CN106701932B - A kind of 21 short tandem repeat composite amplification detection kits using double fluorescence labeling method - Google Patents
A kind of 21 short tandem repeat composite amplification detection kits using double fluorescence labeling method Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention discloses a kind of 21 short tandem repeat composite amplification detection kits using double fluorescence labeling method, comprising: the composite primer group of 21 pairs of specific primers, 21 locus of composite amplification, reaction mixture and hot start Taq polymerase；Labeled primer 5 ' holds first base to be marked with Conventional dye, the short excitation wavelength fluorochrome label of some the intermediate base of primer 5 ' and 3 ', to realize fluorescence energy transfer.Amplification of the invention more stable equilibrium, while having higher sensitivity, can be widely applied to the individual identification of Chinese population, DNA builds library and paternity test, be suitable for all kinds of samples.
Description
Technical field
The invention belongs to molecular genetics fields, and in particular to a kind of 21 short series connection weight using double fluorescence labeling method
Complex sequences composite amplification detection kit and its application.
Background technique
STR genetic marker is also known as microsatellite DNA, is widely present in protokaryon and eukaryotic gene group, is one kind by 2
~6 nucleotide are more than tens to 100 trinucleotide repeat sequences of repetitive unit composition.Different number of core sequence
It is arranged in tandem sequence repeats, and polymorphism is presented in length.According to both ends conservative sequence, design primer carries out PCR, then passes through
The polymorphism of the detection STR genetic marker such as polyacrylamide, agarose gel electrophoresis or Capillary Electrophoresis.
Individual identification DNA fluorescence detection reagent kit mainly using the multiplex PCR composite amplification that is most widely used at present and
Multicolor fluorescence marks capillary electrophoresis detection technology, the DNA Genotyping for biology sample.According to both at home and abroad it is existing and
We oneself research achievement in terms of str locus seat, and according to these locus in crowd's allelic frequency distribution
Feature, we select the locus of polymorphism and heterozygosity with higher in crowd, design and draw near these locus
Object simultaneously marks fluorescein on primer respectively, by multiplexed PCR amplification system, carries out specific expansion to polymorphic STR loci
Increase, then with known length and indicate the molecular weight standards of fluorescein and mix, electrophoresis is simultaneously on genetic analyzer or sequenator
Electrophoresis information is collected, length and genotyping are then carried out respectively to the PCR product of str locus seat, data needed for obtaining reach
To the purpose of individual identification.
Domestic reagent box generally uses FAM, HEX, TAMRA, ROX, ATTO633 to carry out primer and internal standard currently on the market
Label, sequenator is using single laser, wavelength 488nm, and FAM, HEX, TAMRA, ROX and ATTO633 this 5 kinds of fluorescence
Dye excitation wavelength is but distributed in 500nm-650nm.With becoming larger for dye excitation wavelength, equal amount fluorescent mark product, quilt
The fluorescence signal that sequenator laser excitation generates gradually decreases, TAMRA and ROX fluorescence efficiency is well below FAM, HEX.Especially
, ROX dye marker ratio FAM label, same amount of product, signal strength only has a quarter to 1/8th, big in this way
Fluorescence efficiency difference under, the preferable harmonious and higher sensitivity obtained between each fluorescence channel becomes highly difficult.
The present invention uses a kind of double fluorescence labeling method, and fluorescence energy transfer (FRET, fluorescence energy may be implemented
transfer)。
Fluorescence resonance energy transfer is a kind of energy transfer phenomenon generated between two close fluorescent molecules.When one
The fluorescence spectrum of a fluorescent molecule (also known as donor) and the excitation spectrum of another fluorescent molecule (also known as receptor) overlap
When, the excitation of donor fluorescent molecule can induce acceptor molecule and issue fluorescence, while the fluorescence intensity of donor fluorescent molecule itself declines
Subtract.FRET degree and the space length of confession, acceptor molecule are closely related, and FRET can occur for when generally 7~10nm；With away from
From extension, FRET is in be obviously reduced.The efficiency of FRET between donor and receptor, can be by E=1/1+ (R/R0)6Reflect, wherein R
It indicates that the distance between donor and receptor, R0 indicate Fu Shi radius, relies on the overlapping degree of Emission spectrum of donor and Excitation spectra of acceptor,
And the relative bearing of donor and the dipole of receptor energy transfer.
