CN105177125B - 20 STR composite amplification systems of mankind's X chromosome and application - Google Patents
20 STR composite amplification systems of mankind's X chromosome and application Download PDFInfo
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Abstract
The present invention relates to biological technical field, is related to 20 STR composite amplification systems of mankind's X chromosome and application, and the composite amplification system includes 20 pairs of primers, can expand 19 STR bit points simultaneously：DXS6795, DXS6803, DXS6807, DXS9907, DXS7423, GATA172D05, DXS101, DXS9902, DXS7133, DXS6810, GATA31E08, DXS6800, DXS981, DXS10162, DXS6809, GATA165B12, DXS10079, DXS10135, HPRTB and a sex recognition site Amelogenin.The present invention uses multicolored fluorescence labeling, and amplified production size is simple to operate between 85 450bp, and individual identification power is high.The site that composite amplification system provided by the present invention provides has very high compatibility with X STR kits general at present and has newly increased the higher site of some polymorphism informations, can be effectively applied to the auxiliary detection of the related paternity identification of X chromosome, individual identification and X chromosome sex-linked hereditary disease.
Description
Technical field
The present invention relates to biological technical field, is related to 20 STR composite amplification systems of mankind's X chromosome,
Particularly relate to the genetic marker with polymorphism on X chromosome, the glimmering of composite amplification is carried out by PCR
Light detection kit, the system can apply to daily paternity identification, individual identification and the X chromosome related to X chromosome
The auxiliary detection of sex-linked hereditary disease.
Background technology
Human genome STR (STR) is using a few base as core unit, and tandem sequence repeats are formed
DNA genetic markers.Differentiation between not agnate, different crowd even Different Individual is by core unit sequence and repetition
Several difference, this also constitutes STR genetic polymorphism.In genome, average every 15~20kb just has a STR bit point,
The 10% of genome is accounted for, is present in noncoding region and introne more, recurring unit is 2~6bp, number of repetition 10~60 times,
Clip size is in codominant inheritance in 70~500bp, and by Mendel's rule.Therefore STR composite amplifications detection technique can be wide
It is general to be applied to the fields such as legal medical expert's individual identification, paternity test.
In 23 pairs of chromosomes of the mankind, one pair of which is that sex chromosome is X chromosome and Y chromosome.Women's
A pair of X chromosomes can recombinate during egg cell generates as autosome, but in the spermatoblast of male
During generation, X chromosome and Y chromosome can not be recombinated arbitrarily.The X chromosome of father only entails daughter, the X dyes of mother
Colour solid can entail daughter, can also entail son.So this unique mode of inheritance of X chromosome causes it in parental right
There is special application value in identification.For example the sister of parents' missing finds relatives, half-sister's relation is identified, grandmother-grandson
The identification of female's relation, the identification of incest relation, the identification of rape offender and identification of the genetic disease related to X chromosome etc..
With now, on the market compared with the quantity of euchromosome STR kit, the quantity of X-STR kits is only several to be available for
Selection, the related patent numbers of X chromosome are also fewer in addition.
Its STR bit point negligible amounts for being directed to of the existing X-STR kits patent related to X chromosome, and it is more in heredity
There is certain shortcoming in the accuracy of state property, individual identification power and detection, it is therefore necessary to it is more, hereditary more to develop a kind of bit number of points
The X-STR kits that state property is high, individual identification power is strong.
The content of the invention
In order to overcome drawbacks described above, it is an object of the invention to provide one kind can once detect 19 X-STR locus and
20 STR composite amplification systems of mankind's X chromosome of Amelogenin locus, are suitable for use with autosome
STR kits are difficult to the detection of excluding paternity relation case, and fluorescence labeling composite amplification system of the present invention detection is quick, accurate
Really.
A further object of the present invention is to provide mankind's X chromosome STR composite amplification system and dyed in X
The paternity identification that body phase is closed, the application in individual identification.
