CN102703595A - STR (short tandem repeat) sequence high-throughput detection method with base selective controllable extension and detection reagent thereof - Google Patents
STR (short tandem repeat) sequence high-throughput detection method with base selective controllable extension and detection reagent thereof Download PDFInfo
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Abstract
The invention relates to an STR (short tandem repeat) sequence high-throughput detection method with base selective controllable extension and a detection reagent thereof. The basic principle of an STR sequence detection method and a high-throughput DNA (deoxyribonucleic acid) sequencing technology are combined, and the method which runs on a high-throughput sequencing platform for detecting massive STR sequence samples and the corresponding detection reagent are provided. The method comprises the following steps of: directly or indirectly fixing STR sequences of the samples to be detected on a detection chip according to a library preparation method corresponding to a sequencing system, adding the appropriate detection reagent according to the detection flow process, controlling reaction conditions, adopting the base selective controllable extension technical scheme to detect fluorescence intensity signals emitted by all reaction sites where the samples are located, and finally getting the detection results of the massive samples by analyzing fluorescence signal photos of all the detection sites during the whole detection process. The greatest advantage of the method disclosed by the invention is that the number of the samples which can be detected every time is greatly increased, and detection cost and time consumption can be further greatly reduced.
Description
Technical field
The STR sequence high throughput testing method of the controlled extension of a kind of base selectivity of the present invention and detection reagent thereof belong to high-throughput dna sequencing field in the biotechnology, and particularly utilization s-generation high throughput sequencing technologies carries out the detection method of STR sequence.
Background technology
S-generation high-throughput dna sequencing: the carrying out and accomplish of the Human Genome Project and various model animals genome plans produced tremendous influence to the biological study and the medical research in the present age.People can be from the difference of gene level understanding biological phenomena, and disease takes place, development law, and the interaction of medicine and life entity.With regard to gene sequencing, the emphasis of genome times afterwards comprehensively has been measured by the whole genome sequence of single species and has been transferred to a certain species in the comparison of hereditary difference between individual hereditary difference and species on the genomic dna sequence level.At present, aspect the mutational site of seeking new functional gene and disease-related, people still mainly use conventional Sanger dna sequencing method.There is the low and high problem of cost of flux in this method.The expense that first human genomic sequence is measured is approximately 1,000,000,000 dollars, although this expense has been reduced to approximately below 2,000 ten thousand dollars at present, the progress of functional genome still is subject to the dna sequencing technology.For this reason, U.S. Venter foundation proposed the goal in research of 1000 dollars of human genome sequencings in 2003.At the beginning of 2004, the U.S. drops into state-run commune hospital huge fund and supports the dna sequencing Research on New.Their target is at the human complete genome DNA sequencing technologies that develops 100,000 dollars in recent years, and final the attenuating is 1,000 dollars.
Since 2005 454 Life Sciences companies since (the said firm was formally purchased by Roche in 2007) released 454 FLX tetra-sodiums order-checkings platform; Once the leading position of the company of lion's share just begins to have shaken in occupation of order-checking market always in this family of Applied BioSystem (ABI) that released 3730xl dna sequencing appearance; Because their key product capillary array electrophoresis sequenator series has run into two strong rivals; One is exactly 454 sequenators of Roche Holding Ag (Roche), and another is exactly the Solexa genome analysis platform of American I llumina company release in 2006.For this reason, ABI company in 2007 has released the SOLiD sequenator of independent research.These three order-checking platforms are the representative of present high-flux sequence platform.
These platform common characteristic are high sequencing throughput, and with respect to 96 road kapillary order-checkings of tradition order-checking, high-flux sequence is once tested and can be read 400,000 to 4,000,000 sequences.Read length according to the platform difference from 25bp to 450bp, different order-checking platforms once the experiment in, can read the base number that 1G does not wait to 14G, huge like this order-checking ability be traditional sequenator can not compare.