The euchromosome STR kit of same type is shown in Table 1 currently on the market, the present invention and Promega company
The selection of PowerPlex21 locus is completely the same, and 21 locus contain 13 CODIS and newly-increased 4 CODIS
Point, 3 Penta D, the Penta E and D6S1043 and gender in Chinese population with very high polymorphism identify position
Point Amelogenin.Not only all CODIS locus had been contained, but also have selected the locus of 3 high polymorphisms, had been improved whole
Resolution ratio.This 21 locus are also all 21 locus for capableing of typing autosome database at present, so, selection
This 21 locus are the highest schemes of current cost performance.Table 1 and the commercialization euchromosome STR fluorescence detection examination of mainstream on the market
The comparison of agent box locus.Note: runic is 13 CODIS locus, and underscore is 4 newly-increased CODIS locus.
Summary of the invention
In view of the above technical problems, the present invention provides a kind of 21 Short tandem repeatSTRs of the labeling method preparation of fluorescent dye
Sequence composite amplification detection kit, and it is applied to the composite amplification of 21 str locus seats of mankind's autosome, have amplification fast
The characteristics of speed, harmonious good, high sensitivity, meet Chinese population individual identification, paternity test, euchromatic dna builds library demand.
Technical scheme is as follows:
A kind of 21 short tandem repeat composite amplification detection kits using double fluorescence labeling method, comprising:
The composite primer group of 21 pairs of specific primers, 21 locus of composite amplification, reaction mixture and thermal starting Taq
Enzyme；
21 locus of the composite amplification include Amelogenin, D3S1358, vWA, D7S820, CSF1PO,
D8S1179, D21S11, D16S539, TH01, D13S317, TPOX, D18S51, D5S818, FGA, D2S1338, D19S433,
D1S1656, D12S391, Penta D, Penta E and D6S1043；
The composition of the reaction mixture are as follows: MgCl225 DEG C, KCl 125mM of 5mM, Tris-HCl 125mM pH8.3,
DNTPs 0.5mM, BSA 1.5g/L；The dNTPs is the equimolar mixture of dATP, dTTP, dCTP, dGTP；
The sequence of the primer pair of 21 locus of the composite primer group of 21 pairs of specific primers includes SEQ ID
NO.01-SEQ ID NO.42；
Each str locus seat of the composite primer group of 21 pairs of specific primers uses and is located at the locus core weight
The pair of primers of the two sides Fu Qu expands, wherein an at least primer is carried out using double fluorochrome label methods in each pair of primer
Label；
The primer pair of 21 locus of the composite primer group of 21 pairs of specific primers is divided into four groups using double fluorescence
Dye labeling methodologies are marked；Described four groups include first group: D3S1358, vWA, D7S820, CSF1PO, Penta E；SEQ
ID NO.01-SEQ ID NO.10；Second group: D8S1179, D21S11, D16S539, D2S1338, Penta D；SEQ ID
NO.11-SEQ ID NO.20；Third group: D19S433, TH01, D13S317, TPOX, D18S51, D6S1043；SEQ ID
NO.21-SEQ ID NO.32；4th group: Amelogenin, D1S1656, D5S818, D12S391, FGA；SEQ ID NO.33-
SEQ ID NO.42。
Preferably, the double fluorescence labeling method is included in the end of primer 5 ' first base FAM, HEX, TAMRA, ROX
One of be marked；Wherein, the primer marked with FAM, TAMRA and ROX, the intermediate base FAM of primer 5 ' and 3 '
It is marked；The primer marked with HEX, the intermediate base of primer 5 ' and 3 ' are marked with HEX.
21 short tandem repeat composite amplification detection kits of the present invention using double fluorescence labeling method
Application method, comprising the following steps: reagent in kit is mixed with sample DNA, is packed into PCR amplification pipe, is placed in thermal cycle
On instrument, PCR amplification, amplification program are as follows: 95 DEG C of 3min are carried out；94 DEG C of 5s, 60 DEG C of 1min, 26-28 circulation；60 DEG C extend eventually
10min；4-16 DEG C of holding；Amplified production is obtained, amplified production is subjected to fluorescence detection on genetic analyzer, then divided with segment
Analyse softwareThe data detected on analysis gene sequencer；The sample DNA including the use of
The human gene group DNA that any one method is extracted in Chelex100 method, magnetic bead extraction method or Organic extraction method, extracts sample
Source includes blood, blood stain, sperm, saliva, body fluid, hair, muscle or the histoorgan of people, or utilizes the hands-free filter taken
Paper, FTA card, cotton swab, the human blood or Stomatocyte that any one carrier is collected in gauze.
