CN104232751A - Method and system for acquiring autosomal STR gene locus typing result of single individual from mixed seminal stains - Google Patents
Method and system for acquiring autosomal STR gene locus typing result of single individual from mixed seminal stains Download PDFInfo
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Abstract
The invention provides a method and a system for acquiring an autosomal STR gene locus typing result of a single individual from mixed seminal stains. The method comprises the following steps of: acquiring different Y-STR gene loci; acquiring autosomal STR gene locus haplotype typing results of a sufficient amount of Y type spermoblast; merging the autosomal STR gene locus haplotype typing results of the Y type spermoblast, which have same typing results of the different Y-STR gene loci to obtain the autosomal STR gene locus typing result of the single individual from the mixed seminal stains. The invention also provides the system for acquiring the autosomal STR gene locus typing result of the single individual from the mixed seminal stains. The method and system which are provided by the invention can acquire the typing result of the single individual from the mixed seminal stains by comprehensively analyzing autosomal STR and Y-STR typing results, thereby providing a powerful technical support for a public security organ to fast determine a criminal suspect.
Description
Technical field
The present invention relates to a kind of deduction classifying method and system, particularly relate to a kind of method and system obtaining the euchromosome STR locus genotyping result of single individuality from mixing seminal stain.
Background technology
Invading the mixture of the biological evidence extracted criminal-scene masculinity and femininity body fluid often from property, is then the mixture (e.g., mixing seminal stain) of different male sex's body fluid in some cases.And the latter often criminal case scout and the key of detection, at present mainly by the analysis of DNA to different male donors in mixing seminal stain, realize getting rid of and determining suspect.
And now for the testing procedures of mixing seminal stain, such as differential lysis, can only get rid of women's sample, can not be separated the sperm DNA of different male donors, what inspection obtained is the mixing collection of illustrative plates of different sources people, and mixes collection of illustrative plates and often can only be used for getting rid of and can not carrying out identifications individuality.Follow-up needs uses computer software manually to split to collection of illustrative plates, its result depends on different computation models and statistical method, often different experimenters operates and can obtain different results, for in some criminal case more than two people, as three people and above biased sample, such fractionation will be more difficult, usually can not effectively distinguish the DNA of different male donors.
Existing research confirms that conbined usage detection wind lidar (LCM) and LV – PCR method can obtain the euchromosome STR somatotype of single sperm, but because spermatid is formed by reduction division, genetic material only containing half, euchromosome STR somatotype is carried out to spermatid DNA in mixing seminal stain, euchromosome STR Haplotyping A results different in a large number can only be obtained, be difficult to distinguish this Haplotyping A result for which male sex, therefore cannot carry out establishing identity and individual recognition.Y-STR somatotype conventional determines paternal sibship by Y chromosome locus somatotype, get rid of the classifying method of suspect, but the sperm Y-STR locus somatotype of same patroclinous family male individual is the same, therefore be only difficult to carry out establishing identity and individual recognition by Y-STR locus somatotype.
How by conjunction with euchromosome STR and Y-STR genotyping result, effectively obtain the genotyping result of single individuality in mixing seminal stain, and then determine that rapidly suspect provides strong technical support to become and has problem to be solved for public security organ.
Summary of the invention
The invention provides a kind of method obtaining the euchromosome STR locus genotyping result of single individuality from mixing seminal stain, by obtaining difference Y-STR locus, the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for described difference Y-STR locus genotyping result is merged, namely the euchromosome STR genotyping result mixing single individuality in seminal stain is obtained, the genotyping result of single individuality can be obtained from mixing seminal stain DNA according to this result, and then determine rapidly the technical support that suspect provides strong for public security organ.
Present invention also offers a kind of system obtaining the euchromosome STR locus genotyping result of single individuality from mixing seminal stain, by this system by conjunction with euchromosome STR and Y-STR genotyping result, realize the genotyping result obtaining single individual euchromosome STR locus from mixing seminal stain in DNA.
The invention provides a kind of method inferring DNA individuality source in mixing seminal stain, by obtaining difference Y-STR locus, the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for described difference Y-STR locus genotyping result is merged, obtain the euchromosome STR genotyping result mixing each individuality contained in seminal stain, carry out mixing effective deduction in DNA individuality source in seminal stain according to this result, for public security organ determines rapidly the technical support that suspect provides strong.
Present invention also offers a kind of system inferring DNA individuality source in mixing seminal stain, by this system by conjunction with euchromosome STR and Y-STR genotyping result, realize inferring DNA individuality source in mixing seminal stain.