Dna fingerprint identification and STR detection technique: the geneticist Jefferys of Britain University of Leicester in 1984 and co-worker thereof first with isolating people source minisatellite DNA as gene probe, the meaning is it with people's fingerprint equally is that everyone is peculiar.The image of dna fingerprint is a series of stripeds in X-ray film, the spitting image of the barcode on the commodity.Dna fingerprinting; Started the diversified means of detection dna polymorphism (biological Different Individual or different population exist difference on dna structure), analyzed or the like like RFLP (restriction fragment length polymorphism) analysis, tandem repetitive sequence analysis, RAPD (randomly amplified polymorphic DNA).Various analytical procedures all are the basis with the polymorphum of DNA; Generation has height individual specificity's dna fingerprinting; Because dna fingerprinting has the variability of height and stable heredity, and, become the most attractive genetic marker at present still by simple Mendelian's mode heredity.It has following characteristics:
1. the specificity of height: research shows, the probability that two random individuals have a same DNA figure only 3 * 10
-11If compare with two kinds of probes simultaneously, the identical probability of two individuals is less than 5 * 10
-19Whole world population is about 5,000,000,000, and promptly 5 * 10
9Therefore, enzygotic twins children only, otherwise it is identical to have two people's the figure of dna fingerprint hardly.2. stable heredity: DNA is people's a genetic material, it is characterized in that by direct heredity.Analyze to find, in the dna fingerprinting almost each band line can both in one of its parents' collection of illustrative plates, find, this band line meets classical mendelian inheritance, promptly both sides' characteristic is on average transmitted 50% and is given filial generation.3. somatocyte is stable: promptly the dna fingerprint figure of generations such as same individual's different tissues such as blood, muscle, seminal fluid is in full accord.
Doctor Jefferys in 1985 at first is applied to legal medical expert with the dna fingerprint technology and identifies.This technology obtained US Congress's approval as formal court exhibits means in 1989.China police utilize the dna fingerprint technology to track down thousands of routine difficult cases.The dna fingerprint technology has the advantage that many traditional legal medical expert's inspection methods do not possess, and as its seminal stain before 4 years, bloodstain sample, still can extract DNA and perform an analysis; If with the Mitochondrial DNA inspection, the time also will prolong.The evaluation of mummy in thousand in addition; Put to death the remains of tsar Ni Gula period in Russian revolutionary; And fatal crass's late United States Secretary of Commerce Blang and entourage's thereof remains evaluation in the geographic mishap in preceding south recently, all adopted the dna fingerprint technology.
In addition, it is used to individual differentiate, confirm sibship, medical diagnosis and searching and the chain genetic marker of disease in physianthropy; The origin and the evolutionary process that in zoogeny is learned, can be used for verifying animal population; In the species classification, can be used for distinguishing different plant species, the potentiality of distinguishing the different strains of same species are also arranged.In the assignment of genes gene mapping of crop and breeding, also have very widely and use.
STR (short tandem repeat; STR) claim simple repeated sequence (simple sequence repeat again; SSR) or microsatellite DNA (microsatellite DNA), be made up of the core sequence of 2～6bp, multiplicity is usually at 15～30 times.STR extensively is present in the eukaryotic gene group.Because there is interindividual variation in the multiplicity of STR core sequence, has the height polymorphum, thereby be used as during kind that a kind of genetic marker is widely used in plant, animal identifies.In human genome, STR is dispersed in people's the whole genome, and just there is a str locus seat in average per 15～20kb, accounts for 3% of people's gene group.The STR mark also has rich polymorphism, is easy to characteristics such as detection; Therefore being widely used in aspects such as human inheritance's drawing, the assignment of genes gene mapping, linkage analysis, paternity test, criminal's evaluation and human inheritance's research, also is to use method the most widely during dna fingerprint detects.
At present the detection of STR sequence is generally adopted the method for test kit.Its ultimate principle is exactly to utilize the difference of fragment length scope between the locus of the different and amplification of allelotrope length in the locus, adopts high-resolution capillary electrophoresis technique to separate.Usually; Carry out fluorescent mark for primer in each locus; The fluorescence that different gene seat primer mark is different; In conjunction with the mobility of amplified fragments and the color of fluorescence, but applying gene sequenator and adopt corresponding analysis software robotization to detect all information of numerous STR locus of composite amplification.Can find out that present detection means is the basis with the capillary electrophoresis that is similar to first-generation order-checking, the flux of detection is less, can not satisfy the requirement that simultaneously the magnanimity sample is detected simultaneously.