21 short tandem repeat composite amplification detection reagents of the present invention using fluorochrome label method
Box can be applied to the individual identification of Chinese population, paternity test, DNA build library etc..
The beneficial effects of the present invention are:
1, using fluorescent labelling techniques, fluorescence efficiency is high, thus amplification rate faster, it is harmonious more preferable, sensitivity is higher；
2, this kit is the height composite amplification system comprising 21 euchromosome STR locus, 21 locus with
21 locus that China DNA builds library typing are completely the same, and system altitude is compatible；
3, this kit has very strong sample adaptability, i.e., a kind of kit can expand the sample of a variety of samples, no
Sample with sample includes: the people's base extracted using any one in Chelex100 method, magnetic bead extraction method or organic method extraction process
The human blood or Stomatocyte collected by any one carrier such as group DNA and filter paper, FTA card, cotton swab, gauze；
4, this kit has stronger specific amplification and temperature tolerance (annealing temperature by design specific primer
56-62 DEG C of tolerance range of degree ensure that and obtain preferably expanding knot in the PCR amplification instrument Shi Junneng using different brands or quality
Fruit).To dog, pig, ox, sheep, cat, chicken, mouse, rabbit, fish and e. coli dna, 10 kinds of different genera species are detected, and are not expanded
Increase peak, shows that the present invention has good species specificity；
5, the present invention greatly improves TAMRA, ROX by marking the TAMRA and ROX with FRET on primer
Fluorescence efficiency reduces and the gap of FAM, HEX；When capillary electrophoresis detection, compares and use conventional fluorescent dye labeling methodologies
STR composite amplification reagent kit, the present invention has more balanced, faster system, while having higher sensitivity.
Detailed description of the invention
Fig. 1 is that the primer A-H of the intermediate fluorescence difference mark position of embodiment 1 expands the result figure of D13S317；
Fig. 2 is kit allelic ladder figure in embodiment 2；
Fig. 3 is the genotyping result that embodiment 2 expands positive criteria product 9947A；
In Fig. 4 experiment 3, kit Z2015110011F AFLP system of the present invention.
In Fig. 5 experiment 3,21 Direct ID sample Z2015110011F AFLP systems.
In Fig. 6 experiment 3, kit Z2015110071M AFLP system of the present invention.
In Fig. 7 experiment 3,21 Direct ID sample Z2015110071M AFLP systems.
Fig. 8 experiment 4 in, double fluorescence labeling method of the present invention to DNA standard items 9947A 500pg, 250pg, 125pg,
The amplification of five concentration of 62.5pg and 31.25pg.
Fig. 9: in experiment 4, using conventional single fluorescence labeling method to DNA standard items 9947A 500pg, 250pg, 125pg,
The amplification of five concentration of 62.5pg and 31.25pg
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
As shown, the present invention discloses a kind of 21 short tandem repeat composite amplifications using double fluorescence labeling method
Detection kit, comprising:
The composite primer group of 21 pairs of specific primers, 21 locus of composite amplification, reaction mixture and thermal starting Taq
Enzyme；
21 locus of the composite amplification include Amelogenin, D3S1358, vWA, D7S820, CSF1PO,
D8S1179, D21S11, D16S539, TH01, D13S317, TPOX, D18S51, D5S818, FGA, D2S1338, D19S433,
D1S1656, D12S391, Penta D, Penta E and D6S1043；
The composition of the reaction mixture are as follows: MgCl225 DEG C, KCl 125mM of 5mM, Tris-HCl 125mM pH8.3,
DNTPs 0.5mM, BSA 1.5g/L；The dNTPs is the equimolar mixture of dATP, dTTP, dCTP, dGTP；
The sequence of the primer pair of 21 locus of the composite primer group of 21 pairs of specific primers includes SEQ ID
NO.01-SEQ ID NO.42；
Each str locus seat of the composite primer group of 21 pairs of specific primers uses and is located at the locus core weight
The pair of primers of the two sides Fu Qu expands, wherein an at least primer is carried out using double fluorochrome label methods in each pair of primer
Label；
The primer pair of 21 locus of the composite primer group of 21 pairs of specific primers is divided into four groups using double fluorescence
Dye labeling methodologies are marked；Described four groups include first group: D3S1358, vWA, D7S820, CSF1PO, Penta E；SEQ
ID NO.01-SEQ ID NO.10；Second group: D8S1179, D21S11, D16S539, D2S1338, Penta D；SEQ ID
NO.11-SEQ ID NO.20；Third group: D19S433, TH01, D13S317, TPOX, D18S51, D6S1043；SEQ ID
NO.21-SEQ ID NO.32；4th group: Amelogenin, D1S1656, D5S818, D12S391, FGA；SEQ ID NO.33-
SEQ ID NO.42。
Preferably, the double fluorescence labeling method is included in the end of primer 5 ' first base FAM, HEX, TAMRA, ROX
One of be marked；Wherein, the primer marked with FAM, TAMRA and ROX, the intermediate base FAM of primer 5 ' and 3 '
It is marked；The primer marked with HEX, the intermediate base of primer 5 ' and 3 ' are marked with HEX.
