CN105177125A - Human X-chromosome composite amplification system composed of 20 short serially-connected repetitive sequences and applications thereof - Google Patents
Human X-chromosome composite amplification system composed of 20 short serially-connected repetitive sequences and applications thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and relates to a human X-chromosome composite amplification system composed of 20 short serially-connected repetitive sequences and applications thereof. The composite amplification system comprises 20 pairs of primers and is capable of amplifying 19 STR sites: DXS6795, DXS6803, DXS6807, DXS9907, DXS7423, GATA172D05, DXS101, DXS9902, DXS7133, DXS9810, GATA31E08, DXS6800, DXS981, DXS10162, DXS6809, GATA165B12, DXS10079, DXS10135, and HPRTB, and one sex recognition site: Amelogenin. Five-color fluorescence labeling is adopted by the system, the size of amplification products is in a range of 85 to 450 bp, the operation is simple, and the individual recognition performance is high. The sites provided by the provided composite amplification system are highly compatible to the conventional commonly-used X-STR kits, moreover, sites with high polymorphism information are provided, and the provided composite amplification system can be applied to paternity identification related with X-chromosome, individual recognition, and auxiliary detection of X-chromosome sex-linked hereditary diseases.
Description
Technical field
The present invention relates to biological technical field, relate to mankind's X chromosome 20 STR composite amplification systems, be specifically related to utilize genetic marker X chromosome with polymorphism, the fluorescence detection reagent kit being carried out composite amplification by polymerase chain reaction, this system can be applied to the auxiliary detection of the daily paternity identification relevant to X chromosome, individual recognition and X chromosome sex-linked hereditary disease.
Background technology
Human genome STR (STR) is with a few base for core unit, the DNA genetic marker that tandem sequence repeats is formed.Not agnate, the differentiation of different crowd even between Different Individual is the difference by core unit sequence and repeat number, and this also constitutes the genetic polymorphism of STR.In genome, on average every 15 ~ 20kb just has a STR site, accounts for genomic 10%, be present in non-coding region and intron, repeating unit is 2 ~ 6bp more, multiplicity 10 ~ 60 times, clip size at 70 ~ 500bp, and is codominant inheritance by Mendelian's rule.Therefore STR composite amplification detection technique can be widely used in the fields such as legal medical expert's individual recognition, paternity test.
In 23 pairs of karyomit(e)s of the mankind, be wherein X chromosome and Y chromosome for sex chromosome a pair.Can recombinate as autosome in the process that a pair X chromosome of women generates at ovum, but in the process generated at the spermatid of the male sex, X chromosome and Y chromosome can not arbitrarily be recombinated.The X chromosome of father only entails daughter, and the X chromosome of mother can entail daughter, also can entail son.So the mode of inheritance of this uniqueness of X chromosome makes it have special using value in paternity identification.The such as sister of parents' disappearance finds relatives, the qualification of half-sister's relation, grandmother-granddaughter's relation qualification, the qualification of incest relation, the qualification of rape offender and the qualification etc. of the genetic diseases relevant to X chromosome.
Compared with the quantity of present euchromosome STR test kit on the market, the quantity of X-STR test kit only has a few family available, and the patent numbers that X chromosome is relevant is in addition also fewer.
The existing X-STR test kit patent relevant with X chromosome its for STR site comparatively small amt, and certain shortcoming is had in the accuracy of genetic polymorphism, individual recognition power and detection, be therefore necessary to develop the X-STR test kit that a kind of site quantity is many, genetic polymorphism is high, individual recognition power is strong.
Summary of the invention
In order to overcome above-mentioned defect, the object of the present invention is to provide a kind of mankind's X chromosome 20 STR composite amplification systems that once can detect 19 X-STR locus and Amelogenin locus, be applicable to use autosome STR test kit to be difficult to the detection of excluding paternity relation case, and fluorescence labeling composite amplification system of the present invention detection fast, accurately.
Another object of the present invention is to provide the application in paternity identification that mankind's X chromosome STR composite amplification system is correlated with at X chromosome, individual recognition.