In order to realize foregoing invention purpose, the technical solution used in the present invention：Mankind's X chromosome STR
Composite amplification system, it is characterised in that the composite amplification system includes 19 pairs of primers, can expand 19 STR bit points simultaneously：
DXS6795、DXS6803、DXS6807、DXS9907、DXS7423、GATA172D05、DXS101、DXS9902、DXS7133、
DXS6810、GATA31E08、DXS6800、DXS981、DXS10162、DXS6809、GATA165B12、DXS10079、
DXS10135、HPRTB；
The upstream primer sequence of the amplification DXS6795 is as shown in SEQ ID NO1；
The downstream primer sequence of the amplification DXS6795 is as shown in SEQ ID NO2；
The upstream primer sequence of the amplification DXS6803 is as shown in SEQ ID NO3；
The downstream primer sequence of the amplification DXS6803 is as shown in SEQ ID NO4；
The upstream primer sequence of the amplification DXS6807 is as shown in SEQ ID NO5；
The downstream primer sequence of the amplification DXS6807 is as shown in SEQ ID NO6；
The upstream primer sequence of the amplification DXS9907 is as shown in SEQ ID NO7；
The downstream primer sequence of the amplification DXS9907 is as shown in SEQ ID NO8；
The upstream primer sequence of the amplification DXS7423 is as shown in SEQ ID NO9；
The downstream primer sequence of the amplification DXS7423 is as shown in SEQ ID NO10；
The upstream primer sequence of the amplification GATA172D05 is as shown in SEQ ID NO13；
The downstream primer sequence of the amplification GATA172D05 is as shown in SEQ ID NO14；
The upstream primer sequence of the amplification DXS101 is as shown in SEQ ID NO15；
The downstream primer sequence of the amplification DXS101 is as shown in SEQ ID NO16；
The upstream primer sequence of the amplification DXS9902 is as shown in SEQ ID NO17；
The downstream primer sequence of the amplification DXS9902 is as shown in SEQ ID NO18；
The upstream primer sequence of the amplification DXS7133 is as shown in SEQ ID NO19；
The downstream primer sequence of the amplification DXS7133 is as shown in SEQ ID NO20；
The upstream primer sequence of the amplification DXS6810 is as shown in SEQ ID NO21；
The downstream primer sequence of the amplification DXS6810 is as shown in SEQ ID NO22；
The upstream primer sequence of the amplification GATA31E08 is as shown in SEQ ID NO23；
The downstream primer sequence of the amplification GATA31E08 is as shown in SEQ ID NO24；
The upstream primer sequence of the amplification DXS6800 is as shown in SEQ ID NO25；
The downstream primer sequence of the amplification DXS6800 is as shown in SEQ ID NO26；
The upstream primer sequence of the amplification DXS981 is as shown in SEQ ID NO27；
The downstream primer sequence of the amplification DXS981 is as shown in SEQ ID NO28；
The upstream primer sequence of the amplification DXS10162 is as shown in SEQ ID NO29；
The downstream primer sequence of the amplification DXS10162 is as shown in SEQ ID NO30；
The upstream primer sequence of the amplification DXS6809 is as shown in SEQ ID NO31；
The downstream primer sequence of the amplification DXS6809 is as shown in SEQ ID NO32；
The upstream primer sequence of the amplification GATA165B12 is as shown in SEQ ID NO33；
The downstream primer sequence of the amplification GATA165B12 is as shown in SEQ ID NO34；
The upstream primer sequence of the amplification DXS10079 is as shown in SEQ ID NO35；
The downstream primer sequence of the amplification DXS10079 is as shown in SEQ ID NO36；
The upstream primer sequence of the amplification DXS10135 is as shown in SEQ ID NO37；
The downstream primer sequence of the amplification DXS10135 is as shown in SEQ ID NO38；
The upstream primer sequence of the amplification HPRTB is as shown in SEQ ID NO39；
The downstream primer sequence of the amplification HPRTB is as shown in SEQ ID NO40.
Further, mankind's X chromosome STR composite amplification system also includes to expand for a pair
Amelogenin pair of primers, it is respectively：
Amelogenin upstream primer sequence is expanded as shown in SEQ ID NO11；
Amelogenin downstream primer sequence is expanded as shown in SEQ ID NO12.
Foregoing mankind's X chromosome STR composite amplification system, its primer concentration ratio are：
DXS6795SEQ ID NO1-2,0.179 μm of ol/L, DXS6803SEQ ID NO3-4,0.126 μm of ol/L, DXS6807SE-Q
ID NO5-6,0.245 μm of ol/L, DXS9907SEQ ID NO7-8,0.150 μm of ol/L, DXS7423SEQ ID NO9-10,
0.162 μm of ol/L, Amelogenin SEQ ID NO11-12,0.060 μm of ol/L, GATA172D05SEQ ID NO13-14,
0.192 μm of ol/L, DXS101SEQ ID NO15-16,0.190 μm of ol/L, DXS9902SEQ ID NO17-18,0.169 μm of ol/
L, DXS7133SEQ ID NO19-20,0.404 μm of ol/L, DXS6810SEQ ID NO21-22,0.412 μm of ol/L,
GATA31E08SEQ ID NO23-24,0.188 μm of ol/L, DXS6800SEQ ID NO25-26,0.278 μm of ol/L,
DXS981SEQ ID NO27-28,0.130 μm of ol/L, DXS10162SEQ ID NO29-30,0.250 μm of ol/L,
DXS6809SEQ ID NO31-32,0.420 μm of ol/L, GATA165B12SEQ ID NO33-34,0.255 μm of ol/L,
DXS10079SEQ ID NO35-36,0.276 μm of ol/L, DXS10135SEQ ID NO37-38,0.315 μm of ol/L, HPRTB
SEQ ID NO39-40,0.404 μm of ol/L.