21 short tandem repeat composite amplification detection kits of the present invention using double fluorescence labeling method
Application method, comprising the following steps: reagent in kit is mixed with sample DNA, is packed into PCR amplification pipe, is placed in thermal cycle
On instrument, PCR amplification, amplification program are as follows: 95 DEG C of 3min are carried out；94 DEG C of 5s, 60 DEG C of 1min, 26-28 circulation；60 DEG C extend eventually
10min；4-16 DEG C of holding；Amplified production is obtained, amplified production is subjected to fluorescence detection on genetic analyzer, then divided with segment
Analyse softwareThe data detected on analysis gene sequencer；The sample DNA including the use of
The human gene group DNA that any one method is extracted in Chelex100 method, magnetic bead extraction method or Organic extraction method, extracts sample
Source includes blood, blood stain, sperm, saliva, body fluid, hair, muscle or the histoorgan of people, or utilizes the hands-free filter taken
Paper, FTA card, cotton swab, the human blood or Stomatocyte that any one carrier is collected in gauze.
21 short tandem repeat composite amplification detection reagents of the present invention using fluorochrome label method
Box can be applied to the individual identification of Chinese population, paternity test, DNA build library etc..
The determination of embodiment 1, design of primers and the fluorescent marker position on primer sequence
21 str locus seats of system coamplification, be Amelogenin, D3S1358, vWA, D7S820, CSF1PO,
D8S1179, D21S11, D16S539, TH01, D13S317, TPOX, D18S51, D5S818, FGA, D2S1338, D19S433,
D1S1656, D12S391, Penta D, Penta E and D6S1043.
Specific primer is separately designed in the flank of its repetitive sequence to above-mentioned 21 locus.Design of primers uses
Oligo7 software, every primer Tm is close to 60 DEG C.The maximum magnitude of amplified production length be 75-500bp, to each pair of primer into
Row amplification assay simultaneously optimizes, and up to obtaining, peak shape is sharp, the higher amplified peak of peak height.Tested using composite amplification, 56 DEG C-
Between 63 DEG C, non-specific amplification is not generated, has purpose peak, other interactions or cross reaction do not occur.To dog, pig, ox,
Sheep, cat, chicken, mouse, rabbit, fish and e. coli dna are without amplification.Primer sequence is the same as table 2.
What fluorescence labeling method of the present invention utilized is FRET principle, so the degree of FRET and fluorescence are on primer
Position it is related.We have carried out research and actual test to it.To the same primer of D13S317, in different location mark
Remember fluorescence, fluorescent marker may will affect primer combination template at too close 3 ' end, so total length is the primer of 24bp, we
Have chosen 5 ' the intermediate fluorescence of 2-14, end label.5 ' first bases " C " are marked with TAMRA, are indicated with runic.Mark 6-FAM
Base indicated with underscore+bold Italic, be as follows:
A is the primer of conventional single fluorescent marker, and B-I is primer of the intermediate fluorescent marker in different location, expands 0.2-
The DNA of 0.5ng extracts sample.Identical PCR and deposition condition is arranged in A-I, and only fluorescent primer mark mode is different, experiment weight
It is 3 times multiple.In order to avoid fluorescence signal is more than upper limit of detection value, electrophoresis is carried out again after all amplified productions are diluted 10 times.As a result
Show: the average peak height highest of D primer amplification marks the fluorescence efficiency highest at 5 ' the 6th, and the A compared to single fluorescent marker draws
Object can be improved to 9.2 times.As a result such as following table and Fig. 1:
| Fluorescent marker draws | Average detected peak |
| A | 211.2 |
| B | 1712.6 |
| C | 1794.3 |
| D | 1941.1 |
| E | 1601.7 |
| F | 1497.1 |
| G | 1414.7 |
| H | 1244.9 |
| I | 1118.3 |
As can be seen that increasing short wavelength's fluorescent marker between 5 ' -3 ' is remarkably improved the glimmering of long wave dye TAMRA
Light efficiency, the test of ROX is also in this way, details are not described herein again；The position of short wavelength's fluorescent marker influences fluorescence efficiency, for
TAMRA, the fluorescence efficiency highest by 6-FAM label at 5 ' the 6th, in order to keep entire primer system and background fluorescence uniform,
We mark the intermediate fluorescence of fluorescent primers all in the present invention on 5 ' the 6th bit bases, are specifically shown in Table 2.
