CN106701932A - Multiple amplification detection kit for 21 short tandem repeat sequences using novel bifluorescence marking method - Google Patents
Multiple amplification detection kit for 21 short tandem repeat sequences using novel bifluorescence marking method Download PDFInfo
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Abstract
The invention discloses a multiple amplification detection kit for 21 short tandem repeat sequences using a novel bifluorescence marking method. The kit comprises a compound primer group comprising 21 pairs of specific primers, 21 gene bases for compound amplification, a reaction mixed liquid and a warm start Taq enzyme, wherein the first basic group at 5' end of a labeled primer is marked by a conventional dye, and some basic groups at 5' and 3' ends of the primer are marked by a short excitation wavelength fluorescent dye, so that fluorescent energy transfer is achieved. The amplification result disclosed by the invention is more stable and balanced, and meanwhile, the kit is higher in sensitivity, can be widely applied to individual recognition, DNA database building and paternity test of Chinese population, and is suitable for various detection materials.
Description
Technical field
The invention belongs to molecular genetics field, and in particular to a kind of 21 short strings using new double fluorescence labeling method
Connection repetitive sequence composite amplification detection kit and its application.
Background technology
Also known as microsatellite DNA, it is widely present in protokaryon and eukaryotic gene group STR genetic markers, is a class by 2
~6 nucleotides are more than tens to 100 individual trinucleotide repeat sequences of repeat unit composition.Different number of core sequence
Arranged in tandem sequence repeats, and length is presented polymorphism.According to two ends conservative sequence, primer is designed, enter performing PCR, then passed through
Polyacrylamide, agarose gel electrophoresis or Capillary Electrophoresis etc. detect the polymorphism of STR genetic markers.
Individual identification DNA fluorescence detection reagent kits mainly using the multiplex PCR composite amplification that is most widely used at present and
Multicolor fluorescence marks capillary electrophoresis detection technology, for the DNA Genotypings of biology sample.According to both at home and abroad it is existing and
Our oneself achievement in research in terms of str locus seat, and according to these locus in crowd's allelic frequency distribution
Feature, we select the locus with polymorphism and heterozygosity higher in crowd, are designed near these locus and drawn
Thing simultaneously marks fluorescein on primer respectively, by multiplexed PCR amplification system, specific expansion is carried out 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, then the PCR primer to str locus seat carries out length and genotyping respectively, data needed for obtaining reach
To the purpose of individual identification.
Domestic reagent box is typically carried out using FAM, HEX, TAMRA, ROX, ATTO633 to primer and internal standard in the market
Mark, sequenator uses single laser, and wavelength is 488nm, and FAM, HEX, TAMRA, ROX and ATTO633 this 5 kinds of fluorescence
Dye excitation wavelength is but distributed in 500nm-650nm.As the change of dye excitation wavelength is big, equal amount fluorescent mark product, quilt
The fluorescence signal that sequenator laser excitation is produced is gradually decreased, TAMRA and ROX fluorescence efficiencies are well below FAM, HEX.Especially
, ROX dye markers are marked than FAM, and same amount of product, signal intensity only has a quarter to 1/8th, so big
Fluorescence efficiency difference under, obtain that the preferable harmonious and higher sensitivity between each fluorescence channel becomes is highly difficult.
The present invention uses a kind of new double fluorescence labeling method, it is possible to achieve fluorescence energy transfer (FRET, fluorescence
energy transfer)。
Fluorescence Resonance Energy transfer is a kind of energy transfer phenomenon produced apart between two close fluorescence molecules.When one
The fluorescence spectrum of individual fluorescence molecule (being also called donor) overlaps with the excitation spectrum of another fluorescence molecule (being also called acceptor)
When, the excitation energy of donor fluorescent molecule induces acceptor molecule and sends fluorescence, while the donor fluorescent molecule fluorescence intensity of itself declines
Subtract.FRET degree is closely related with the space length of confession, acceptor molecule, and FRET can occur during generally 7~10nm；With away from
From extension, FRET is in be obviously reduced.The efficiency of FRET between donor and acceptor, can be by E=1/1+ (R/R0)6Reflection, wherein R
The distance between donor and acceptor are represented, R0 represents Fu Shi radiuses, relies on the overlapping degree of Emission spectrum of donor and Excitation spectra of acceptor,
And the relative bearing of the dipole of donor and acceptor energy transfer.