In order to realize foregoing invention object, the technical solution used in the present invention: mankind's X chromosome STR composite amplification system, it is characterized in that, this composite amplification system comprises 19 pairs of primers, 19 STR sites of can simultaneously increasing: DXS6795, DXS6803, DXS6807, DXS9907, DXS7423, GATA172D05, DXS101, DXS9902, DXS7133, DXS6810, GATA31E08, DXS6800, DXS981, DXS10162, DXS6809, GATA165B12, DXS10079, DXS10135, HPRTB;
The upstream primer sequence of described amplification DXS6795 is as shown in SEQIDNO1;
The downstream primer sequence of described amplification DXS6795 is as shown in SEQIDNO2;
The upstream primer sequence of described amplification DXS6803 is as shown in SEQIDNO3;
The downstream primer sequence of described amplification DXS6803 is as shown in SEQIDNO4;
The upstream primer sequence of described amplification DXS6807 is as shown in SEQIDNO5;
The downstream primer sequence of described amplification DXS6807 is as shown in SEQIDNO6;
The upstream primer sequence of described amplification DXS9907 is as shown in SEQIDNO7;
The downstream primer sequence of described amplification DXS9907 is as shown in SEQIDNO8;
The upstream primer sequence of described amplification DXS7423 is as shown in SEQIDNO9;
The downstream primer sequence of described amplification DXS7423 is as shown in SEQIDNO10;
The upstream primer sequence of described amplification GATA172D05 is as shown in SEQIDNO13;
The downstream primer sequence of described amplification GATA172D05 is as shown in SEQIDNO14;
The upstream primer sequence of described amplification DXS101 is as shown in SEQIDNO15;
The downstream primer sequence of described amplification DXS101 is as shown in SEQIDNO16;
The upstream primer sequence of described amplification DXS9902 is as shown in SEQIDNO17;
The downstream primer sequence of described amplification DXS9902 is as shown in SEQIDNO18;
The upstream primer sequence of described amplification DXS7133 is as shown in SEQIDNO19;
The downstream primer sequence of described amplification DXS7133 is as shown in SEQIDNO20;
The upstream primer sequence of described amplification DXS6810 is as shown in SEQIDNO21;
The downstream primer sequence of described amplification DXS6810 is as shown in SEQIDNO22;
The upstream primer sequence of described amplification GATA31E08 is as shown in SEQIDNO23;
The downstream primer sequence of described amplification GATA31E08 is as shown in SEQIDNO24;
The upstream primer sequence of described amplification DXS6800 is as shown in SEQIDNO25;
The downstream primer sequence of described amplification DXS6800 is as shown in SEQIDNO26;
The upstream primer sequence of described amplification DXS981 is as shown in SEQIDNO27;
The downstream primer sequence of described amplification DXS981 is as shown in SEQIDNO28;
The upstream primer sequence of described amplification DXS10162 is as shown in SEQIDNO29;
The downstream primer sequence of described amplification DXS10162 is as shown in SEQIDNO30;
The upstream primer sequence of described amplification DXS6809 is as shown in SEQIDNO31;
The downstream primer sequence of described amplification DXS6809 is as shown in SEQIDNO32;
The upstream primer sequence of described amplification GATA165B12 is as shown in SEQIDNO33;
The downstream primer sequence of described amplification GATA165B12 is as shown in SEQIDNO34;
The upstream primer sequence of described amplification DXS10079 is as shown in SEQIDNO35;
The downstream primer sequence of described amplification DXS10079 is as shown in SEQIDNO36;
The upstream primer sequence of described amplification DXS10135 is as shown in SEQIDNO37;
The downstream primer sequence of described amplification DXS10135 is as shown in SEQIDNO38;
The upstream primer sequence of described amplification HPRTB is as shown in SEQIDNO39;
The downstream primer sequence of described amplification HPRTB is as shown in SEQIDNO40.
Further, mankind's X chromosome STR composite amplification system also comprises the pair of primers of the Amelogenin that can increase for a pair, is respectively:
The upstream primer sequence of amplification Amelogenin is as shown in SEQIDNO11;
The downstream primer sequence of amplification Amelogenin is as shown in SEQIDNO12.