The primer information of the present invention of table 2.
Note: the base of 5 ' fluorescent markers of overstriking font, underscore are the base of intermediate fluorescent marker, corresponding fluorescence
Element is corresponding identical overstriking or underscore label.
The optimization and determination of 2 amplification system of embodiment
And, series of tests has been done to primer concentration and amplification component to keep amplification balanced, finally determined primer dosage and
Expand the component of buffer solution.
Kit includes following composition:
2.5 × buffer components and its concentration are as follows: MgCl25mM, Tris-HCl 125mM (pH8.3,25 DEG C), KCl
125mM, dNTPs 0.5mM, BSA 1.5g/L.DNTPs is four kinds of deoxyribonucleotides (dATP, dTTP, dCTP, dGTP)
Equimolar mixture.Marker Orange-500 is prepared by 201610988458.1 method of patent.The present invention also provides be used for
The allelic ladder of analysis, each locus primer expand oppositional allele respectively, and amplified production is composed.
Fig. 2 is Allellic ladder electrophorogram.DNA standard items DNA 9947A is purchased from U.S. promega company.The purchase of thermal starting enzyme
From the precious biology in Dalian.
This kit can be expanded well in 56-62 DEG C of annealing region, and amplification system is in various reaction thermal cycles
On instrument, such as ABI9700, ABI2720, Bio-Rad T100, rich day Life Pro, it is available preferably using program below
Result: 95 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 5 seconds, and 60 DEG C of annealing extend 1 minute, which runs 26-28 circulation；
60 DEG C keep the temperature 10 minutes；4-16 DEG C of heat preservation.
Using 9947A standard items DNA as template, according to the form below sample-adding:
With 95 DEG C of program 3 minutes on ABI9700；94 DEG C 5 seconds, 60 DEG C 1 minute operation 26 circulation；60 degree 10 minutes
Amplification.
+ 10 μ L deionized formamide of+0.5 μ L molecular weight internal standard of 1 μ L amplified production sample-adding, with 1kv on ABI3130XL,
1s sample introduction, 15KV voltage carry out electrophoresis 40min.As a result it is analyzed with GeneMapper software.Fig. 3 is 9947A augmentation detection
As a result map, it is consistent with theoretical parting.
Embodiment 3, the consistency more of the invention with other commercial kit partings
In order to detect the consistency of the present invention with other commercial kit partings, the present embodiment is had chosen
MicroreaderTM21 Direct ID System are as reference object, respectively using the present invention and 21 Direct ID reagents
Box expands 1316 samples (wherein independent individuals 663), and product is by 3100 genetic analyzer capillary electricity of ABI
Swimming carries out parting, and analyses and compares to genotyping result.21Direct ID kit is operated according to its specification, the present invention
Using steps are as follows:
3.1. polymerase chain reaction (PCR) expands
1) buffer, primer mixture, Taq enzyme are taken, is made into mixed liquor according to following table, packing is anti-to PCR after oscillation mixes
Ying Guanzhong, every 25 μ L of pipe.
The present invention:
21 Direct ID:
| Component | Dosage (μ L) |
| MicroreaderTM1.25×Buffer B | 20 |
| MicroreaderTM21-D 5×Primer Mix | 5 |
| MicroreaderTMTaq DNA Polymerase II | 0.5 |
| Template DNA (from blood filter paper or FTA card) | 1 1.2mm diameter |
| React final volume | 25.5uL |
2) according to the present invention and 21 Direct ID specifications, setting thermal cycler (9700 DNA cloning instrument of ABI) is joined
Then PCR reaction tube is put into beginning amplification gene segment in instrument by number.
The present invention: 95 DEG C 3 minutes；94 DEG C 5 seconds, 60 DEG C 1 minute operation 26 circulation；60 degree 10 minutes, 4-16 DEG C is held
Continuation of insurance temperature, until taking out sample.