A kind of method obtaining the euchromosome STR locus genotyping result of single individuality from mixing seminal stain provided by the invention, described mixing seminal stain is the mixing seminal stain from two or more individuality, and the method comprises:
1) obtain the mixing genotyping result of the Y-STR locus of mixing seminal stain DNA, determine genotyping result at least one Y-STR locus all discrepant between each individuality from this genotyping result, i.e. difference Y-STR locus;
2) from described mixing seminal stain, be separated the Y type spermatid of q.s, obtain the genotyping result of the described difference Y-STR locus of each Y type spermatid, euchromosome STR locus and sex determining gene seat Amelogenin respectively;
The genotyping result of wherein said euchromosome STR locus is the Haplotyping A result of this locus, and the Y type spermatid of described q.s refers at least 10 the Y type spermatids containing each individuality in this mixing seminal stain in the spermatid be separated;
3) respectively the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for described difference Y-STR locus genotyping result is merged, namely obtain the euchromosome STR genotyping result mixing single individuality in seminal stain.
In the solution of the present invention, described mixing seminal stain comes from the mixing seminal stain of two or more without the individuality of paternal genetic connection.
Further, described individuality is the individuality from Chinese population, and described Y-STR locus is DYS456, DYS390, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448.
Further, described euchromosome STR locus is the str locus seat that can carry out individual recognition, such as str locus seat D5S818, the combination of D21S11, D7S820, D3S1358, VWA, D8S1179, D16S539, D6S1043, D13S317 and D12S391.These locus are that the living environment, ethnic origin etc. that applicant passes through Chinese population is comprehensively analyzed, investigate the phenotypic characteristic difference of the national population in each department, comprise resemblance, physical signs etc., document and network data base investigation is carried out, the combination of the specific locus that the basis of existing research obtains for these differences.
In a specific embodiment of the present invention, the process obtaining described locus genotyping result comprise adopt with described locus one to one amplimer increase to obtain amplified production to it, and the genotypic step of described locus is obtained by this amplified production, wherein, the amplimer mixing the Y-STR locus of DNA in seminal stain is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.28 in sequence table; The amplimer of described difference Y-STR locus is the two or more pieces nucleotide sequence being selected from SEQ ID No.1 to SEQ ID No.28; Described euchromosome STR locus, and the amplimer of sex determining gene seat Amelogenin is the nucleotide sequence of SEQ ID No.29 to SEQ ID No.50 in sequence table.
In the solution of the present invention, the mixing genotyping result obtaining the Y-STR locus of mixing seminal stain DNA can adopt this area conventional means, such as, adopt differential lysis to obtain.Further, the Y type spermatid being separated q.s from described mixing seminal stain can adopt the single celled method of separated sperm from seminal fluid of this area routine, and such as detection wind lidar method, micro-suction spindle draw method or flow cytometric sorting etc.Further, respectively the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for described difference Y-STR locus genotyping result is carried out merging referring to and first Y type spermatid identical for difference Y-STR locus genotyping result is classified as one group, then will be classified as the euchromosome STR locus haplotype genotyping result of Y type spermatid of a group comprehensively for euchromosome STR locus genotyping result, this euchromosome STR locus genotyping result is the euchromosome STR genotyping result of single individuality in mixing seminal stain.
Further, in the solution of the present invention, the Y-STR locus adopted is the Y-STR locus between the different male individuals of Chinese population with high degree of specificity through applicant's lot of experiment validation, from the genotyping result of these Y-STR locus, all discrepant described difference Y-STR locus of genotyping result between each individuality can be determined.And based on the primer sequence for these Y-STR locus provided in the application, described difference Y-STR locus can be obtained by conventional means for different male individual those skilled in the art, certain those skilled in the art also can be used according to practical situation the primer sequence that above-mentioned primer basis carries out improving or finely tuning and obtain and increase and somatotype to described Y-STR locus, thus obtain described difference Y-STR locus.
Further, the amplimer of described difference Y-STR locus is the two or more pieces nucleotide sequence being selected from SEQ ID No.1 to SEQ ID No.28, refer in the application's scheme, according to the difference of male individual in mixing seminal stain, one or more difference Y-STR locus can be obtained, according to the concrete difference Y-STR locus obtained, those skilled in the art can select primer sequence for this difference Y-STR locus to implement the scheme of the application from the primer sequence for Y-STR locus that the application provides, the means design that can certainly exist according to other this areas is for the primer of these locus, as long as the amplification of corresponding gene seat can be realized.In the process of scheme implementing the application, only can use a difference Y-STR locus, also can use 1-5 difference Y-STR locus, those skilled in the art can select as required.