Aforesaid mankind's X chromosome STR composite amplification system, its primer concentration ratio is: DXS6795SEQIDNO1-2,0.179 μm of ol/L, DXS6803SEQIDNO3-4,0.126 μm of ol/L, DXS6807SE-QIDNO5-6,0.245 μm of ol/L, DXS9907SEQIDNO7-8,0.150 μm of ol/L, DXS7423SEQIDNO9-10,0.162 μm of ol/L, AmelogeninSEQIDNO11-12,0.060 μm of ol/L, GATA172D05SEQIDNO13-14,0.192 μm of ol/L, DXS101SEQIDNO15-16,0.190 μm of ol/L, DXS9902SEQIDNO17-18,0.169 μm of ol/L, DXS7133SEQIDNO19-20,0.404 μm of ol/L, DXS6810SEQIDNO21-22,0.412 μm of ol/L, GATA31E08SEQIDNO23-24,0.188 μm of ol/L, DXS6800SEQIDNO25-26,0.278 μm of ol/L, DXS981SEQIDNO27-28,0.130 μm of ol/L, DXS10162SEQIDNO29-30,0.250 μm of ol/L, DXS6809SEQIDNO31-32,0.420 μm of ol/L, GATA165B12SEQIDNO33-34,0.255 μm of ol/L, DXS10079SEQIDNO35-36,0.276 μm of ol/L, DXS10135SEQIDNO37-38,0.315 μm of ol/L, HPRTBSEQIDNO39-40,0.404 μm of ol/L.
Mankind's X chromosome STR 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 the 4th group.
5 ' end band of a primer of every a pair Auele Specific Primer centering has fluorescein-labelled, and described fluorescein-labelled mode is: DXS6795, DXS6803, DXS6807, DXS9907, DXS7423 adopt FAM mark; Amelogenin, GATA172D05, DXS101, DXS9902, DXS7133, DXS6810 adopt HEX mark; GATA31E08, DXS6800, DXS981, DXS10162, DXS6809 adopt TAMRA mark; GATA165B12, DXS10079, DXS10135, HPRTB adopt ROX mark.The interior mark that this composite amplification system detects adopts fluorescent orange element marker Orange mark.
Amplification system comprises primer mixture, enzyme reaction buffer solution, genomic dna, described enzyme reaction buffer solution comprises warm start archaeal dna polymerase, Brij58, dNTP, DMSO, Tris-HCl, BSA, DTT, and enzyme reaction buffer solution is not frozen in the Conservation environment of-20 DEG C.Be that 25 μ l calculate with amplification system, primer mixture is (5*Primermix) 5 μ l, enzyme reaction buffer solution (2.5*BufferD) 10 μ l, genomic dna (extracting template DNA, DS sample etc.) 1-3 μ l.Involved in the present invention to DNA sample can derive from the blood, blood stain, seminal fluid, seminal stain, saliva, body fluid, hair, muscle, tissue, nail etc. of people.Described DNA sample available reagent box method, phenol chloroform extraction method, Chelex-100 method, paramagnetic particle method carry out DNA extraction process.
The formula of described enzyme reaction buffer solution is: KCl0.1M; MgCl20.0015mM; Tris-HCl0.05M; BSA0.16mg/ml; DNTP0.2mM; Brij580.001M; DTT0.02M; Betaine0.1mol/L; DMSO0.40%, per-cent is the percent by volume that DMSO stoste accounts for enzyme reaction buffer solution, when namely configuring 100ml enzyme reaction buffer solution, adds 0.4mlDMSO stoste; Warm start archaeal dna polymerase 0.25U.
The amplification program of described composite amplification system is: the first step sex change: 96 DEG C, 2 minutes; Thermal cycling: 94 DEG C 30 seconds; 60 DEG C, 70 seconds, carry out 27-30 circulation altogether; Extend 60 DEG C eventually, 30 minutes; Insulation: 15 DEG C.Amplified production of the present invention through capillary electrophoresis, thus need carry out fragment analysis.
The genetic data statistics of 19 X-STR of the present invention is shown in result (table 1).
Table 1: the genetic data cartogram of 19 X-STR of the present invention
Wherein Marker: locus site, Cytogeneticlocalisation: karyomit(e) Distance positioning, Physicallocalisation [Mb]: physical distance is located, the abbreviation of PIC:Polymorphisminformationcontent, polymorphism information content, the abbreviation of HET:Heterozygotie, heterozygosity, be used for weighing gene polynorphisms, the abbreviation of MEC:Meanpaternityexclusionchange, average parentage exclusion probability, the abbreviation of PD:PowerofDiscrimination, individual recognition capability.