Mankind's X chromosome STR's is grouped into：DXS6795, DXS6803, DXS6807, DXS9907, DXS7423 first
Group；Amelogenin, GATA172D05, DXS101, DXS9902, DXS7133, DXS6810 are second group；GATA31E08、
DXS6800, DXS981, DXS10162, DXS6809 are the 3rd group；GATA165B12, DXS10079, DXS10135, HPRTB are
4th group.
5 ' ends of one primer of every a pair of specific primers centering mark with fluorescein, described fluorescein mark side
Formula is：DXS6795, DXS6803, DXS6807, DXS9907, DXS7423 are marked using FAM；Amelogenin、
GATA172D05, DXS101, DXS9902, DXS7133, DXS6810 are marked using HEX；GATA31E08、DXS6800、
DXS981, DXS10162, DXS6809 are marked using TAMRA；GATA165B12, DXS10079, DXS10135, HPRTB are used
ROX is marked.The internal standard of the composite amplification system detection is marked using fluorescent orange element label Orange.
Amplification system includes primer mixture, enzyme reaction buffer solution, genomic DNA, and described enzyme reaction buffer solution includes
Thermal starting archaeal dna polymerase, Brij58, dNTP, DMSO, Tris-HCl, BSA, DTT, preservation ring of the enzyme reaction buffer solution at -20 DEG C
Do not frozen in border.Calculated by 25 μ l of amplification system, primer mixture is (5*Primer mix) 5 μ l, enzyme reaction buffer solution
(2.5*Buffer D) 10 μ l, genomic DNA (extraction template DNA, DS sample etc.) 1-3 μ l.The DNA that the present invention relates to
Sample can be from the blood of people, blood stain, seminal fluid, seminal stain, saliva, body fluid, hair, muscle, tissue, nail etc..The DNA samples
Product available reagent box method, phenol chloroform method, Chelex-100 methods, paramagnetic particle method carry out DNA extraction processs.
The formula of described enzyme reaction buffer solution is：KCl 0.1M；MgCl20.0015mM；Tris-HCl 0.05M；BSA
0.16mg/ml；dNTP 0.2mM；Brij580.001M；DTT 0.02M；Betaine 0.1mol/L；DMSO0.40%, percentage
Than the percentage by volume that enzyme reaction buffer solution is accounted for for DMSO stostes, that is, when configuring 100ml enzyme reaction buffer solutions, add 0.4ml
DMSO stostes；Thermal starting archaeal dna polymerase 0.25U.
The amplification program of described composite amplification system is：The first step is denatured：96 DEG C, 2 minutes；Thermal cycle：94 DEG C 30 seconds；
60 DEG C, 70 seconds, 27-30 circulation is carried out altogether；60 DEG C, 30 minutes of extension eventually；Insulation：15℃.The present invention amplified production need through
Capillary Electrophoresis is crossed, so as to carry out fragment analysis.
19 X-STR of the present invention genetic data statistics is shown in result (table 1).
Table 1：19 X-STR of the present invention genetic data statistical form
Wherein Marker:Locus site, Cytogenetic localisation：Chromosome Distance positioning,
Physical localisation[Mb]：Physical distance positions, PIC：Polymorphism information content's
Abbreviation, polymorphism information content, HET：Heterozygotie abbreviation, heterozygosity, for weighing gene polynorphisms, MEC：
Mean paternity exclusion change abbreviation, average parentage exclusion probability, PD：Power of
Discrimination abbreviation, personal recognition capability.
The present invention compared with prior art, has advantages below：19 X chromosome STR bit point ratios involved in the present invention
Most of commercial kit bit number of points is more currently on the market, can better meet public security, judicial authority for X-
The high request of STR detections.According to the statistics to 1248 samples of Chinese Han Population, it is seen that：Site selected by the present invention have compared with
High polymorphism information, individual identification rate are higher.Genomic DNA can be obtained by extracting the modes such as template DNA, DS sample
, template wide adaptation range.The specificity of primer is higher, no nonspecific products generation and signal stabilization.
Brief description of the drawings
Fig. 1 be enzyme reaction buffer solution (2.5*Buffer D) Optimal Experimental in Brij58 concentration comparative result；
Fig. 2 be enzyme reaction buffer solution (2.5*Buffer D) Optimal Experimental in DMSO concentration comparative result；
Fig. 3 .1 and Fig. 3 .2 are the optimization component expanding effect figure given by the present invention；
Fig. 4 .1 and Fig. 4 .2 are the test experience result of species specificity involved in the present invention；
Fig. 5 .1 and Fig. 5 .2 are the expanding effect collection of illustrative plates of several samples；
Fig. 6 .1 and Fig. 6 .2 are the AFLP system of XX sex-reversal syndrome cases；
Fig. 7 .1,7.2,7.3 and 7.4 are that half-sister's parental right relation identifies AFLP system.