Present invention also offers a kind of method obtaining the euchromosome STR locus genotyping result of single individuality from hybrid dna, described hybrid dna is from the mixture cell of two or more male individual, and the method comprises:
1) obtain the mixing genotyping result of the Y-STR locus of hybrid dna, determine genotyping result at least one Y-STR locus all discrepant between each individuality from this genotyping result, i.e. difference Y-STR locus;
2) from described mixture cell, be separated the somatocyte of q.s, obtain the genotyping result of each somatic described difference Y-STR locus, euchromosome STR locus and sex determining gene seat Amelogenin respectively;
The somatocyte of described q.s refers at least 10 individual cells containing each individuality in the cell be separated;
3) respectively somatic euchromosome STR locus haplotype genotyping result identical for described difference Y-STR locus genotyping result is combined, namely obtain the euchromosome STR genotyping result mixing single individuality in seminal stain.
A kind of system obtaining the euchromosome STR locus genotyping result of single individuality from mixing seminal stain provided by the invention, comprises and obtains difference Y-STR locus system, somatotype system, euchromosome STR somatotype system;
Described acquisition difference Y-STR locus system, for obtaining the mixing genotyping result of the Y-STR locus of mixing seminal stain DNA, determines genotyping result at least one Y-STR locus all discrepant between each individuality from this genotyping result, i.e. difference Y-STR locus;
Described somatotype system is used for the single Y type spermatid being separated q.s from described mixing seminal stain, obtain the genotyping result of the described difference Y-STR locus of each Y type spermatid, euchromosome STR locus and sex determining gene seat Amelogenin respectively, the genotyping result of wherein said euchromosome STR locus is the Haplotyping A result of this locus, and the single Y type spermatid of described q.s refers at least 10 the Y type spermatids containing each individuality in this mixing seminal stain in the spermatid be separated;
Described euchromosome STR somatotype system is used for being merged by the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for described difference Y-STR locus genotyping result respectively, namely obtains the euchromosome STR genotyping result mixing single individuality in seminal stain.
Further, obtain in the system of euchromosome STR locus genotyping result of single individuality described from mixing seminal stain, described individuality is the individuality from Chinese population, described Y-STR locus is DYS456, DYS390, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448.
Further, described euchromosome STR locus is the str locus seat that can carry out individual recognition, such as str locus seat D5S818, the combination of D21S11, D7S820, D3S1358, VWA, D8S1179, D16S539, D6S1043, D13S317 and D12S391.
A kind of method inferring DNA individuality source in mixing seminal stain provided by the invention, described mixing seminal stain is the mixing seminal stain from two or more individuality, and the method comprises:
1) obtain the mixing genotyping result of the Y-STR locus of above-mentioned mixing seminal stain DNA, from this genotyping result, determine genotyping result at least one Y-STR locus all discrepant between each individuality, i.e. difference Y-STR locus;
2) from described mixing seminal stain, be separated the Y type spermatid of q.s, obtain described difference Y-STR locus, the euchromosome STR locus of each Y type spermatid respectively, and the genotyping result of sex determining gene seat Amelogenin;
The genotyping result of wherein said euchromosome STR locus is the Haplotyping A result of this locus, the Y type spermatid of described q.s refers at least 10 the Y type spermatids containing each individuality in this mixing seminal stain in the spermatid be separated, and described euchromosome STR locus is the combination of the euchromosome STR locus that can carry out individual recognition;
3) respectively the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for difference Y-STR locus genotyping result is merged, obtain the euchromosome STR locus genotyping result mixing single individuality in seminal stain;
4) the individuality source of DNA in mixing seminal stain is inferred according to the genotyping result of the euchromosome STR locus of above-mentioned single individuality.
Further, described individuality is the individuality from Chinese population, and described Y-STR locus is DYS456, DYS390, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448; Described euchromosome STR locus is D5S818, D21S11, D7S820, D3S1358, VWA, D8S1179, D16S539, D6S1043, D13S317 and D12S391.
Further, in the solution of the present invention, the process obtaining described locus genotyping result comprise adopt with described locus one to one amplimer increase to obtain amplified production to it, and the genotypic step of described locus is obtained by this amplified production, wherein, the amplimer mixing the Y-STR locus of DNA in seminal stain is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.28 in sequence table; The amplimer of described difference Y-STR locus is the two or more pieces nucleotide sequence being selected from SEQ ID No.1 to SEQ ID No.28; Described euchromosome STR locus, and the amplimer of sex determining gene seat Amelogenin is the nucleotide sequence of SEQ ID No.29 to SEQ ID No.50 in sequence table.