The present invention compared with prior art, have the following advantages: 19 X chromosome STR sites involved in the present invention are more than existing commercially most commercial kit site quantity, public security can be better met, high request that juridical authorities is detected for X-STR.According to the statistics to Chinese Han Population 1248 samples, visible: the site selected by the present invention has more much higher state property information, individual recognition rate is higher.Genomic dna obtains by extracting the mode such as template DNA, DS sample, template wide accommodation.The specificity of primer is higher, generates and signal stabilization without nonspecific products.
Accompanying drawing explanation
Fig. 1 is the concentration comparative result of Brij58 in the optimization experiment of enzyme reaction buffer solution (2.5*BufferD);
Fig. 2 is the concentration comparative result of DMSO in the optimization experiment of enzyme reaction buffer solution (2.5*BufferD);
The optimization component expanding effect figure of Fig. 3 .1 and Fig. 3 .2 given by the present invention;
Fig. 4 .1 and Fig. 4 .2 is the test experience result of species specificity involved in the present invention;
Fig. 5 .1 and Fig. 5 .2 is the expanding effect collection of illustrative plates of several sample;
Fig. 6 .1 and Fig. 6 .2 is the AFLP system of XX sex-reversal syndrome case;
Fig. 7 .1,7.2,7.3 and 7.4 is half-sister's parental right relation qualification AFLP system.
Embodiment
Below in conjunction with Figure of description and embodiment, technical scheme of the present invention is described in further details.
The composite amplification system of the Patent Publication that the locus dot information, the X chromosome that compare now common on the market X-STR test kit is correlated with and test kit of the present invention are enumerated in lower list 2.
Table 2 existing X-STR composite amplification system and amplification system of the present invention contrast
Although at present the office such as police and judicial is very strong for the demand of X-STR test kit, the test kit related in above table or composite amplification system or because price or because cannot commercialization use in the public security of reality, the administration of justice and each laboratory, institutes generally rate be not very high.So developing an X-STR test kit that site quantity is many, genetic polymorphism is high, individual recognition power is strong is have very bright and clear market outlook.
20 the STR composite amplification system designs of embodiment 1 human chromosomal
1. design of primers
Described primer adopts the software designs such as PrimePremier5 and NCBIBlast to form, should guarantee during design primer that the Tm value of each primer is in the scope of (60 ± 2) DEG C, amplification efficiency is close and guarantee that the amplified production of each pair of primer varies in size and is enough to distinguish in capillary electrophoresis.After having designed, with the interaction between software analysis primer dimer and different primers such as AutoDimer, if having interaction can produce nonspecific products or dimeric needs redesigns, until obtain satisfactory primer sequence.
Select people's source DNA template, single amplification is carried out respectively with above-mentioned 20 pairs of primers, amplified production is placed in the agarose gel electrophoresis of 1.5%, PCR system and amplification condition is adjusted to obtain the common amplification condition of 20 pairs of primers according to electrophoresis result, the effect of final expectation is: under same system and amplification condition, and all primer pairs all can occur bright and more single object band.If there is primer to can not meet above-mentioned condition, then redesign primer.
2. fluoroscopic marker system is set up
Have selected FAM, HEX, TAMRX, ROX and set up four look fluoroscopic marker system, be divided into four groups to carry out fluorescent mark designed 20 pairs of primers.Often group adopts a kind of fluorescent mark, is FAM, HEX, TAMRX, ROX respectively.After obtaining fluorescent dye primer, the non-fluorescence combination of primers matched with it, then carry out single amplification respectively, amplified production is placed on 3500 genetic analyzers and carries out capillary electrophoresis, assess the amplification efficiency of often pair of primer according to the result of capillary electrophoresis detection.Thereafter, by same fluorescently-labeled 4 to or 5 to or the mixing of the 6 pairs of primers be placed in same pipe and increase, amplified production is placed on 3500 genetic analyzers and carries out capillary electrophoresis, determine the amplification efficiency of often pair of primer according to the result that capillary electrophoresis detects, and judge this 4 couple or 5 to or 6 pairs of primers mixing amplifications whether cause non-specific.Finally, capillary electrophoresis result according to single amplification and combination amplification tentatively determines the add-on of each primer pair, 20 pairs of primer mixing are placed in same pipe increase, adjust respective concentration according to the electrophoresis result of composite amplification again, make that the amplification efficiency of each primer pair (reacting on the peak height of electrophoresis result) is basically identical to be as the criterion.The 20 pairs of primer sequences finally determined and concentration ratio (primer concentration value is the primer concentration in PCR amplification system) are in table 3.For above-mentioned 20 gene locuss design Auele Specific Primer, the length range of its amplified production is between 85-450bp.Detected components Plays thing of the present invention adopts the orange substance markers of Orange, can separate the size detecting each locus site of sample in mark zone clearly.