The euchromosome STR kit of same type is shown in Table 1 in the market, of the invention and Promega companies
The selection of PowerPlex21 locus is completely the same, and 21 locus contain 13 CODIS and newly-increased 4 CODIS
Point, 3 Penta D, Penta E and D6S1043 with polymorphism very high in Chinese population, and a sex identification position
Point Amelogenin.Both all CODIS locus had been contained, 3 locus of polymorphism high had been have selected again, improve overall
Resolution ratio.This 21 locus are also all of 21 locus for being capable of typing autosome database at present, so, selection
This 21 locus are current cost performance highest schemes.Table 1 and the commercialization euchromosome STR fluoroscopic examination examination of main flow on the market
Agent box locus is contrasted.Note：Runic is 13 CODIS locus, and underscore is 4 newly-increased CODIS locus.
The content of the invention
For above-mentioned technical problem, the present invention provides 21 short series connection prepared by a kind of labeling method of novel fluorescence dyestuff
Repetitive sequence composite amplification detection kit, and 21 composite amplifications of str locus seat of mankind's autosome are applied to, with expansion
The characteristics of fast, harmony is good, sensitivity is high is speeded, Chinese population individual identification is met, paternity test, euchromatic dna builds storehouse
Demand.
Technical scheme is as follows：
A kind of 21 STR composite amplification detection kits using new double fluorescence labeling method, bag
Include：
21 couples of the composite primer groups of specific primer, 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 D6S 1043；
The composition of the reaction mixture is：MgCl25mM, Tris-HCl 125mM pH8.325 DEG C, KCl 125mM,
DNTPs 0.5mM, BSA 1.5g/L；The dNTPs is the equimolar mixture of dATP, dTTP, dCTP, dGTP；
21 sequences of the primer pair of locus of the composite primer group of 21 pairs of specific primers include SEQ ID
NO.01-SEQ ID NO.42；
Each str locus seat of the composite primer group of 21 pairs of specific primers is using respectively positioned at the locus core weight
The pair of primers amplification of Fu Qu both sides, at least one primer uses new pair of fluorochrome label method wherein in each pair primer
It is marked；
The primer pair of 21 locus of the composite primer group of 21 pairs of specific primers is divided into four groups using new double
Fluorochrome label method is 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；3rd 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 new double fluorescence labeling method be included in primer 5 ' hold first base FAM, HEX, TAMRA,
One kind in ROX is marked；Wherein, the primer for being marked with FAM, TAMRA and ROX, a base is used in the middle of primer 5 ' and 3 '
FAM is marked；The primer marked with HEX, a base is marked with HEX in the middle of primer 5 ' and 3 '.
21 STR composite amplifications detection examination using new double fluorescence labeling method of the present invention
The application method of agent box, comprises the following steps：Reagent in kit is mixed with sample DNA, loads PCR amplification pipes, be placed in heat
On circulating instrument, enter performing PCR amplification, amplification program is：95℃ 3min；94 DEG C of 5s, 60 DEG C of 1min, 26-28 circulation；60℃
Extend 10min eventually；4-16 DEG C of holding；Amplified production is obtained, amplified production fluoroscopic examination is carried out on genetic analyzer, then use
Fragment analysis softwareThe data detected on analysis gene sequencer；The sample DNA using
Chelex100 methods, magnetic bead extraction method or the human gene group DNA that any one method is extracted in Organic extraction method, extract sample
Source includes blood, blood stain, seminal fluid, saliva, body fluid, hair, muscle or the histoorgan of people, or using the hands-free filter for taking
Any one carrier is collected in paper, FTA cards, cotton swab, gauze human blood or Stomatocyte.
21 STR composite amplifications detection of use novel fluorescence dye labeling methodologies of the present invention
Kit, can be applied to the individual identification of Chinese population, paternity test, DNA and builds the aspects such as storehouse.