In another embodiment of the present invention, can analyze by genetic analyzer the genotype that described amplified production obtains described locus.Further, described genetic analyzer can be the genetic analyzer that those skilled in the art's routine uses, such as ABI3130 or ABI3500 type genetic analyzer.Pass through
the genotype of locus described in this pcr amplification product analyzed by ID-X software or other GeneMapper software etc.
Further, described pcr amplification primer can for having the pcr amplification primer (as shown in table 2 and 4) of fluorescence.In embodiments of the present invention, by described genetic analyzer, the length that will be increased by the primer recorded in actual product length, coloured product and the table 2 and 4 that amplify, coloured product are compared, as long as the product length amplified actual is equal or close with 4 amplification lengths recorded with table 2, can deviation be there is in the two, as long as within the scope of those skilled in the art's acceptable, or be confirmed to be the fragment that will increase by order-checking.
A kind of system inferring DNA individuality source in mixing seminal stain provided by the invention, comprises and obtains difference Y-STR locus system, somatotype system, euchromosome STR somatotype system and deduction system,
Described acquisition difference Y-STR locus system is for obtaining the mixing genotyping result of the Y-STR locus of DNA in above-mentioned mixing seminal stain, genotyping result at least one Y-STR locus all discrepant between each individuality is determined, i.e. difference Y-STR locus from this genotyping result;
Described somatotype system is used for the Y type spermatid being separated q.s from described mixing seminal stain, obtain the described difference Y-STR locus of each Y type spermatid respectively, euchromosome STR locus, and the genotyping result of sex determining gene seat Amelogenin, the genotyping result of wherein said euchromosome STR locus is the Haplotyping A result of this locus, the Y type spermatid of described q.s refers at least 10 the Y type spermatids containing each individuality in this mixing seminal stain in the spermatid be separated, described euchromosome STR locus is the combination of the euchromosome STR locus that can carry out individual recognition,
Described euchromosome STR somatotype system is used for being merged by the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for difference Y-STR locus genotyping result respectively, obtains the euchromosome STR locus genotyping result mixing single individuality in seminal stain;
Described deduction system is used for the individuality source of inferring DNA in mixing seminal stain according to the genotyping result of the euchromosome STR locus of above-mentioned single individuality.
Further, in the system of the present invention, described individuality is the individuality from Chinese population, and described Y-STR locus is DYS456, DYS390, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448; Described euchromosome STR locus is D5S818, D21S11, D7S820, D3S1358, VWA, D8S1179, D16S539, D6S1043, D13S317 and D12S391.
Further, in the system of the present invention, the process obtaining described locus genotyping result comprise adopt with described locus one to one amplimer increase to obtain amplified production to it, and the genotypic step of described locus is obtained by this amplified production, wherein, the amplimer mixing the Y-STR locus of DNA in seminal stain is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.28 in sequence table; The amplimer of described difference Y-STR locus is the two or more pieces nucleotide sequence being selected from SEQ ID No.1 to SEQ ID No.28; Described euchromosome STR locus, and the amplimer of sex determining gene seat Amelogenin is the nucleotide sequence of SEQ ID No.29 to SEQ ID No.50 in sequence table.
In the solution of the present invention, the information of described 14 Y-STR locus is as shown in table 1 below:
Table 1
The primer sequence of described 14 Y-STR locus and correspondence thereof is as shown in table 2, and described primer can be the primer with mark, as shown in table 2:
Table 2
In the solution of the present invention, the information of described 10 euchromosome STR locus and 1 sex determination locus Amelogenin is as shown in table 3:
Table 3
Above-mentioned 10 autosomal locuses are that applicant filters out the composite amplification excellent in efficiency existed in Chinese population, 10 autosomal locuses that specificity is higher through great many of experiments and bioinformatic analysis.Sex determining gene seat Amelogenin is for sex identification, if male sex's result is XY, if women, result is XX.
Preferred amplimer sequence provided by the invention, by the online software design of such as Autoprimer.Amplimer and corresponding locus information as shown in table 4 below, PCRU represents upstream primer, and PCRL represents downstream primer;
Table 4
The present invention program has the following advantages:
1) method of the present invention can realize the deduction in the individuality source of DNA in multiple individuality mixing seminal stain.Do not need to use computer software to carry out subsequent disposal, genotyping result repeatability is high, accurately.
2) can pass through fluorescent mark, utilize conventional genetic analyzer, according to the molecular weight of the gene that will increase and the difference of fluorescence color, obtain detected result intuitively, and this result is through sequence verification, accuracy is 100%.
3) scheme provided by the invention effectively can determine the deduction in the individuality source of DNA multiple individuality mixing seminal stain from gene level, enormously simplify qualification detecting site body fluid or the tissue-derived step of its mottling, can for clear and definite case property, determine that suspect and conviction and sentence etc. provide scientific basis accurately.