Table 320 pair primer sequence and concentration ratio table
Involved in the present invention to DNA sample can derive from the blood, blood stain, seminal fluid, seminal stain, saliva, body fluid, hair, muscle, tissue, nail etc. of people.Described DNA sample available reagent box method, phenol chloroform extraction method, Chelex-100 method, paramagnetic particle method carry out DNA extraction process.
Amplified production of the present invention can detect with the genetic analyzer of ABI series.Detected result can be analyzed in the data analysis software such as Genemapper, obtains corresponding STR somatotype collection of illustrative plates and data.
Embodiment 2 is to the genotype tests of some samples
1. the collection (blood sample is donated by volunteer) of blood sample
2.DNA extracts
Chelex-100 method is adopted to extract genomic dna (with reference to " ForensicDNAProtocol " .HumanaPress, 1998) anticoagulated whole blood of 0.5-5 μ l or the blood cake of (1-3mm) * (2-5mm) is got as in 500 μ l centrifuge tubes, vibration mixing Chelex solution makes Chelex fully suspend, often pipe adds 195 μ lChelex-100 (5%) solution and 5 μ l Proteinase Ks (20mg/ml) vibration mixing, 56 degrees Celsius of insulations two hours or after spending the night, take out vibration 2 minutes, 13000rpm is heated after 10 minutes centrifugal 5 minutes in boiling water, carefully pipette in 150 μ l supernatants to new centrifuge tube.
3. reaction system
PCR reaction mixture is made in the following manner by after each reaction reagent (damping fluid, primer mixture, genomic dna etc.) vibration mixing, amplification system cumulative volume is 25 μ l, comprise primer mixture (5*Primermix) 5 μ l, wherein primer concentration is shown in embodiment 1, enzyme reaction buffer solution (2.5*BufferD) 10 μ l, genomic dna 2 μ l (extract template DNA, DS sample etc.), ddH
2o8 μ l.
The enzyme reaction buffer solution (2.5*BufferD) used in composite amplification system involved in the present invention has special formula (table 4), a kind of warm start enzyme (MRTaq II) and PCR reaction buffer premixed liquid, and it can not freeze in the Conservation environment of-20 DEG C, there is antifreeze function.
| Composition | Unit | Concentration in damping fluid |
| KCl | M | 0.1 |
| MgCl 2 | M | 0.0015 |
| Tris-HCl | M | 0.05 |
| BSA (bovine serum albumin) | mg/ml | 0.16 |
| dNTP | mM | 0.2 |
| Brij58 (Soxylat A 25-7) | mM | 0.001 |
| DTT (dithiothreitol (DTT)) | M | 0.02 |
| Betaine (trimethyl-glycine) | M | 0.1 |
| DMSO (dimethyl sulfoxide (DMSO)) | ％ | 0.40％ |
| MR TaqⅡ | U | 0.25 |
Table 4: enzyme reaction buffer solution (2.5*BufferD) is filled a prescription
4.PCR response procedures
The pcr amplification program (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 detects
By the 2.5:100 mixing in proportion of mark and methane amide in Orange500, get 12.5 μ l mixtures and join 96 orifice plates, add amplified production sample or allelotrope standard substance 1 μ l again, mixing leaves standstill several minutes, 95 DEG C of sex change 3min, ice bath 3min immediately, puts on ABI3500 sequenator after centrifugal, prepare to detect.