Summary of the invention
The objective of the invention is to provide to above-mentioned weak point the STR sequence high throughput testing method and the detection reagent thereof of the controlled extension of a kind of base selectivity, is a kind of high-throughput based on s-generation dna sequencing technology, low cost, quick STR sequence detecting method and detection reagent thereof.The present invention proposes the synthetic sequencing technologies of the controlled extension of a kind of base selectivity; Increased substantially the pattern detection flux of STR sequence; In the identical time, existing relatively detection means, the sample number of detection can reach the lifting of several orders of magnitude; Correspondingly, the detection cost of single sample has also obtained significantly reducing.Through the new detection method of design corresponding to the STR sequence of s-generation sequencing technologies; Realization is incorporated into the high-throughput characteristics of s-generation order-checking in the detection technique of STR sequence, thereby makes STR sequential detection technology become possibility in great extensive applications such as identification, legal medical expert's detection, cracking of cases and individual people's gene identity information acquisitions.
The STR sequence high throughput testing method of the controlled extension of a kind of base selectivity of the present invention and detection reagent thereof are to take following technical scheme to realize
:
The step of the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity is:
(1) the choosing and increasing of locus:, choose the STR sequence of respective numbers, as detected object according to the different detection needs; Through round pcr, the above-mentioned STR sequence of choosing is come out from the corresponding gene seat amplification of human DNA sample.
The locus at said STR sequence place is ACTBP2; APOAI1; CYAR04; FABP; FOLP23; GABARB1; PLA2A1; TFIIDA; CD4; CSF1PO; F13A1; F13B; FES/FPS; FGA (FIBRA); HPRTB; LPL; Penta D; Penta E; SE33; TH01; TPOX; VWA; D1S1656; D2S441; D2S1338; D3S1358; D5S818; D6S1043; D7S820; D8S1179; D10S1248; D12S391; D13S317; D14S1434; D16S539; D18S51; D19S433; D21S11; D22S1045; DYS19; DYS385 a/b; DYS388; DYS389 I/II; DYS390; DYS391; DYS392; DYS393; DYS434; DYS437; DYS438; DYS439; DYS447; DYS448; DYS456; DYS458; DYS460; DYS464; DYS635; Y-GATA-A4; Y-GATA-A7.1; Y-GATA-A7.2; Y-GATA-A10; Y-GATA-H4; Amelogenin; In the locus relevant such as D22-GATA198B05 one or more with identification.
After having chosen the STR sequence that needs to detect, select relevant detection base site and design the detection of packets scheme that the polygene seat detects simultaneously according to the sequence information of core sequence repetition unit in the STR sequence.
(2) preparation in STR sequence library on the detection chip: same a kind of STR single stranded sequence copy that unique sample is only arranged on each reaction site of chip.Above-mentioned STR single stranded sequence copy can be directly fixed on the planar solid substrate surface of chip; Also can pass through small spheroid, earlier the single stranded sequence copy is fixed on the surface of microballoon indirectly, and then will fix the surface that microballoon that single stranded sequence copies is fixed in planar substrates.
Described small spheroid can be selected magnetic bead or non magnetic microballon for use, and its particle diameter is generally less than and equals 10 microns.
Direct method taked by STR single stranded sequence copy or indirect method is fixed on chip surface, needs to prepare type according to the order-checking library that the high-flux sequence appearance that is used for detection chip adopts separately.
(3) detection of STR core sequence multiplicity: before extension carries out, will read out sample information and locus information under the STR sequence on each reaction site on the chip earlier; Through the method for hybridization, the extension that will extend initial primers and 3 ' end sealing stops primer and is combined on the single stranded sequence of reaction site in the detection chip; Adding by 3 kinds with need to detect base mismatch right nucleotide monomer and another kind of and the detection reagent that needs to detect base pairing, has the fluorophor that can shear, nucleotide monomer that 3 ' end is controlled sealing and archaeal dna polymerase etc. constitute, carry out extension; After accomplishing, an above-mentioned extension detects the fluorescent signal of dot matrix on the microarray, and then that the extension of fluorescence shearing and recovery monomer 3 ' end is active; Then continue to add the above-mentioned detection reagent that constitutes by four kinds of nucleotide monomers and archaeal dna polymerase etc., carry out the next round extension, till all reaction site on the chip all can not detect fluorescent signal.