The beneficial effects of the invention are as follows：
1st, using novel fluorescence labelling technique, fluorescence efficiency is high, thus amplification rate faster, it is harmonious more preferably, sensitivity
It is higher；
2nd, this kit be comprising 21 height composite amplification systems of euchromosome STR locus, 21 locus with
21 locus that China DNA builds storehouse typing are completely the same, and system altitude is compatible；
3rd, this kit has very strong sample adaptability, i.e., a kind of kit can expand the sample of various samples, no
Sample with sample includes：Using people's base that any one is extracted in Chelex100 methods, magnetic bead extraction method or organic method extraction process
Because of the human blood or Stomatocyte of any one carrier collection such as group DNA and filter paper, FTA cards, cotton swab, gauze；
4th, by designing specific primer, with stronger specific amplification and temperature tolerance, (annealing is warm for this kit
56-62 DEG C of tolerance range of degree, it is ensured that preferably amplification knot can be obtained in the PCR amplification instrument using different brands or quality
Really).To dog, pig, ox, sheep, cat, chicken, mouse, rabbit, fish and e. coli dna, 10 kinds of different genera species are detected, without expansion
Increase peak, show that the present invention has good species specificity；
5th, the present invention drastically increases TAMRA, ROX by TAMRA and ROX of the mark with FRET on primer
Fluorescence efficiency, reduces the gap with FAM, HEX；During capillary electrophoresis detection, compared to use conventional fluorescent dye labeling methodologies
STR composite amplification reagent kits, the present invention has more balanced, faster system, while having sensitivity higher.
Brief description of the drawings
Fig. 1 is the result figure of the primer A-H amplifications D13S317 of the middle fluorescence of embodiment 1 difference mark position；
Fig. 2 is kit allelic ladder figure in embodiment 2；
Fig. 3 is the genotyping result of the amplification positive criteria product of embodiment 2 9947A；
Fig. 4 is kit Z2015110011F AFLP systems of the present invention in experiment 3；
Fig. 5 is 21 Direct ID sample Z2015110011F AFLP systems in experiment 3；
Fig. 6 is kit Z2015110071M AFLP systems of the present invention in experiment 3；
Fig. 7 is 21 Direct ID sample Z2015110071M AFLP systems in experiment 3；
Fig. 8 for experiment 4 in double fluorescence labeling method of the present invention to DNA standard items 9947A 500pg, 250pg, 125pg,
Five amplifications of concentration of 62.5pg and 31.25pg；
Fig. 9 in experiment 4 using conventional list fluorescence labeling method to DNA standard items 9947A 500pg, 250pg, 125pg,
Five amplifications of concentration of 62.5pg and 31.25pg.
Specific embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
As illustrated, the present invention discloses a kind of 21 STRs using new double fluorescence labeling method being combined
Amplification detection kit, including：
21 couples of the composite primer groups of specific primer, 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 D6S 1043；
The composition of the reaction mixture is：MgCl25mM, Tris-HCl 125mM pH8.325 DEG C, KCl 125mM,
DNTPs 0.5mM, BSA 1.5g/L；The dNTPs is the equimolar mixture of dATP, dTTP, dCTP, dGTP；
21 sequences of the primer pair of locus of the composite primer group of 21 pairs of specific primers include SEQ ID
NO.01-SEQ ID NO.42；
Each str locus seat of the composite primer group of 21 pairs of specific primers is using respectively positioned at the locus core weight
The pair of primers amplification of Fu Qu both sides, at least one primer uses new pair of fluorochrome label method wherein in each pair primer
It is marked；
The primer pair of 21 locus of the composite primer group of 21 pairs of specific primers is divided into four groups using new double
Fluorochrome label method is 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；3rd 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。
The new double fluorescence labeling method is included in the end of primer 5 ' first base FAM, HEX, TAMRA, ROX
One kind is marked；Wherein, the primer for being marked with FAM, TAMRA and ROX, a base is carried out with FAM in the middle of primer 5 ' and 3 '
Mark；The primer marked with HEX, a base is marked with HEX in the middle of primer 5 ' and 3 '.
21 STR composite amplifications detection examination using new double fluorescence labeling method of the present invention
The application method of agent box, comprises the following steps：Reagent in kit is mixed with sample DNA, loads PCR amplification pipes, be placed in heat
On circulating instrument, enter performing PCR amplification, amplification program is：95℃ 3min；94 DEG C of 5s, 60 DEG C of 1min, 26-28 circulation；60℃
Extend 10min eventually；4-16 DEG C of holding；Amplified production is obtained, amplified production fluoroscopic examination is carried out on genetic analyzer, then use
Fragment analysis softwareThe data detected on analysis gene sequencer；The sample DNA using
Chelex100 methods, magnetic bead extraction method or the human gene group DNA that any one method is extracted in Organic extraction method, extract sample
Source includes blood, blood stain, seminal fluid, saliva, body fluid, hair, muscle or the histoorgan of people, or using the hands-free filter for taking
Any one carrier is collected in paper, FTA cards, cotton swab, gauze human blood or Stomatocyte.