Embodiment
Technical scheme is described in further details with reference to Figure of description and embodiment.
Following table 2 enumerates locus site information, the X chromosome for comparing now common X-STR kits on the market
The composite amplification system of related Patent Publication and the kit of the present invention.
The existing X-STR composite amplification systems of table 2 contrast with amplification system of the present invention
Although the offices such as current police and judicial are very strong for the demand of X-STR kits, it is related in above table
The kit or composite amplification system that arrive or because price or because can not commercialization in the public security of reality, the administration of justice and each
Generally rate is not very high for the use in institutes laboratory.So developing, a bit number of points is more, genetic polymorphism is high, individual identification
The strong X-STR kits of power are that have very bright and clear market prospects.
1 human chromosomal of embodiment, 20 STR composite amplification system designs
1. design of primers
The primer is formed using the Software for Design such as Prime Premier5 and NCBI Blast, should be true when designing primer
The Tm values of each primer are protected in the range of (60 ± 2) DEG C, amplification efficiency approaches and ensures the amplified production size of each pair of primer not
Together and it is enough to distinguish in Capillary Electrophoresis.After the completion of design, with the software analysis such as AutoDimer primer dimer and difference
Interaction between primer, need to redesign if interaction can produce nonspecific products or dimer,
Until obtaining satisfactory primer sequence.
From people's source DNA template, single amplification is carried out with above-mentioned 20 pairs of primers respectively, amplified production is placed in 1.5%
Agarose gel electrophoresis, PCR system and amplification condition are adjusted to obtain the common amplification bar of 20 pairs of primers according to electrophoresis result
Part, final intended effect are：Under same system and amplification condition, all primer pairs can occur bright and more single
Purpose band.If primer can not meet above-mentioned condition, then primer is redesigned.
2. fluoroscopic marker system is established
It has selected FAM, HEX, TAMRX, ROX and establish four color fluoroscopic marker systems, 20 pairs of designed primers are divided into four
Group carries out fluorescence labeling.Every group uses a kind of fluorescence labeling, is FAM, HEX, TAMRX, ROX respectively.Obtain fluorescent dye primer
Afterwards, the non-fluorescence primer to be matched with it combines, and then carries out single amplification respectively, amplified production is placed in into 3500 genetic analyses
Capillary Electrophoresis is carried out on instrument, the amplification efficiency of each pair primer is assessed according to the result of capillary electrophoresis detection.Thereafter, will be same
4 couple of one fluorescence labeling or 5 pairs or 6 pairs of primers mixing are placed in same pipe and expanded, and amplified production is placed in into 3500 genetic analyses
Capillary Electrophoresis is carried out on instrument, the amplification efficiency of each pair primer is determined according to the result of capillary electrophoresis detection, and judge
Whether 4 couple or 5 pairs or 6 pairs of primers mixing amplifications cause non-specificity.Finally, according to single amplification and the capillary of combination amplification
Electrophoresis tube result primarily determines that the addition of each primer pair, 20 pairs of primers are mixed to be placed in same pipe expanded, further according to multiple
The electrophoresis result for closing amplification adjusts respective concentration, makes the amplification efficiency (reacting on the peak height of electrophoresis result) of each primer pair
It is basically identical to be defined.(primer concentration value is drawing in PCR amplification system to the 20 pairs of primer sequences and concentration ratio finally determined
Thing concentration) it is shown in Table 3.Specific primer is designed for above-mentioned 20 gene locis, the length range of its amplified production is 85-
Between 450bp.The detected components Plays thing of the present invention uses the orange substance markers of Orange, can clearly mark and distinguish inspection
The size in each locus site of test sample sheet.
3 20 pairs of primer sequences of table and concentration ratio table
The DNA sample that the present invention relates to can derive from blood, blood stain, seminal fluid, seminal stain, saliva, body fluid, the hair of people
Hair, muscle, tissue, nail etc..The DNA sample available reagent box method, phenol chloroform method, Chelex-100 methods, paramagnetic particle method
Carry out DNA extraction processs.
The amplified production of the present invention can be detected with the genetic analyzer of ABI series.Testing result can be
Analyzed on the DAS such as Genemapper, obtain corresponding STR partings collection of illustrative plates and data.
Embodiment 2 detects to the Genotyping of some sample
1. the collection of blood sample (blood sample is donated by volunteer)
2.DNA is extracted
Using the extraction genomic DNA (reference of Chelex-100 methods《Forensic DNA Protocol》.Humana
Press, 1998) taking 0.5-5 μ l anticoagulated whole blood or (1-3mm) * (2-5mm) blood cake, vibration is mixed as in 500 μ l centrifuge tubes
Even Chelex solution makes Chelex fully suspend, and often pipe adds 195 μ l Chelex-100 (5%) solution and 5 μ l Proteinase Ks
(20mg/ml) vibration mixes, and after 56 degrees Celsius are incubated two hours or stay overnight, takes out vibration 2 minutes, is heated 10 minutes in boiling water
13000rpm is centrifuged 5 minutes afterwards, carefully pipettes 150 μ l supernatants into new centrifuge tube.