21 Direct ID:95 DEG C 2 minutes；94 DEG C 5 seconds, 60 DEG C 70 seconds operation 28 circulation；60 degree 30 minutes, 4-16
DEG C lasting heat preservation, until taking out sample.
3.2. after amplified reaction, reaction tube is taken out, carries out electrophoresis and detection with ABI3100 genetic analyzer.
1) it takes (+10 μ L deionized formamide of 0.5 μ L molecular weight internal standard) × (sample number) to be made into after mixed liquor mixes to dispense,
Every 10 μ L of pipe.
2) 1 μ L amplified production or allelic ladder (Allelic Ladder) are separately added into again, brief centrifugation is by liquid
Be collected into 95 DEG C of centrifuge tube tube bottom sample to be denaturalized 4 minutes, then rapid cooled on ice 4 minutes, make DNA be denaturalized completely and
Keep denatured state that sample is put into the sample tray of Genetic Analyser.
3) be arranged instrument parameter (sample introduction voltage 1kV of the present invention, sample injection time 1 second；21 Direct ID sample introduction voltages
3kv, 10s), start electrophoresis detection about after forty minutes, electrophoresis terminates, and obtains figure with GeneMapper software analysis experimental data
Spectrum and genotyping result.
The comparison of 3.3 genotyping results
The present invention and 21 Direct ID include 20 euchromosome STR sites and an Amelogenin gender position
Point.20 often contaminate in STR bit point, and the two has 19 sites identical, D3S1358, vWA, D7S820, CSF1PO, D8S1179,
D21S11, D16S539, TH01, D13S317, TPOX, D18S51, D5S818, FGA, D2S1338, D19S433, D12S391,
Penta D, Penta E, D6S1043.The parting in above-mentioned 19 sites and Amelogenin is compared.
By the way that genotyping result is compared, 52640 equipotential bases of 1316 samples (wherein independent individuals 663)
Cause, the present invention and the genotyping result of 21 two kits of Direct ID are completely the same.Two examinations are used respectively to wherein 2 samples
The amplification of agent box is illustrated, and amplification figure is shown in Fig. 4-Fig. 7.
Embodiment 4, the present invention with conventional labels method amplification system remolding sensitivity compared with
The sensitivity of DNA amplification template of the present invention is tested, is compared with the primer of conventional single fluorescent marker.Point
Two groups of primer ponds of double fluorescence labeling and single fluorescent marker of the invention are not prepared, primer concentration is assembled by respectively most having ready conditions,
Specifically it see the table below.
By embodiment 2 configure amplification system, be added doubling dilution DNA standard items 9947A (500pg, 250pg, 125pg,
62.5pg and 31.25pg), by two sets of primers, respectively optimum program carries out amplification program, specific as follows:
Double fluorescence labeling primer sets: 95 DEG C 3 minutes；94 DEG C 5 seconds, 60 DEG C 1 minute operation 26 circulation；60 degree 10 points
Clock, 4-16 DEG C of lasting heat preservation, until taking out sample.
Single fluorescent dye primer group: 95 DEG C 3 minutes；94 DEG C 10 seconds, 60 DEG C 1 minute, 72 degree 1 minute, operation 28 follow
Ring；60 degree 30 minutes, 4-16 DEG C of lasting heat preservation, until taking out sample.
After amplified reaction, reaction tube is taken out, carries out electrophoresis and detection with ABI3100 genetic analyzer.
1) it takes (+10 μ L deionized formamide of 0.5 μ L molecular weight internal standard) × (sample number) to be made into after mixed liquor mixes to dispense,
Every 10 μ L of pipe.
2) 1 μ L amplified production or allelic ladder (Allelic Ladder) are separately added into again, brief centrifugation is by liquid
Be collected into 95 DEG C of centrifuge tube tube bottom sample to be denaturalized 4 minutes, then rapid cooled on ice 4 minutes, make DNA be denaturalized completely and
Keep denatured state that sample is put into the sample tray of Genetic Analyser.
3) be arranged instrument parameter (double fluorescence labeling sample introduction voltage 1kV, sample injection time 1 second；Single fluorescent marker sample introduction voltage
3kv, 10s), start electrophoresis detection about after forty minutes, electrophoresis terminates, and obtains figure with GeneMapper software analysis experimental data
Spectrum and genotyping result.