6. data analysis
Import raw data, at the File menu setecting Addsampletoproject of homepage, find sample file, filesselected presss from both sides, and clicks addtolist, and click add, namely sample file is presented at Project window; Selection analysis parameter.Definition analysismethod, panel, sizestandard.Browse the raw data of sample electrophoresis, choose the filename of certain sample, under " sample " menu, select " Rawdata ".Moving tracing line, makes cursor be parked on the right side of primer peak (first orange interior mark peak before), and the numerical value shown in now window lower left corner X-axis is as the starting point in analysismethod analytical parameters; Click green analysis button, occur saveproject dialog box, preserve after name, namely software start processing data, analyzed rear lower left corner display analysiscompleted.Adopt GeneMapper
rthe data that ID-X software analysis obtains also generate collection of illustrative plates.
Embodiment 3 is for the optimization experiment of enzyme reaction buffer solution (2.5*BufferD)
KCl in the concentration gradient of enzyme reaction buffer solution is: 0.075M, 0.1M, 0.125M tri-concentration gradients.MgCl
2in the concentration gradient of enzyme reaction buffer solution be: 1mM, 1.5mM, 2mM tri-concentration gradients.Brij58 in the concentration gradient of enzyme reaction buffer solution is: 0.001mM, 0.0015mM, 0.002mM tri-concentration gradients.DTT in the concentration gradient of enzyme reaction buffer solution is: 0.015M, 0.02M, 0.025M tri-concentration gradients.Betaine in the concentration gradient of enzyme reaction buffer solution is: 0.05M, 0.1M, 0.15M tri-concentration gradients.DMSO in the concentration gradient of enzyme reaction buffer solution is: 0.3%, 0.4%, 0.5% 3 concentration gradient.MRTaq II in the concentration gradient of enzyme reaction buffer solution is: 0.15U, 0.25U, 0.35U tri-concentration gradients.Orthogonal experiment for the design of components different concns of above-mentioned enzyme reaction buffer solution carries out composite amplification, the optimum combination of experimental result determination composite amplification system.Accompanying drawing 1 lists the concentration comparative result of wherein Brij58.Accompanying drawing 2 lists the concentration comparative result of wherein DMSO.Accompanying drawing 3.1 and 3.2 lists optimization component (component ratio namely in embodiment 2) the expanding effect figure given by the present invention.Institute's drawings attached of accompanying drawing 1-7 is GeneMapper
rthe data collection of illustrative plates that ID-X software analysis obtains.
Embodiment 4: for the test experience of species specificity, have chosen the DNA profiling (phenol chloroform) of different plant species, sees whether composite amplification system of the present invention has the advantage of species specificity.Have chosen pig, ox, sheep, mouse, chicken, duck, fish and human genome DNA as detected object, the composite amplification system that result the present invention relates to can amplify the X-STR somatotype of human genome DNA efficiently, and other species all do not have amplified production.Fig. 4 .1 and 4.2 lists the test experience result of species specificity involved in the present invention.
Embodiment 5: utilize composite amplification system of the present invention to detect different sample.Involved in the present invention to DNA sample can derive from the blood, blood stain, seminal fluid, seminal stain, saliva, body fluid, hair, muscle, tissue, nail etc. of people.Described DNA sample available reagent box method, phenol chloroform extraction method, Chelex-100 method, paramagnetic particle method carry out DNA extraction process.Accompanying drawing 5.1 and 5.2 lists the expanding effect collection of illustrative plates of wherein several sample.Proof utilizes various sample all can realize effectively amplification, detects quick and precisely, applied widely.
Embodiment 6: utilize composite amplification system of the present invention to identify XX sex-reversal syndrome case together.
This sample playing case is the blood filter paper sample of a pair father and son, and the secondal sexual character of son shows as the male sex, but comes to nothing when utilizing the micro-deleted test kit of Y to detect.Find that the somatotype in the Amel site of the son of this case is XX when carrying out the detection of X-STR site, genotype is women, on X chromosome, there are 2 allelotrope (table 6) in some STR site, and in this case, the allelotrope of son and the allelotrope of father can matched.Its father is that normal male is individual, has 1 X chromosome and 1 Y chromosome.So son inherits an X chromosome from its father in this case, it is a typical sex-reversal syndrome case (Fig. 6 .1 and 6.2).
The X-STR somatotype of table 6:XX sex-reversal syndrome case father and son
| 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.