3. reaction system
It will be made into the following manner after each reaction reagent (buffer solution, primer mixture, genomic DNA etc.) vibration mixing
PCR reaction mixtures, amplification system cumulative volume are 25 μ l, including the μ l of primer mixture (5*Primer mix) 5, wherein primer are dense
Degree is shown in embodiment 1, enzyme reaction buffer solution (2.5*Buffer D) 10 μ l, the μ l of genomic DNA 2 (extraction template DNA, DS sample
Deng), ddH2O8μl。
It is special that the enzyme reaction buffer solution (2.5*Buffer D) used in composite amplification system involved in the present invention has
Formula (table 4), be a kind of thermal starting enzyme (MR Taq II) and PCR reaction buffer premixed liquids, and it is in -20 DEG C of preservation
It will not be frozen in environment, there is antifreeze function.
| Composition | Unit | Concentration in buffer solution |
| KCl | M | 0.1 |
| MgCl2 | M | 0.0015 |
| Tris-HCl | M | 0.05 |
| BSA (bovine serum albumin) | mg/ml | 0.16 |
| dNTP | mM | 0.2 |
| Brij58 (APEO) | mM | 0.001 |
| DTT (dithiothreitol (DTT)) | M | 0.02 |
| Betaine (glycine betaine) | M | 0.1 |
| DMSO (dimethyl sulfoxide (DMSO)) | % | 0.40% |
| MR TaqⅡ | U | 0.25 |
Table 4：Enzyme reaction buffer solution (2.5*Buffer D) is formulated
4.PCR response procedures
The PCR amplification programs (table 5) that composite amplification system involved in the present invention uses.
Table 5：The amplification program of composite amplification system of the present invention
5. capillary electrophoresis detection
By Orange500 internal standards and formamide in proportion 2.5:100 mixing, take 12.5 μ l mixtures to be added to 96 orifice plates,
The amplified production sample either μ l of allele reference material 1 are added, mixing is stood several minutes, and 95 DEG C are denatured 3min, immediately ice
3min is bathed, is put into after centrifugation on ABI3500 sequenators, prepares detection.
6. data analysis
Initial data is imported, in the File menu setecting Add sample to project of homepage, finds sample text
Part, filesselected folder, clicks on add to list, clicks on add, and sample file is shown in Project windows；Selection analysis is joined
Number.Define analysis method, panel, size standard.The initial data of sample electrophoresis is browsed, chooses certain sample
Filename, under " sample " menu select " Raw data ".Moving tracing line, cursor is set to be parked in (first on the right side of primer peak
Before individual orange internal standard peak), the numerical value now to be shown in the X-axis of the window lower left corner is used as analysis method analytical parameters
In starting point；Green analysis button is clicked on, save project dialog boxes occurs, is preserved after name, software is start to process
Data, the lower left corner shows analysis completed after the completion of analysis.Using GeneMapperRID-X software analysis obtains
Data simultaneously generate collection of illustrative plates.
Embodiment 3 is directed to the Optimal Experimental of enzyme reaction buffer solution (2.5*Buffer D)
KCl is in the concentration gradient of enzyme reaction buffer solution：Tri- concentration gradients of 0.075M, 0.1M, 0.125M.MgCl2
The concentration gradient of enzyme reaction buffer solution is：Tri- concentration gradients of 1mM, 1.5mM, 2mM.Concentration of the Brij58 in enzyme reaction buffer solution
Gradient is：Tri- concentration gradients of 0.001mM, 0.0015mM, 0.002mM.DTT is in the concentration gradient of enzyme reaction buffer solution：
Tri- concentration gradients of 0.015M, 0.02M, 0.025M.Betaine is in the concentration gradient of enzyme reaction buffer solution：0.05M、0.1M、
Tri- concentration gradients of 0.15M.DMSO is in the concentration gradient of enzyme reaction buffer solution：0.3%th, 0.4%, 0.5% 3 concentration ladder
Degree.MR Taq II are in the concentration gradient of enzyme reaction buffer solution：Tri- concentration gradients of 0.15U, 0.25U, 0.35U.For above-mentioned
The orthogonal experiment of the component design various concentrations of enzyme reaction buffer solution carries out composite amplification, and experimental result determines compound expansion
The optimum combination of increasing system.Accompanying drawing 1 lists wherein Brij58 concentration comparative result.Accompanying drawing 2 lists the dense of wherein DMSO
Spend comparative result.Accompanying drawing 3.1 and 3.2 lists the optimization component (component ratio i.e. in embodiment 2) given by the present invention
Expanding effect figure.Accompanying drawing 1-7 all accompanying drawings are GeneMapperRThe data collection of illustrative plates that ID-X software analysis obtains.