Harmonious and whole peak height is not so good as the primer of double fluorescence labeling between the color of the primer amplification of conventional list fluorescent marker.It adopts
With the primer of double fluorescence labeling, due to the raising of signal, the relatively conventional single fluorescent dye primer dosage of primer dosage will be lacked.Especially
The big fluorescent dye primer of both fluorescence emission wavelengths of TAMRA, ROX, conventional list labeled primer dosage are that double labelling primer is used
The several times of amount, however amplified signal intensity and harmony are still not as good as double labelling primer.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Sequence table
<110>Jiangsu Su Bo biomedicine limited liability company
<120>a kind of 21 short tandem repeat composite amplification detection reagents using novel double fluorescence labeling method
Box
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<213>artificial sequence (Artificial Sequence)
<400> 6
gggtatgata gaacacttgt catagtttag aa 32
<210> 7
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ccggaggtaa aggtgtctta aagtg 25
<210> 8
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atttcctgtg tcagaccctg ttctaa 26
<210> 9
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ttgtgtaaga agtgttaaag gggatc 26
<210> 10
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ctgaaacagg agaatcactt gaacc 25
<210> 11
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
cacggcctgg caacttatat gtattt 26
<210> 12
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
tcactgtatc gtatcccatt gcg 23
<210> 13
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ccataaatat gtgagtcaat tccccaagt 29
<210> 14
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
taggaggtag atagactgga tagatagacg 30
<210> 15
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
tgcagaaaag cactgaaaga agaatc 26
<210> 16
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
tggtaaaaca gcctacagag tgattc 26
<210> 17
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
cctgtctcac cccttttcct 20
<210> 18
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
taaagacttc atggtcctga ctacag 26
<210> 19
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
ctgaagtgag ccatgatcac acc 23
<210> 20
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
tgggttgtct tattgatgtg taagaatt 28
<210> 21
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
aatcttctct ctttcttcct ctctcc 26
<210> 22
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
gcactccagc ctgggcaac 19
<210> 23
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
attcaaaggg tatctgggct ctgg 24
<210> 24
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
gattatccag cctggcccac ac 22
<210> 25
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
caatgtgaat attgggatgg gttg 24
<210> 26
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
gcagcccaaa aagacagaca 20
<210> 27
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ctcccagctc tcacaacccc 20
<210> 28
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
ccttctgtcc ttgtcagcgt ttatttg 27
<210> 29
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
acttctctgg tgtgtggaga tg 22
<210> 30
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
agcctgggta acacagtgag 20
<210> 31
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
agagctcaga ttctctgccc 20
<210> 32
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
tcctcccact tctaaaacac ct 22
<210> 33
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
ctgggctctg taaagaatag tg 22
<210> 34
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
catcagagct taaactggga 20
<210> 35
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
tacaattaaa cacacacaca cctatct 27
<210> 36
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
tggtagagat ggaagaaaat cccc 24
<210> 37
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
ttaatagcaa gtatgtgaca agggtga 27
<210> 38
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
tcagaggaat gctttagtgc tttttag 27
<210> 39
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
agggaagatg aaaaaagaga ctgtatt 27
<210> 40
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
gactgagcca tgctcctagt 20
<210> 41
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
tttccaaaag tcaaatgccc cataggtt 28
<210> 42
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
tggatatgct gtactttttc tatgactttg cg 32
Claims (3)
1. a kind of 21 short tandem repeat composite amplification detection kits using double fluorescence labeling method, feature exist
In, comprising:
Composite primer group, reaction mixture and the heat that 21 pairs of specific primers for 21 locus of composite amplification are constituted open
Dynamic Taq enzyme；
By 21 locus of composite amplification be Amelogenin, D3S1358, vWA, D7S820, CSF1PO, D8S1179,