Accompanying drawing explanation
The fluorescently-labeled Y of Fig. 1 and X-type sperm figure, show brilliant white, and X-specific probe and Xp11.1-q11.1 area hybridization shows light gray at the Yq12 area hybridization of Y-specific probe and Y chromosome.
Fig. 2 A shows the mixing somatotype collection of illustrative plates of the Y-STR locus obtained by existing method.Fig. 2 B, 2C and 2D are the monosperm Y-STR somatotype collection of illustrative plates of three volunteers using existing method to obtain.
Respective sperm YA-STR somatotype (namely comprising the somatotype of difference Y-STR locus, euchromosome STR locus and the sex determining gene seat Amelogenin) collection of illustrative plates of three volunteers that Fig. 3 A, 3B and 3C use the present invention program to obtain from the mixing seminal stain of three volunteers.
Embodiment
The sample used in the embodiment of the present invention 1 collects semen sample under three healthy volunteer's informed consents.This research is ratified by the Ethics Committee of Chinese Material Evidence Identification Center, Ministry of Public Security, totally 4 parts:
Method therefor is ordinary method if no special instructions, agents useful for same consumptive material and instrument as shown in the table:
Embodiment 1, verify and of the present inventionly a kind ofly from mixing seminal stain, obtain the accuracy of the method and system of the euchromosome STR locus genotyping result of single individuality
A kind of system inferring the individuality source of DNA in mixing seminal stain of the present invention, comprises and obtains difference Y-STR locus system, somatotype system, euchromosome STR somatotype system.
Get 2ml seminal fluid respectively to three volunteers to drip respectively on filter paper, be made as single seminal stain sample, totally 3 parts; Each volunteer is dripped on filter paper after getting the mixing of 1mL seminal fluid respectively, and be made as mixing seminal stain sample, totally 1 part, sample at room temperature dries for subsequent use.
One, utilize system and method for the present invention, from above-mentioned 1 part of mixing seminal stain sample, obtain the euchromosome STR locus genotyping result of single individuality, process is as follows:
1, the acquisition difference Y-STR locus system in described system is utilized to obtain the mixing genotyping result of the Y-STR locus of mixing seminal stain sample DNA, genotyping result at least one Y-STR locus all discrepant between each individuality is determined from this genotyping result, i.e. difference Y-STR locus, comprising:
Utilize prior art such as differential lysis in conjunction with genetic analyzer, such as ABI3130/3500XL obtains the mixing genotyping result of the Y-STR locus of mixing seminal stain DNA, as shown in Figure 2 A, from this result, determine all discrepant DYS392 locus of genotyping result between three individualities, it can be used as difference Y-STR locus; Also obtain the monosperm Y-STR genotyping result of three volunteers as a reference by the unicellular separation method of inspection in conjunction with genetic analyzer, as shown in Fig. 2 B-Fig. 2 D simultaneously.
2, utilize the somatotype system in described system from described mixing seminal stain, be separated the Y type spermatid of q.s, then the genotyping result of the described difference Y-STR locus of each Y type spermatid, euchromosome STR locus and sex determining gene seat Amelogenin is obtained respectively
The genotyping result of wherein said euchromosome STR locus is the Haplotyping A result of this locus, and the Y type spermatid of described q.s refers at least 10 the Y type spermatids containing each individuality in this mixing seminal stain in the spermatid be separated;
The Y type spermatid of 2.1 separation q.s, comprises the following steps
1) the appropriate seminal stain sample of clip, be resuspended in 60 μ LCarnoyShi stationary liquids (methyl alcohol: acetic acid is 3:1), draw 20 μ L cell suspensions and be added on pen film slide (Carl Zeiss Inc. through ultraviolet disinfection, Germany) on, and be placed in ThermoBriteslide hybridization instrument (StatSpin, Inc., Norwood, MA) upper 56 DEG C hatch 2 hours after, then slide glass to be dipped in 0.5M NaOH solution 4 minutes.
2) Fish in situ hybridization
According to FISH pretreatment reagent kit (Abbott Molecular Inc., Des Plaines, IL) specification sheets process slide, first protein enzyme solution 37 DEG C hatches 12 minutes, then, after under room temperature, PBS soaks 5 minutes, soak 1 minute in 70% ethanolic soln.After air-dry, use
x SpectrumOrange
tMy SpectrumGreen
tMdNA Probe Kit (Abbott Molecular Inc.) carries out FISH hybridization, and washing and hybridization step are as described in C.Murray etc.