Have 2 couples of mother and daughters in present case, advocate that the daughter in these two couples of mother and daughters is half-blooded sister, the same father of its hypothesis is not alive, therefore only to use X-STR site to detect.Detected result (table 7 and Fig. 7 .1-7.4) can determine that father's gene type of the daughter in these two couples of mother and daughters is identical substantially, so not getting rid of is half-blooded sisterhood.
Table 7: half-sister's parental right relation identified gene somatotype
Above-described embodiment is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention; some improvement and equivalent replacement can also be made; these are weighed the present invention and require to improve and be equal to the technical scheme after replacing, and all fall into protection scope of the present invention.
Claims (9)
1. mankind's X chromosome STR composite amplification system, it is characterized in that, this composite amplification system comprises 19 pairs of primers, 19 STR sites of can simultaneously increasing: DXS6795, DXS6803, DXS6807, DXS9907, DXS7423, GATA172D05, DXS101, DXS9902, DXS7133, DXS6810, GATA31E08, DXS6800, DXS981, DXS10162, DXS6809, GATA165B12, DXS10079, DXS10135, HPRTB;
The upstream primer sequence of described amplification DXS6795 is as shown in SEQIDNO1;
The downstream primer sequence of described amplification DXS6795 is as shown in SEQIDNO2;
The upstream primer sequence of described amplification DXS6803 is as shown in SEQIDNO3;
The downstream primer sequence of described amplification DXS6803 is as shown in SEQIDNO4;
The upstream primer sequence of described amplification DXS6807 is as shown in SEQIDNO5;
The downstream primer sequence of described amplification DXS6807 is as shown in SEQIDNO6;
The upstream primer sequence of described amplification DXS9907 is as shown in SEQIDNO7;
The downstream primer sequence of described amplification DXS9907 is as shown in SEQIDNO8;
The upstream primer sequence of described amplification DXS7423 is as shown in SEQIDNO9;
The downstream primer sequence of described amplification DXS7423 is as shown in SEQIDNO10;
The upstream primer sequence of described amplification GATA172D05 is as shown in SEQIDNO13;
The downstream primer sequence of described amplification GATA172D05 is as shown in SEQIDNO14;
The upstream primer sequence of described amplification DXS101 is as shown in SEQIDNO15;
The downstream primer sequence of described amplification DXS101 is as shown in SEQIDNO16;
The upstream primer sequence of described amplification DXS9902 is as shown in SEQIDNO17;
The downstream primer sequence of described amplification DXS9902 is as shown in SEQIDNO18;
The upstream primer sequence of described amplification DXS7133 is as shown in SEQIDNO19;
The downstream primer sequence of described amplification DXS7133 is as shown in SEQIDNO20;
The upstream primer sequence of described amplification DXS6810 is as shown in SEQIDNO21;
The downstream primer sequence of described amplification DXS6810 is as shown in SEQIDNO22;
The upstream primer sequence of described amplification GATA31E08 is as shown in SEQIDNO23;
The downstream primer sequence of described amplification GATA31E08 is as shown in SEQIDNO24;
The upstream primer sequence of described amplification DXS6800 is as shown in SEQIDNO25;
The downstream primer sequence of described amplification DXS6800 is as shown in SEQIDNO26;
The upstream primer sequence of described amplification DXS981 is as shown in SEQIDNO27;
The downstream primer sequence of described amplification DXS981 is as shown in SEQIDNO28;
The upstream primer sequence of described amplification DXS10162 is as shown in SEQIDNO29;
The downstream primer sequence of described amplification DXS10162 is as shown in SEQIDNO30;
The upstream primer sequence of described amplification DXS6809 is as shown in SEQIDNO31;
The downstream primer sequence of described amplification DXS6809 is as shown in SEQIDNO32;
The upstream primer sequence of described amplification GATA165B12 is as shown in SEQIDNO33;
The downstream primer sequence of described amplification GATA165B12 is as shown in SEQIDNO34;
The upstream primer sequence of described amplification DXS10079 is as shown in SEQIDNO35;
The downstream primer sequence of described amplification DXS10079 is as shown in SEQIDNO36;
The upstream primer sequence of described amplification DXS10135 is as shown in SEQIDNO37;
The downstream primer sequence of described amplification DXS10135 is as shown in SEQIDNO38;
The upstream primer sequence of described amplification HPRTB is as shown in SEQIDNO39;
The downstream primer sequence of described amplification HPRTB is as shown in SEQIDNO40.