Embodiment 4：For the test experience of species specificity, the DNA profiling (phenol chloroform) of different plant species is have chosen,
Whether see the composite amplification system of the present invention has the advantage of species specificity.It has chosen pig, ox, sheep, mouse, chicken, duck, fish and people
Type genomic dna can efficiently amplify human gene as detection object, as a result composite amplification system of the present invention
Group DNA X-STR partings, other species do not have amplified production.It is special that Fig. 4 .1 and 4.2 list species involved in the present invention
The test experience result of the opposite sex.
Embodiment 5：Different samples are detected using the composite amplification system of the present invention.The DNA sample that the present invention relates to
Can be from the blood of people, blood stain, seminal fluid, seminal stain, saliva, body fluid, hair, muscle, tissue, nail etc..The DNA sample can
DNA extraction processs are carried out with RNA isolation kit, phenol chloroform method, Chelex-100 methods, paramagnetic particle method.Accompanying drawing 5.1 and 5.2 lists
The expanding effect collection of illustrative plates of wherein several samples.Prove realize effectively amplification using various samples, detection quick and precisely, is applicable
Scope is wide.
Embodiment 6：XX sex-reversal syndromes case together is identified using the composite amplification system of the present invention.
The sample for playing case is the blood filter paper sample of a pair of fathers and sons, and the secondary sex characters of son shows as male, but sharp
Come to nothing when being detected with the micro-deleted kits of Y.The Amel sites of the son of the case are found when carrying out X-STR site primers
Parting be XX, genotype is women, and some STR bit points have 2 allele (table 6) on X chromosome, son in the case
Allele and the allele of father can be with matcheds.Its father is normal male individual, has 1 X chromosome and 1
Y chromosome.So son inherits an X chromosome at its father in the case, it is a typical sex-reversal syndrome
Case (Fig. 6 .1 and 6.2).
Table 6：The X-STR partings of XX sex-reversal syndrome case father and sons
| Site | Father's genotype | Son's genotype |
| DXS6795 | 11 | 11 |
| DXS6803 | 11 | 11；12 |
| DXS6807 | 14 | 11；14 |
| DXS9907 | 13 | 13 |
| DXS7423 | 15 | 15 |
| AMEL | X；Y | X |
| DXS981 | 12.3 | 12.3；14 |
| DXS101 | 24 | 24；28 |
| DXS9902 | 12 | 10；12 |
| DXS7133 | 10 | 9；10 |
| DXS6810 | 19 | 18；19 |
| GATA31E08 | 9 | 9 |
| DXS6800 | 16 | 16 |
| GATA172D05 | 10 | 10 |
| DXS10162 | 18 | 18 |
| DXS6809 | 34 | 34 |
| GATA165B12 | 10 | 9；10 |
| DXS10079 | 21 | 20；21 |
| DXS10135 | 20 | 20；23 |
| HPRTB | 13 | 13 |
Embodiment 7：Half-sister's parental right relation is identified.
There are 2 couples of mother and daughters in present case, advocate that the daughter in these two pair mother and daughter is half-blooded sister, it is assumed same
Father is not alive, therefore may only be detected using X-STR sites.Testing result (table 7 and Fig. 7 .1-7.4) substantially can be with
Determine that father's Genotyping of the daughter in these two pair mother and daughter is identical, so it is half-blooded sisterhood to be not excluded for.
Table 7：Half-sister's parental right relation identifies Genotyping
Above-described embodiment is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill of the art
For personnel, under the premise without departing from the principles of the invention, some improvement and equivalent substitution can also be made, these are to the present invention
Power require be improved with the technical scheme after equivalent substitution, each fall within protection scope of the present invention.