3) detection wind lidar
Adopt PALM laser capture microdissection capture systems (Carl Zeiss Inc.) 400 times of amplifying observations under fluorescent microscope, X-type sperm is light grey signal, Y type sperm is brilliant white signal (see Fig. 1), catches single Y type sperm, and collects AG480F
on the single reflecting point of slide (Advalytix AG, Germany).
4) 3) in each reflecting point add the spermatid lysis buffer of 0.75 μ L, then add 5 μ L mineral oil (Advalytix AG).Slide is placed in upper 56 DEG C of AmpliSpeed circulating instrument (Advalytix AG) and hatches 40 minutes, then boils 10 minutes, obtains sperm lysate.
2.2 obtain the Y type spermatid be separated comprise 1 difference Y-STR locus DYS392,10 euchromosome STR locus, and 1 sex determination locus Amelogenin is at the genotyping result of interior totally 12 locus, comprising:
1) adopt with described locus one to one amplimer increase to obtain amplified production (various primer sequence provided by the invention is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) to it,
Wherein said euchromosome STR locus is the str locus seat D5S818 that can carry out individual recognition, the combination of D21S11, D7S820, D3S1358, VWA, D8S1179, D16S539, D6S1043, D13S317 and D12S391; The amplimer of described difference Y-STR locus DYS392 is two rule nucleotide sequences of SEQ ID No.19 to SEQ ID No.20; Described euchromosome STR locus, and the amplimer of sex determining gene seat Amelogenin is the nucleotide sequence of SEQ ID No.29 to SEQ ID No.50 in sequence table.Use the fluorescent PCR amplimer shown in table 2 and 4, the configuration YA-STR reaction mixture according to table 5:
Table 5
Draw the YA-STR reaction mixture of 0.75 microlitre prepared according to table 5, joined AG480F
in sperm lysate on the single reflecting point of slide, then start pcr amplification, pcr amplification program can be such as: 95 DEG C of 11min, 30 circulations (94 DEG C 1 minute, 61 DEG C 1.25 minutes, 72 DEG C 1.25 minutes), 60 DEG C extend 60 points of kinds, 4 DEG C of preservations, obtain pcr amplification product.
2) genetic analyzer is utilized to obtain the genotype of described locus by the described amplified production obtained
The pcr amplification product obtained is utilized by ABI3130/3500XL type genetic analyzer
iD-X software detects, to obtain the genotyping result of 12 locus described in above-mentioned 1 part of mixing seminal stain sample.
3, described euchromosome STR somatotype system is utilized to be merged by the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for described difference Y-STR locus genotyping result respectively, namely the euchromosome STR genotyping result mixing single individuality in seminal stain is obtained, as shown in following table 6-8.
The YA-STR genotyping result figure of the single spermatid of three volunteers that Fig. 3 A, 3B and 3C use the present invention program to obtain from the mixing seminal stain of three volunteers.In this figure above-mentioned, mark has the peak of square frame to be effective peak below, the repeat number of digitized representation amplified production and fragment length in square frame, the height at peak represents the abundance of product, the color at peak is the color that fluorescence produces after exciting, wherein, blue peak representative is through the amplified production of FAM dye marker, green peak representative is through the amplified production of HEX dye marker, the representative of red peak is through the amplified production (in Fig. 3 A, 3B and 3C, every width figure from top to bottom three row peaks is respectively blue, green, red) of ROX dye marker.In different analysers, the light that fluorescence dye produces after exciting may from itself with color different, this is understandable for a person skilled in the art.
Adopt the genotyping result that the application's scheme obtains, (adopt the repeat number of amplified production and fragment length to represent) as shown shown in 6-8 can find out with comparing of prior art genotyping result, consistence reaches 100%, illustrate that method and system of the present invention has high accuracy, the euchromosome STR locus genotyping result obtaining single individuality from mixing seminal stain can be realized.
Embodiment 2 verifies a kind of accuracy inferring the method and system of the system in DNA individuality source in mixing seminal stain of the present invention
In the present embodiment, described mixing seminal stain is with embodiment 1, in this mixing seminal stain, the individuality source of DNA is known, but it is unknown to set its individual source in the implementation process of the embodiment of the present application 2, the individuality source of the method and system in DNA individuality source in mixing seminal stain to DNA in this mixing seminal stain is inferred to adopt the application to infer.
First, obtain single individual euchromosome STR locus somatotype in mixing seminal stain, this process is with embodiment 1, then, utilize the deduction system in the system inferring DNA individuality source in mixing seminal stain, infer the individuality source of DNA in mixing seminal stain according to the genotyping result of the euchromosome STR locus of above-mentioned single individuality.