21 STR composite amplifications detection of use novel fluorescence dye labeling methodologies of the present invention
Kit, can be applied to the individual identification of Chinese population, paternity test, DNA and builds the aspects such as storehouse.
The determination of embodiment 1, design of primers and the fluorescence labeling position on primer sequence
21 str locus of system coamplification seat, is 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 is used
Oligo7 softwares, every primer Tm is close to 60 DEG C.The maximum magnitude of amplified production length is 75-500bp, and each pair primer is entered
Row amplification assay simultaneously optimizes, until obtaining the amplified peak that peak shape is sharp, peak height is higher.Again by composite amplification test, 56 DEG C-
Between 63 DEG C, non-specific amplification, purposeful peak are not produced, other do not occur and interacts or cross reaction.To dog, pig, ox,
Sheep, cat, chicken, mouse, rabbit, fish and e. coli dna are without amplification.Primer sequence is with table 2.
What novel fluorescence labeling method of the present invention was utilized is FRET principles, so the degree of FRET is being drawn with fluorescence
Position on thing is relevant.We have carried out research with actual test to it.To the same primer of D13S317, in different positions
Tagging fluorescence, fluorescence labeling may influence primer combination masterplate at too close 3 ' end, so primer of the total length for 24bp,
We have chosen fluorescence in the middle of the mark of 2-14,5 ' end.5 ' first bases " C " are marked with TAMRA, are represented with runic.Mark 6-
Base underscore+the bold Italic of FAM represents, such as following table：
A is the primer of conventional list fluorescence labeling, and B-I is primer of the middle fluorescence labeling in diverse location, expands 0.2-
The DNA of 0.5ng extracts sample.A-I sets identical PCR and deposition condition, and only fluorescent primer mark mode is different, experiment weight
It is multiple 3 times.In order to avoid fluorescence signal exceedes upper limit of detection value, electrophoresis is carried out again after all amplified productions are diluted into 10 times.As a result
Show：The average peak height highest of D primers amplification, is marked at the fluorescence efficiency highest of 5 ' the 6th, and the A compared to single fluorescence labeling draws
Thing, can improve to 9.2 times.Result such as following table and Fig. 1：
| Fluorescence labeling 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 fluorescence labeling between 5 ' -3 ' is remarkably improved the glimmering of long wave dye TAMRA
Light efficiency, the test of ROX is also in this way, here is omitted；The position influence fluorescence efficiency of short wavelength's fluorescence labeling, for
TAMRA, 6-FAM is marked at the fluorescence efficiency highest of 5 ' the 6th, in order that whole primer system and background fluorescence are homogeneous,
All be marked at the middle fluorescence of all fluorescent primers in the present invention on 5 ' the 6th bit bases by we, is specifically shown in Table 2.
The primer information of the present invention of table 2.
Note：The base of 5 ' fluorescence labelings of overstriking font, underscore is the base of middle fluorescence labeling, corresponding fluorescence
Element is correspondence identical overstriking or underscore mark.
The optimization and determination of the amplification system of embodiment 2
To make amplification balanced and, series of tests has been done to primer concentration and amplification component, finally determine primer dosage and
Expand the component of cushioning liquid.
Kit includes following composition：
2.5 × buffer components and its concentration be：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 are prepared by the method for patent 201610988458.1.Present invention also offers being used for
The allelic ladder of analysis, each locus primer expands oppositional allele respectively, and amplified production is combined.
Fig. 2 is Allellic ladder electrophoresis patterns.DNA standard items DNA 9947A are purchased from promega companies of the U.S..Thermal starting enzyme is purchased
It is precious biological from 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, can be obtained preferably using following program
Result：95 DEG C of predegenerations 3 minutes；94 DEG C are denatured 5 seconds, and 60 DEG C of annealing extend 1 minute, and the step runs 26-28 circulation；
60 DEG C are incubated 10 minutes；4-16 DEG C of insulation.
With 9947A standard items DNA as template, according to the form below sample-adding：
| Component | Dosage (μ L) |
| 2.5×PCR Mix | 10 |
| 5×Primers | 5 |
| 5U/ μ L thermal starting Taq archaeal dna polymerases | 1 |
| 9947A | 0.5 |
| Nuclease free deionization H2O | It is 25uL to mend to cumulative volume |
With program 95 DEG C 3 minutes on ABI9700；94 DEG C 5 seconds, 60 DEG C 1 minute operation 26 circulation；60 degree 10
Minute amplification.