Claims (9)
1. mankind's X chromosome STR composite amplification system, it is characterised in that the composite amplification system includes 19 pairs
Primer, 19 STR bit points can be expanded simultaneously：DXS6795、DXS6803、DXS6807、DXS9907、DXS7423、
GATA172D05、DXS101、DXS9902、DXS7133、DXS6810、GATA31E08、DXS6800、DXS981、DXS10162、
DXS6809、GATA165B12、DXS10079、DXS10135、HPRTB；
The upstream primer sequence of the amplification DXS6795 is as shown in SEQ ID NO1；
The downstream primer sequence of the amplification DXS6795 is as shown in SEQ ID NO2；
The upstream primer sequence of the amplification DXS6803 is as shown in SEQ ID NO3；
The downstream primer sequence of the amplification DXS6803 is as shown in SEQ ID NO4；
The upstream primer sequence of the amplification DXS6807 is as shown in SEQ ID NO5；
The downstream primer sequence of the amplification DXS6807 is as shown in SEQ ID NO6；
The upstream primer sequence of the amplification DXS9907 is as shown in SEQ ID NO7；
The downstream primer sequence of the amplification DXS9907 is as shown in SEQ ID NO8；
The upstream primer sequence of the amplification DXS7423 is as shown in SEQ ID NO9；
The downstream primer sequence of the amplification DXS7423 is as shown in SEQ ID NO10；
The upstream primer sequence of the amplification GATA172D05 is as shown in SEQ ID NO13；
The downstream primer sequence of the amplification GATA172D05 is as shown in SEQ ID NO14；
The upstream primer sequence of the amplification DXS101 is as shown in SEQ ID NO15；
The downstream primer sequence of the amplification DXS101 is as shown in SEQ ID NO16；
The upstream primer sequence of the amplification DXS9902 is as shown in SEQ ID NO17；
The downstream primer sequence of the amplification DXS9902 is as shown in SEQ ID NO18；
The upstream primer sequence of the amplification DXS7133 is as shown in SEQ ID NO19；
The downstream primer sequence of the amplification DXS7133 is as shown in SEQ ID NO20；
The upstream primer sequence of the amplification DXS6810 is as shown in SEQ ID NO21；
The downstream primer sequence of the amplification DXS6810 is as shown in SEQ ID NO22；
The upstream primer sequence of the amplification GATA31E08 is as shown in SEQ ID NO23；
The downstream primer sequence of the amplification GATA31E08 is as shown in SEQ ID NO24；
The upstream primer sequence of the amplification DXS6800 is as shown in SEQ ID NO25；
The downstream primer sequence of the amplification DXS6800 is as shown in SEQ ID NO26；
The upstream primer sequence of the amplification DXS981 is as shown in SEQ ID NO27；
The downstream primer sequence of the amplification DXS981 is as shown in SEQ ID NO28；
The upstream primer sequence of the amplification DXS10162 is as shown in SEQ ID NO29；
The downstream primer sequence of the amplification DXS10162 is as shown in SEQ ID NO30；
The upstream primer sequence of the amplification DXS6809 is as shown in SEQ ID NO31；
The downstream primer sequence of the amplification DXS6809 is as shown in SEQ ID NO32；
The upstream primer sequence of the amplification GATA165B12 is as shown in SEQ ID NO33；
The downstream primer sequence of the amplification GATA165B12 is as shown in SEQ ID NO34；
The upstream primer sequence of the amplification DXS10079 is as shown in SEQ ID NO35；
The downstream primer sequence of the amplification DXS10079 is as shown in SEQ ID NO36；
The upstream primer sequence of the amplification DXS10135 is as shown in SEQ ID NO37；
The downstream primer sequence of the amplification DXS10135 is as shown in SEQ ID NO38；
The upstream primer sequence of the amplification HPRTB is as shown in SEQ ID NO39；
The downstream primer sequence of the amplification HPRTB is as shown in SEQ ID NO40.
2. mankind's X chromosome STR composite amplification system according to claim 1, it is characterised in that also
Including a pair of primers that can expand Amelogenin sites, it is respectively：
Amelogenin upstream primer sequence is expanded as shown in SEQ ID NO11；
Amelogenin downstream primer sequence is expanded as shown in SEQ ID NO12.
3. mankind's X chromosome STR composite amplification system according to claim 2, its primer concentration ratio
It is：DXS6795 SEQ ID NO1-2,0.179 μm of ol/L, DXS6803 SEQ ID NO3-4,0.126 μm of ol/L, DXS6807
SEQ ID NO5-6,0.245 μm of ol/L, DXS9907 SEQ ID NO7-8,0.150 μm of ol/L, DXS7423 SEQ ID NO9-
10,0.162 μm of ol/L, Amelogenin SEQ ID NO11-12,0.060 μm of ol/L, GATA172D05 SEQ ID NO13-
14,0.192 μm of ol/L, DXS101 SEQ ID NO15-16,0.190 μm of ol/L, DXS9902 SEQ ID NO17-18,0.169
μm ol/L, DXS7133 SEQ ID NO19-20,0.404 μm of ol/L, DXS6810 SEQ ID NO21-22,0.412 μm of ol/L,
GATA31E08 SEQ ID NO23-24,0.188 μm of ol/L, DXS6800 SEQ ID NO25-26,0.278 μm of ol/L,
DXS981 SEQ ID NO27-28,0.130 μm of ol/L, DXS10162 SEQ ID NO29-30,0.250 μm of ol/L, DXS6809
SEQ ID NO31-32,0.420 μm of ol/L, GATA165B12 SEQ ID NO33-34,0.255 μm of ol/L, DXS10079 SEQ
ID NO35-36,0.276 μm of ol/L, DXS10135 SEQ ID NO37-38,0.315 μm of ol/L, HPRTB SEQ ID NO39-
40,0.404 μm of ol/L.