D21S11, D16S539, TH01, D13S317, TPOX, D18S51, D5S818, FGA, D2S1338, D19S433, D1S1656,
D12S391, Penta D, Penta E and D6S1043；
The composition of the reaction mixture are as follows: MgCl225 DEG C of 5mM, Tris-HCl 125mM pH8.3, KCl 125mM, dNTPs
0.5mM, BSA 1.5g/L；The dNTPs is the equimolar mixture of dATP, dTTP, dCTP, dGTP；
The sequence for expanding 21 pairs of specific primers of 21 locus is SEQ ID NO.01-SEQ ID NO.42；
In 21 locus by composite amplification, each locus uses and is located at the locus core duplicate block two sides
Pair of primers amplification, wherein there is a primer to be marked in each pair of primer using double fluorochrome label methods；It is described to be used for
21 pairs of specific primers for expanding 21 locus are divided into four groups and are marked using double fluorochrome label methods；Described four groups
Including first group of primer sets: for the primer pair SEQ ID NO.01-SEQ ID NO.02 of amplifying locus D3S1358, being used for
The primer pair SEQ ID NO.03-SEQ ID NO.04 of amplifying locus vWA, the primer pair for amplifying locus D7S820
SEQ ID NO.05-SEQ ID NO.06, the primer pair SEQ ID NO.07-SEQ ID for amplifying locus CSF1PO
NO.08, the primer pair SEQ ID NO.09-SEQ ID NO.10 for amplifying locus PentaE；Second group of primer sets: it is used for
The primer pair SEQ ID NO.11-SEQ ID NO.12 of amplifying locus D8S1179, the primer for amplifying locus D21S11
Primer pair SEQ ID NO.15-SEQ ID to SEQ ID NO.13-SEQ ID NO.14, for amplifying locus D16S539
NO.16 is used for amplifying locus for the primer pair SEQ ID NO.17-SEQ ID NO.18 of amplifying locus D2S1338
The primer pair SEQ ID NO.19-SEQ ID NO.20 of Penta D；Third group primer sets: for amplifying locus D19S433's
Primer pair SEQ ID NO.21-SEQ ID NO.22, the primer pair SEQ ID NO.23-SEQ ID for amplifying locus TH01
NO.24 is used for amplifying locus for the primer pair SEQ ID NO.25-SEQ ID NO.26 of amplifying locus D13S317
Primer pair the SEQ ID NO.27-SEQ ID NO.28, the primer pair SEQ ID for amplifying locus D18S51 of TPOX
NO.29-SEQ ID NO.30, the primer pair SEQ ID NO.31-SEQ ID NO.32 for amplifying locus D6S1043；The
Four groups of primer sets: for the primer pair SEQ ID NO.33-SEQ ID NO.34 of amplifying locus Amelogenin, for expanding
The primer pair SEQ ID NO.35-SEQ ID NO.36 of locus D1S1656, the primer pair for amplifying locus D5S818
SEQ ID NO.37-SEQ ID NO.38, the primer pair SEQ ID NO.39-SEQ ID for amplifying locus D12S391
NO.40, the primer pair SEQ ID NO.41-SEQ ID NO.42 for amplifying locus FGA；Using double in first group of primer sets
5 ' first base in end of the primer that fluorochrome label method is marked are marked with FAM, and the base at 5 ' the 6th, ends is used
FAM is marked；5 ' first alkali in end of the primer being marked in second group of primer sets using double fluorochrome label methods
Base is marked with HEX, and the base at 5 ' the 6th, ends is marked with HEX；Using double fluorochrome labels in third group primer sets
5 ' first base in end of the primer that method is marked are marked with TAMRA, and the base at 5 ' the 6th, ends is marked with FAM
Note；5 ' the first base ROX in end of the primer being marked in the 4th group of primer sets using double fluorochrome label methods into
The base of line flag, 5 ' the 6th, ends is marked with FAM.
2. 21 short tandem repeat composite amplifications according to claim 1 using double fluorescence labeling method detect examination
The application method of agent box, which comprises the following steps: reagent in kit is mixed with sample DNA, is packed into PCR expansion
Increase pipe, be placed on thermal cycler, carries out PCR amplification, amplification program are as follows: 95 DEG C of 3min；94 DEG C of 5s, 60 DEG C of 1min, 26-28 are followed
Ring；60 DEG C extend 10min eventually；4-16 DEG C of holding；Amplified production is obtained, amplified production is subjected to fluorescence inspection on genetic analyzer
It surveys, then uses fragment analysis softwareThe data detected on analysis gene sequencer；The sample DNA packet
It includes using the human gene group DNA that any one method is extracted in Chelex100 method, magnetic bead extraction method or Organic extraction method, or
The human blood or Stomatocyte collected using any one carrier in the hands-free filter paper taken, FTA card, cotton swab, gauze.
3. 21 short tandem repeat composite amplifications according to claim 1 using double fluorescence labeling method detect examination
Agent box builds the application in library in the individual identification of Chinese population, paternity test, DNA.
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| CN116083595B (en) * | 2022-09-30 | 2023-11-14 | 江苏苏博生物医学科技南京有限公司 | Kit and method for detecting 33 short tandem repeat complex amplifications containing sigma gene locus |
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