The μ L deionized formamides of+0.5 μ L molecular weight internal standard of 1 μ L amplified productions+10 are loaded, with 1kv on ABI3130XL,
1s sample introductions, 15KV voltages carry out electrophoresis 40min.Result is analyzed with GeneMapper softwares.Fig. 3 is 9947A augmentation detections
As a result collection of illustrative plates, consistent with theoretical parting.
Embodiment 3, the uniformity with other commercial kit partings relatively more of the invention
In order to detect the uniformity of the present invention and other commercial kit partings, the present embodiment have chosen
MicroreaderTM21 Direct ID System use the present invention and 21 Direct ID reagents respectively as reference object
Box is expanded to 1316 samples (wherein independent individuals 663), and product is by the genetic analyzer capillaries of ABI 3100 electricity
Swimming carries out parting, and comparison is analyzed to genotyping result.21 Direct ID kits are operated according to its specification, the present invention
It is as follows using step：
3.1. PCR (PCR) amplification
1) buffer solution, primer mixture, Taq enzyme are taken, mixed liquor is made into according to following table, vibration dispenses anti-to PCR after mixing
Ying Guanzhong, often μ L of pipe 25.
The present invention：
| Component | Dosage (μ L) |
| 2.5×PCR Mix | 10 |
| 5×Primers | 5 |
| 5U/ μ L thermal starting Taq archaeal dna polymerases | 1 |
| Masterplate DNA (from blood filter paper or FTA cards) | 1 1.2mm apertures blood stain |
| Nuclease free deionization H2O | It is 25uL to mend to cumulative volume |
21 Direct ID：
| Component | Dosage (μ L) |
| MicroreaderTM 1.25×Buffer B | 20 |
| MicroreaderTM 21-D 5×Primer Mix | 5 |
| MicroreaderTM Taq DNA Polymerase II | 0.5 |
| Masterplate DNA (from blood filter paper or FTA cards) | 1 1.2mm diameter |
| Reaction final volume | 25.5uL |
2) according to the present invention and 21 Direct ID specifications, thermal cycler (ABI 9700DNA amplification instruments) parameter is set,
Then PCR reaction tubes are put into instrument and start amplification gene fragment.
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 of 30 minutes, 4-16
DEG C lasting insulation, until taking out sample.
3.2. after amplified reaction terminates, reaction tube is taken out, electrophoresis and detection is carried out with ABI3100 genetic analyzers.
1) take (the μ L deionized formamides of 0.5 μ L molecular weight internal standard+10) × (sample number) and be made into packing after mixed liquor is mixed,
Every μ L of pipe 10.
2) 1 μ L amplified productions 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 and be denatured 4 minutes, then rapid cooled on ice 4 minutes, make DNA be denatured completely and
Keep denatured state that sample is put into the sample tray of Genetic Analyser.
3) instrument parameter (sample introduction voltage 1kV of the present invention, sample injection time 1 second are set；21Direct ID sample introduction voltages
3kv, 10s), start electrophoresis detection after about 40 minutes, electrophoresis terminates, and figure is obtained with GeneMapper software analysis experimental datas
Spectrum and genotyping result.
3.3 genotyping results are contrasted
The present invention and 21 Direct ID are comprising 20 euchromosome STR sites and an Amelogenin sexes position
Point.In 20 normal dye STR bit points, 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.Parting to above-mentioned 19 sites and Amelogenin is compared.
Compare by genotyping result, 1316 52640 equipotential bases of sample (wherein independent individuals 663)
Cause, the present invention is completely the same with the genotyping result of 21 two kits of Direct ID.To wherein 2 samples respectively with two examinations
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
Sensitivity to DNA amplification masterplate of the present invention is tested, and the primer with conventional list fluorescence labeling is contrasted.Point
Two groups of primer ponds of double fluorescence labeling of the invention and single fluorescence labeling are not prepared, and primer concentration is assembled by each most having ready conditions,
Specifically see the table below.
By embodiment 2 configure amplification system, add doubling dilution DNA standard items 9947A (500pg, 250pg, 125pg,
62.5pg and 31.25pg), by two sets of primers, each optimum program is carried 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
Minute, 4-16 DEG C of lasting insulation, 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, run 28
Circulation；60 degree 30 minutes, 4-16 DEG C of lasting insulation, until taking out sample.