Use conventional inspection systems simultaneously, such as sequencing system, again the single spermatid of q.s is collected (such as from three volunteers, each individuality at least 10), obtain the genotyping result of above-mentioned 12 locus, the genotyping result that obtained with the present embodiment by this genotyping result (adopt of the present invention infer mix the system that in seminal stain, DNA individuality is originated) compares, and result is as shown in table 9:
Table 9
Illustrate adopt invention deduction mixing seminal stain in DNA individuality source system and method can realize to mixing seminal stain in DNA individuality source deduction, thus for clear and definite case property, determine that suspect and conviction and sentence etc. provide scientific basis accurately.
Claims (10)
1. from mixing seminal stain, obtain a method for the euchromosome STR locus genotyping result of single individuality, described mixing seminal stain is the mixing seminal stain from two or more individuality, and it is characterized in that, the method comprises:
1) obtain the mixing genotyping result of the Y-STR locus of mixing seminal stain DNA, determine genotyping result at least one Y-STR locus all discrepant between each individuality from this genotyping result, i.e. difference Y-STR locus;
2) from described mixing seminal stain, be separated the Y type spermatid of q.s, obtain the genotyping result of the described difference Y-STR locus of each Y type spermatid, euchromosome STR locus and sex determining gene seat Amelogenin respectively;
The genotyping result of wherein said euchromosome STR locus is the Haplotyping A result of this locus, and the Y type spermatid of described q.s refers at least 10 the Y type spermatids containing each individuality in this mixing seminal stain in the spermatid be separated;
3) respectively the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for described difference Y-STR locus genotyping result is merged, namely obtain the euchromosome STR genotyping result mixing single individuality in seminal stain.
2. method according to claim 1, is characterized in that, described individuality is the individuality from Chinese population, described Y-STR locus is DYS456, DYS390, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448; Described euchromosome STR locus is the str locus seat D5S818 that can carry out individual recognition, the combination of D21S11, D7S820, D3S1358, VWA, D8S1179, D16S539, D6S1043, D13S317 and D12S391.
3. method according to claim 2, it is characterized in that, the process obtaining described locus genotyping result comprise adopt with described locus one to one amplimer increase to obtain amplified production to it, and obtain the genotypic step of described locus by this amplified production
Wherein, the amplimer mixing the Y-STR locus of DNA in seminal stain is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.28 in sequence table;
The amplimer of described difference Y-STR locus is the two or more pieces nucleotide sequence being selected from SEQ ID No.1 to SEQ ID No.28;
Described euchromosome STR locus, and the amplimer of sex determining gene seat Amelogenin is the nucleotide sequence of SEQ ID No.29 to SEQ ID No.50 in sequence table.
4. from mixing seminal stain, obtain a system for the euchromosome STR locus genotyping result of single individuality, it is characterized in that, described system comprises acquisition difference Y-STR locus system, somatotype system, euchromosome STR somatotype system;
Described acquisition difference Y-STR locus system, for obtaining the mixing genotyping result of the Y-STR locus of mixing seminal stain DNA, determines genotyping result at least one Y-STR locus all discrepant between each individuality from this genotyping result, i.e. difference Y-STR locus;
Described somatotype system is used for the Y type spermatid being separated q.s from described mixing seminal stain, obtain the genotyping result of the described difference Y-STR locus of each Y type spermatid, euchromosome STR locus and sex determining gene seat Amelogenin respectively, the genotyping result of wherein said euchromosome STR locus is the Haplotyping A result of this locus, and the Y type spermatid of described q.s refers at least 10 the Y type spermatids containing each individuality in this mixing seminal stain in the spermatid be separated;
Described euchromosome STR somatotype system is used for being merged by the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for described difference Y-STR locus genotyping result respectively, namely obtains the euchromosome STR genotyping result mixing single individuality in seminal stain.
5. infer the method in DNA individuality source in mixing seminal stain, described mixing seminal stain is the mixing seminal stain from two or more individuality, and it is characterized in that, the method comprises:
1) obtain the mixing genotyping result of the Y-STR locus of above-mentioned mixing seminal stain DNA, from this genotyping result, determine genotyping result at least one Y-STR locus all discrepant between each individuality, i.e. difference Y-STR locus;
2) from described mixing seminal stain, be separated the Y type spermatid of q.s, obtain described difference Y-STR locus, the euchromosome STR locus of each Y type spermatid respectively, and the genotyping result of sex determining gene seat Amelogenin;
The genotyping result of wherein said euchromosome STR locus is the Haplotyping A result of this locus, the Y type spermatid of described q.s refers at least 10 the Y type spermatids containing each individuality in this mixing seminal stain in the spermatid be separated, and described euchromosome STR locus is the combination of the euchromosome STR locus that can carry out individual recognition;
3) respectively the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for difference Y-STR locus genotyping result is merged, obtain the euchromosome STR locus genotyping result mixing single individuality in seminal stain;
4) the individuality source of DNA in mixing seminal stain is inferred according to the genotyping result of the euchromosome STR locus of above-mentioned single individuality.