2. mankind's X chromosome STR composite amplification system according to claim 1, is characterized in that, also comprises the primer in Amelogenin site of increasing for a pair, is respectively:
The upstream primer sequence of amplification Amelogenin is as shown in SEQIDNO11;
The downstream primer sequence of amplification Amelogenin is as shown in SEQIDNO12.
3. mankind's X chromosome STR composite amplification system according to claim 2, its primer concentration ratio is: DXS6795SEQIDNO1-2,0.179 μm of ol/L, DXS6803SEQIDNO3-4,0.126 μm of ol/L, DXS6807SEQIDNO5-6,0.245 μm of ol/L, DXS9907SEQIDNO7-8,0.150 μm of ol/L, DXS7423SEQIDNO9-10,0.162 μm of ol/L, AmelogeninSEQIDNO11-12,0.060 μm of ol/L, GATA172D05SEQIDNO13-14,0.192 μm of ol/L, DXS101SEQIDNO15-16,0.190 μm of ol/L, DXS9902SEQIDNO17-18,0.169 μm of ol/L, DXS7133SEQIDNO19-20,0.404 μm of ol/L, DXS6810SEQIDNO21-22,0.412 μm of ol/L, GATA31E08SEQIDNO23-24,0.188 μm of ol/L, DXS6800SEQIDNO25-26,0.278 μm of ol/L, DXS981SEQIDNO27-28,0.130 μm of ol/L, DXS10162SEQIDNO29-30,0.250 μm of ol/L, DXS6809SEQIDNO31-32,0.420 μm of ol/L, GATA165B12SEQIDNO33-34,0.255 μm of ol/L, DXS10079SEQIDNO35-36,0.276 μm of ol/L, DXS10135SEQIDNO37-38,0.315 μm of ol/L, HPRTBSEQIDNO39-40,0.404 μm of ol/L.
4. mankind's X chromosome STR composite amplification system according to claim 1, it is characterized in that, mankind's X chromosome STR 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 the 4th group.
5. the mankind's X chromosome STR composite amplification system according to claim 1 or 4, it is characterized in that 5 ' end band of a primer of every a pair Auele Specific Primer centering has fluorescein-labelled, described fluorescein-labelled mode is: DXS6795, DXS6803, DXS6807, DXS9907, DXS7423 adopt FAM mark; Amelogenin, GATA172D05, DXS101, DXS9902, DXS7133, DXS6810 adopt HEX mark; GATA31E08, DXS6800, DXS981, DXS10162, DXS6809 adopt TAMRA mark; GATA165B12, DXS10079, DXS10135, HPRTB adopt ROX mark.
6. mankind's X chromosome STR composite amplification system according to claim 1, it is characterized in that: amplification system comprises primer mixture, enzyme reaction buffer solution, genomic dna, described enzyme reaction buffer solution comprises warm start archaeal dna polymerase, Brij58, dNTP, DMSO, Betaine, Tris-HCl, BSA, DTT, and enzyme reaction buffer solution is not frozen in the Conservation environment of-20 DEG C.
7. mankind's X chromosome STR composite amplification system according to claim 6, is characterized in that the formula of described enzyme reaction buffer solution is: KCl0.1M; MgCl20.0015mM; Tris-HCl0.05M; BSA0.16mg/ml; DNTP0.2mM; Brij580.001M; DTT0.02M; Betaine0.1mol/L; DMSO0.40%, per-cent is the percent by volume that DMSO stoste accounts for enzyme reaction buffer solution; Warm start archaeal dna polymerase 0.25U.
8. mankind's X chromosome STR composite amplification system according to claim 1, is characterized in that: the amplification program of described composite amplification system is: the first step sex change: 96 DEG C, 2 minutes; Thermal cycling: 94 DEG C 30 seconds; 60 DEG C, 70 seconds, carry out 27-30 circulation altogether; Extend 60 DEG C eventually, 30 minutes; Insulation: 15 DEG C.
9. mankind's X chromosome STR composite amplification system according to claim 1 be correlated with at X chromosome paternity identification, application in individual recognition.
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