After amplified reaction terminates, reaction tube is taken out, electrophoresis and detection are carried out with ABI3100 genetic analyzers.
1) take (the μ L deionized formamides of 0.5 μ L molecular weight internal standard+10) × (sample number) and be made into packing after mixed liquor is mixed,
Every μ L of pipe 10.
2) 1 μ L amplified productions 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 and be denatured 4 minutes, then rapid cooled on ice 4 minutes, make DNA be denatured completely and
Keep denatured state that sample is put into the sample tray of Genetic Analyser.
3) instrument parameter (double fluorescence labeling sample introduction voltage 1kV, sample injection time 1 second are set；Single fluorescence labeling sample introduction voltage
3kv, 10s), start electrophoresis detection after about 40 minutes, electrophoresis terminates, and figure is obtained with GeneMapper software analysis experimental datas
Spectrum and genotyping result.
Harmonious and overall peak height is not as the primer of double fluorescence labeling between the color of the primer amplification of conventional list fluorescence labeling.Adopt
With the primer of double fluorescence labeling, due to the raising of signal, the more conventional single fluorescent dye primer consumption of primer consumption will lack.Especially
The big fluorescent dye primer of both fluorescence emission wavelengths of TAMRA, ROX, conventional list labeled primer consumption is that double labelling primer is used
The several times of amount, but amplified signal intensity and harmony are still not as good as double labelling primer.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in specification and implementation method
With, it can be applied to various suitable the field of the invention completely, for those skilled in the art, can be easily
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
Claims (4)
1. a kind of 21 STR composite amplification detection kits using new double fluorescence labeling method, its feature
It is, including：
21 pairs of the composite primer groups of specific primer, composite amplification 21 locus, reaction mixture and hot start Taq polymerases；
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 is：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；
21 sequences of the primer pair of locus of the composite primer group of 21 pairs of specific primers include SEQ ID NO.01-
SEQ ID NO.42；
Each str locus seat of the composite primer group of 21 pairs of specific primers is using respectively positioned at the locus core duplicate block
The pair of primers amplification of both sides, at least one primer is carried out using new pair of fluorochrome label method wherein in each pair primer
Mark；
The primer pair of 21 locus of the composite primer group of 21 pairs of specific primers is divided into four groups using new pair of fluorescence
Dye labeling methodologies are marked；Described four groups include first group：D3S1358, vWA, D7S820, CSF1PO, PentaE；SEQ
ID NO.01-SEQ ID NO.10；Second group：D8S1179, D21S11, D16S539, D2S1338, Penta D；SEQ ID
NO.11-SEQ ID NO.20；3rd 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。
2. 21 STR composite amplifications using new double fluorescence labeling method according to claim 1 are examined
Test agent box, it is characterised in that：The new double fluorescence labeling method be included in primer 5 ' hold first base FAM, HEX,
One kind in TAMRA, ROX is marked；Wherein, the primer for being marked with FAM, TAMRA and ROX, in the middle of primer 5 ' and 3 '
Base is marked with FAM；The primer marked with HEX, a base is marked with HEX in the middle of primer 5 ' and 3 '.
3. 21 STR composite amplifications using new double fluorescence labeling method according to claim 1 are examined
The application method of test agent box, it is characterised in that comprise the following steps：Reagent in kit is mixed with sample DNA, is loaded
PCR amplification pipes, are placed on thermal cycler, enter performing PCR amplification, and amplification program is：95℃3min；94 DEG C of 5s, 60 DEG C of 1min, 26-
28 circulations；60 DEG C extend 10min eventually；4-16 DEG C of holding；Amplified production is obtained, amplified production is carried out on genetic analyzer
Fluoroscopic examination, then use fragment analysis softwareThe data detected on analysis gene sequencer；The sample
The product DNA human genomes that any one method is extracted using Chelex100 methods, magnetic bead extraction method or in Organic extraction method
DNA (source for extracting sample includes blood, blood stain, seminal fluid, saliva, body fluid, hair, muscle or the histoorgan of people), or
The human blood or Stomatocyte collected using any one carrier in the hands-free filter paper for taking, FTA cards, cotton swab, gauze.
4. 21 STR composite amplifications of use novel fluorescence dye labeling methodologies according to claim 1
Detection kit, it is characterised in that：The kit can be applied to the individual identification of Chinese population, paternity test, DNA and build storehouse etc.
Aspect.
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