1211554、rs1510523、rs1800498、rs1876482、rs4466287、rs4756、rs4918664、rs590086、
rs67302、rs7226659、rs723220、rs748144、rs7516782、rs7528721、rs7752055、rs7927633、
rs8072587、rs8081883、rs8137373、rs931247、rs2056126、rs12142199、rs12419341、
rs1366220、rs1399272、rs1453858、rs1475840、rs1557553、rs16891982、rs2080161、
Rs2267666, rs2289266, rs3176921, rs3827760, rs4749305, rs728404, rs741272 and
rs830599。
2. detecting system according to claim 1, which is characterized in that the detecting system includes 42 first ancestor's SNP sites
Primer information.
3. system according to claim 1, which is characterized in that the amplification system of the PCR of the system includes Water（HPLC
grade）, PCR Buffer, 25mM MgCl2,25mM dNTP Mix, PCR primer Mix, PCR Enzyme, SAP reaction system
Including Water（HPLC grade）, SAP Buffer, SAP Enzyme, the iPLEX reaction systems of the system include Water
（HPLC grade）, iPLEX GOLD Buffer, iPLEX Termination mix, iPLEX extension primers mixture and
iPLEX Enzyme。
4. according to any one of the claim 1-3 detecting systems, which is characterized in that in the detecting system PCR amplification system,
A concentration of 25 μM of a concentration of 25mM of MgCl2, dNTP Mix, a concentration of 0.1mM of every primer, PCR primer Mix concentration
For 5U/ml, a concentration of 5U/ml of PCR Enzyme, in SAP reaction systems, a concentration of 1.7U/ μ l of SAP Enzyme.
5. according to the detection method of any one of the claim 1-4 systems, the detection architecture is suitable for various types of inspections
Material, can be not only used for ancestors' information of extrapolated sample people from source, and can disclose the genetic structure of different groups, infer group
Genetic background information；It is as follows：
（1）The preparation of sample：The absorbance of sample DNA requires OD260/OD280 in 1.7-1.9, DNA concentration>10ng/µl；
（2）The dilution of primer：By primer dry powder being ranked sequentially according to synthesis of synthesis, 12000rpm is centrifuged 1 minute；Addition 1
A concentration of 100 μM to PCR primer of × TE buffer, extension primer are 500 μM；Vortex oscillation, centrifugation, stands overnight molten to its
Preparation primer Mix, spare after solution；
（3）The preparation of primer Mix
1）PCR primer diluted concentration is 100 μM, prepares final concentration of 0.5 μM of PCR primers Mix；
2）The preparation of extension primer Mix；
3）The preparation of PCR system：1.8 μ l Water are taken respectively（HPLC grade）, 1 0.5 μM of μ l PCR primers Mix, 0.5 μ l
10 x PCR Buffer, 0.4 μ l 25mM MgCl2,0.1 μ l 25mM dNTP Mix, 0.2 μ l 5U/ μ l PCR Enzyme
PCR Mix are prepared to 1.5ml pipes, 1 μ l DNA are then added, be vortexed concussion and centrifugation；
4）PCR response procedures：95 DEG C of 2min of pre-degeneration, then 95 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 60sec totally 45
Cycle, 72 DEG C of 5min, last 4 DEG C of preservations；
（4）SAP mixes the preparation of liquid
1）1.53 μ l ultra-pure waters, 0.17 μ l SAP Buffer, 0.30 μ l SAP Enzyme (1.7U/ μ l) preparations are taken respectively
SAP mixes liquid（The system is single hole）；
2）Each hole is added the mixed liquid of 2 μ l SAP and plate is sealed with film after the completion, and be vortexed concussion and centrifugation；
3）The plate sealed is put into PCR instrument and carries out following procedure：37 DEG C of 40 min, 85 DEG C of 5 min, 4 DEG C of preservations；
（5）Extension
1）0.619 μ l sterile deionized waters, 0.2 μ l iPLEX GOLD Buffer, 0.2 μ l iPLEX are taken respectively
Termination mix, 0.94 μ l iPLEX extension primer mixtures, 0.041 μ l iPLEX Enzyme（The system is single
Hole）It prepares iPLEX and extends mixed liquid；
2）The above-mentioned iPLEX of 2 μ l are added to extend mixed liquid and mix, be vortexed concussion and centrifugation；
3）Thermal cycle is carried out according to following procedure：step 1：94℃ 30s；step 2: 95℃ 5s, 52℃ 5 s, 80℃
5s, 52 DEG C of 5s, 80 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s are repeated
40 cycles；step 3: 72℃ 3min；step 4:4 DEG C of long-term preservations；
（6）Desalting processing
1）Clean resin（Resin）It paves on 384/6mg dimple plate, air-dries minimum 10 minutes；
2）16 μ l water are added in each hole for having sample of sample plane, then plate is sealed with film, be vortexed concussion 15min,
Then 2000rpm centrifuges 5min；
3）6 mg cleaning resins are added（Resin）：Gently by sample plane reversion high up in the air, it is placed on the dimple for having put resin
On plate, then dimple plate are inverted together with sample plane（Two allegros are not horizontally moveable in the process）, resin is allowed to fall
To in hole；Plate is sealed with film, is placed on circulator reverse shake up 15 minutes；Plate is centrifuged 5 minutes with 3200g；
（7）Start MassARRAY Nanodispenser RS1000 point sample instruments, the extension products after purifying resin are moved 384
On hole SpectroCHIP (Sequenom) chip；
（8）SpectroCHIP chips after point sample are used into matrix solid-dispersion flying time mass spectrum analysis,
Testing result understands the molecular weight peaks of Mass Spectrometer Method using 4.0 softwares of TYPER automatically, and conversion is shown as corresponding to SNP site
Molecular weight mass spectrum peak figure.
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| CN110257489A (en) * | 2019-06-17 | 2019-09-20 | 南方医科大学 | A kind of detection technique system of 30 Multiple-allele SNP sites based on the sequencing of two generations |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020146735A1 (en) * | 2001-03-15 | 2002-10-10 | Bruker Daltonik Gmbh | Method and apparatus for mass spectrometric genotyping |
| CN107419017A (en) * | 2017-07-25 | 2017-12-01 | 公安部物证鉴定中心 | The method and system for unknown source individual infer in five continents border group source |
-
2018
- 2018-05-06 CN CN201810423363.4A patent/CN108531617A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020146735A1 (en) * | 2001-03-15 | 2002-10-10 | Bruker Daltonik Gmbh | Method and apparatus for mass spectrometric genotyping |
| CN107419017A (en) * | 2017-07-25 | 2017-12-01 | 公安部物证鉴定中心 | The method and system for unknown source individual infer in five continents border group source |
Non-Patent Citations (1)
| Title |
|---|
| 李亚男等: "43个SNP遗传标记复合检测体系的建立及其法医学应用", 《法医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257489A (en) * | 2019-06-17 | 2019-09-20 | 南方医科大学 | A kind of detection technique system of 30 Multiple-allele SNP sites based on the sequencing of two generations |
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