6. method according to claim 5, is characterized in that, described individuality is the individuality from Chinese population, described Y-STR locus is DYS456, DYS390, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448; Described euchromosome STR locus is D5S818, D21S11, D7S820, D3S1358, VWA, D8S1179, D16S539, D6S1043, D13S317 and D12S391.
7. method according to claim 6, it is characterized in that, the process obtaining described locus genotyping result comprise adopt with described locus one to one amplimer increase to obtain amplified production to it, and obtain the genotypic step of described locus by this amplified production
Wherein, the amplimer mixing the Y-STR locus of DNA in seminal stain is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.28 in sequence table;
The amplimer of described difference Y-STR locus is the two or more pieces nucleotide sequence being selected from SEQ ID No.1 to SEQ ID No.28;
Described euchromosome STR locus, and the amplimer of sex determining gene seat Amelogenin is the nucleotide sequence of SEQ ID No.29 to SEQ ID No.50 in sequence table.
8. infer the system in DNA individuality source in mixing seminal stain, it is characterized in that, comprise and obtain difference Y-STR locus system, somatotype system, euchromosome STR somatotype system and deduction system,
Described acquisition difference Y-STR locus system is for obtaining the mixing genotyping result of the Y-STR locus of DNA in above-mentioned mixing seminal stain, genotyping result at least one Y-STR locus all discrepant between each individuality is determined, i.e. difference Y-STR locus from this genotyping result;
Described somatotype system is used for the Y type spermatid being separated q.s from described mixing seminal stain, obtain the described difference Y-STR locus of each Y type spermatid respectively, euchromosome STR locus, and the genotyping result of sex determining gene seat Amelogenin, the genotyping result of wherein said euchromosome STR locus is the Haplotyping A result of this locus, the Y type spermatid of described q.s refers at least 10 the Y type spermatids containing each individuality in this mixing seminal stain in the spermatid be separated, described euchromosome STR locus is the combination of the euchromosome STR locus that can carry out individual recognition,
Described euchromosome STR somatotype system is used for being merged by the euchromosome STR locus haplotype genotyping result of Y type spermatid identical for difference Y-STR locus genotyping result respectively, obtains the euchromosome STR locus genotyping result mixing single individuality in seminal stain;
Described deduction system is used for the individuality source of inferring DNA in mixing seminal stain according to the genotyping result of the euchromosome STR locus of above-mentioned single individuality.
9. system according to claim 8, is characterized in that, described individuality is the individuality from Chinese population, described Y-STR locus is DYS456, DYS390, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448; Described euchromosome STR locus is D5S818, D21S11, D7S820, D3S1358, VWA, D8S1179, D16S539, D6S1043, D13S317 and D12S391.
10. system according to claim 9, it is characterized in that, the process obtaining described locus genotyping result comprise adopt with described locus one to one amplimer increase to obtain amplified production to it, and obtain the genotypic step of described locus by this amplified production
Wherein, the amplimer mixing the Y-STR locus of DNA in seminal stain is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.28 in sequence table;
The amplimer of described difference Y-STR locus is the two or more pieces nucleotide sequence being selected from SEQ ID No.1 to SEQ ID No.28;
Described euchromosome STR locus, and the amplimer of sex determining gene seat Amelogenin is the nucleotide sequence of SEQ ID No.29 to SEQ ID No.50 in sequence table.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105018597A (en) * | 2015-05-27 | 2015-11-04 | 宁波海尔施基因科技有限公司 | Kit for multiplex amplification of 34 loci of human genomic DNA |
| CN112725332A (en) * | 2021-02-04 | 2021-04-30 | 广州高盛智造科技有限公司 | Mixed fine spot extraction process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018597A (en) * | 2015-05-27 | 2015-11-04 | 宁波海尔施基因科技有限公司 | Kit for multiplex amplification of 34 loci of human genomic DNA |
| CN105018597B (en) * | 2015-05-27 | 2018-04-17 | 宁波海尔施基因科技有限公司 | A kind of composite amplification reagent kit of 34 locus of human gene group DNA |
| CN112725332A (en) * | 2021-02-04 | 2021-04-30 | 广州高盛智造科技有限公司 | Mixed fine spot extraction process |
| CN112725332B (en) * | 2021-02-04 | 2023-08-22 | 广州高盛智造科技有限公司 | Mixed fine spot extraction process |
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