4. mankind's X chromosome STR composite amplification system according to claim 1, it is characterised in that people
Class X chromosome STR's is grouped into：DXS6795, DXS6803, DXS6807, DXS9907, DXS7423 are first group；
Amelogenin, GATA172D05, DXS101, DXS9902, DXS7133, DXS6810 are second group；GATA31E08、
DXS6800, DXS981, DXS10162, DXS6809 are the 3rd group；GATA165B12, DXS10079, DXS10135, HPRTB are
4th group.
5. mankind's X chromosome STR composite amplification system according to claim 1 or 4, it is characterised in that
5 ' ends of one primer of every a pair of specific primers centering mark with fluorescein, and described fluorescein mark mode is：
DXS6795, DXS6803, DXS6807, DXS9907, DXS7423 are marked using FAM；Amelogenin、GATA172D05、
DXS101, DXS9902, DXS7133, DXS6810 are marked using HEX；GATA31E08、DXS6800、DXS981、DXS10162、
DXS6809 is marked using TAMRA；GATA165B12, DXS10079, DXS10135, HPRTB are marked using ROX.
6. mankind's X chromosome STR composite amplification system according to claim 1, it is characterised in that：Expand
Increasing system includes primer mixture, enzyme reaction buffer solution, genomic DNA, and described enzyme reaction buffer solution is gathered including thermal starting DNA
Synthase, Brij58, dNTP, DMSO, Betaine, Tris-HCl, BSA, DTT, Conservation environment of the enzyme reaction buffer solution at -20 DEG C
In do not freeze.
7. mankind's X chromosome STR composite amplification system according to claim 6, it is characterised in that described
The formula of enzyme reaction buffer solution be：KCl 0.1M；MgCl2 0.0015mM；Tris-HCl 0.05M；BSA 0.16mg/ml；
dNTP 0.2mM；Brij58 0.001M；DTT 0.02M；Betaine 0.1mol/L；DMSO 0.40%, percentage are DMSO former
Liquid accounts for the percentage by volume of enzyme reaction buffer solution；Thermal starting archaeal dna polymerase 0.25U.
8. mankind's X chromosome STR composite amplification system according to claim 1, it is characterised in that：Institute
The amplification program for the composite amplification system stated is：The first step is denatured：96 DEG C, 2 minutes；Thermal cycle：94 DEG C 30 seconds；60 DEG C, 70
Second, 27-30 circulation is carried out altogether；60 DEG C, 30 minutes of extension eventually；Insulation：15℃.
9. mankind's X chromosome STR composite amplification system according to claim 1 is in X chromosome correlation
Application in paternity identification, individual identification.
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| CN105969905B (en) * | 2016-07-27 | 2018-10-16 | 中国科学院北京基因组研究所 | The detection kit of the 38 STR allele based on high-flux sequence |
| CN106222258B (en) * | 2016-07-27 | 2018-10-16 | 中国科学院北京基因组研究所 | The detection kit of the 35 STR allele based on high-flux sequence |
| CN105969906B (en) * | 2016-07-27 | 2018-06-22 | 北京智鉴源技术有限公司 | The detection kit of the 30 STR allele based on high-flux sequence |
| CN106086210B (en) * | 2016-08-03 | 2018-08-31 | 中国科学院北京基因组研究所 | The detection kit of the 40 STR allele based on high-flux sequence |
| CN106048053B (en) * | 2016-08-03 | 2018-08-31 | 中国科学院北京基因组研究所 | The detection kit of the 38 STR allele based on high-flux sequence |
| CN107012225B (en) * | 2017-04-20 | 2020-10-09 | 司法鉴定科学研究院 | STR locus detection kit and detection method based on high-throughput sequencing |
| CN108060233B (en) * | 2017-12-13 | 2021-04-30 | 苏州阅微基因技术有限公司 | Fluorescence multiplex amplification system and kit for 30Y chromosome STR loci and application |
| CN108384842B (en) * | 2018-02-27 | 2021-04-27 | 宁波海尔施基因科技有限公司 | Composite PCR amplification method for identifying species and human individual of unknown biological sample suspected to be human |
| CN108179199B (en) * | 2018-02-28 | 2020-09-22 | 公安部物证鉴定中心 | Kit for detecting X-STR locus based on second-generation sequencing technology and special primer combination thereof |
| CN108676856B (en) * | 2018-04-17 | 2022-07-22 | 深圳华大法医科技有限公司 | Human X chromosome STR locus detection system, DNA sample analysis method and primer pair |
| CN110863056A (en) * | 2018-08-27 | 2020-03-06 | 深圳华大法医科技有限公司 | Method, reagent and application for accurately typing human DNA |
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