When reading the affiliated information of STR sequence, sample identification and read method that need be different according to the separation property selection that whether has sample on the chip.If chip has the sample property separated, need use the affiliated locus of combination fluorescent method distinguishing sequence during detection, detected result need obtain the information of sample according to the numbering inquiry of sample areas on the chip.If chip does not have the sample property separated, need to introduce the information that the nucleic acid identification fragment writes down sample information and affiliated locus, need carry out nucleic acid identification fragment process of reading during detection.
The method that combination fluorescence is distinguished the affiliated locus of STR sequence is: the specific sequence fragment of selecting suitable quantity; It is modified last some kinds of fluorescence; The different fluorescence results that produce during with the STR sequence hybridization through these specific sequence fragments distinguish the STR sequence type that connects on each reaction site of chip.
The purpose of selectivity extension method is to skip the base site that need not to detect.If current extension site is not the base site that needs detection, then extension can not ended, and proceeds; If current extension site is the base site that needs detection, extension is ended.
(4) analysis of fluorescent signal:, confirm the repeat number of the STR core sequence of locus to be detected through the detected fluorescence occurrence number of each reaction site and the fluorescence intensity of STR microarray on the record chip; Through the fluorescence intensity of same reaction site in each extension result analyzed; Judge whether the STR sequence place locus that connects on this reaction site has two different allelotrope, and analyze two allelotrope STR sequences core sequence multiplicity separately.Whether final detected result has two not isoalleles for STR sequence place locus, and the core sequence multiplicity of STR sequence, comprises unique perhaps two kinds of different core sequence multiplicity.
The detected result that finally obtains can apply to the different detection field, as: the independent detected result of Amelogenin locus can be used for the sex detection of unknown sample; Detected result to Amelogenin, D18S1364, D12S391, D13S325, D6S1043, D2S1772, D11S2368, D8S1132, D7S3048, these 10 locus of D22-GATA198B05 can be used for paternity test, judges the biology affinity relation between different samples; Detected result to D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, vWA, D8S1179, TPOX, FGA, D19S433, D12S391, D6S1043, these 20 locus of D2S1338; Can be used for the dna fingerprint detection technique and carry out individual identification, thereby further be applied to great fields such as the foundation of crime DB, legal medical expert's detection, cracking of cases and individual people's gene identity information acquisition.
The detection reagent of the STR sequence high throughput testing method of the controlled extension of described a kind of base selectivity comprises extends the biochemical reagents that need use when various biochemical reactions carry out in monomer reagent, archaeal dna polymerase, extension primer reagent, reaction buffer, fluorescence shearing reagent, extension recovery reagent, these testing processes of sex change liquid; Wherein extend monomer reagent and comprised four kinds of different nucleotide monomer dNTP, wherein N is in A, T, C, four kinds of bases of G; Three kinds in four kinds of nucleotide monomers is common nucleotide monomer with detecting the unpaired monomer in base site; Remaining a kind of with detect base site paired monomer through following modification: the monomer marked fluorescence that can shear, and monomeric 3 ' the terminal modified chemical group that controlled extension is arranged; The mixed system of four kinds of nucleotide monomers is as extending monomer reagent, and encapsulation is preserved separately; Whether 5 ' the end that order-checking stopped primer when the archaeal dna polymerase that uses during detection need be according to experimental design is modified with blocking group, selects the polysaccharase of respective type.Said extension primer reagent, reaction buffer, fluorescence are sheared reagent, are extended and recover reagent, sex change liquid reagent, all select biochemical reagents commonly used in the biological experiment for use.Before the detection reaction, the respective detection reagent of capacity is joined in the reaction system according to ratio and the order that experimental standard requires.
Beneficial effect of the present invention: the present invention compared with prior art has following advantage:
1. great advantage of the present invention is to have invented the synthetic sequencing technologies of the controlled extension of a kind of base selectivity; And it is used in the detection of STR sequence; Make STR sequential detection and s-generation high throughput sequencing technologies adapt; Greatly improve each detectable sample size, thereby greatly reduced the cost and the consumed time of detection.What adopt during existing technology for detection is the capillary electrophoresis that first-generation order-checking is used; Only can detect ten several samples at every turn; And detection technique of the present invention can detect tens thousand of hundreds thousand of even samples based on s-generation high throughput sequencing technologies at every turn, has improved at least 3 one magnitude.The raising that detects flux can greatly promote the application of STR sequential detection technology in great fields such as identification, the foundation of crime DB, legal medical expert's detection, cracking of cases and individual people's gene identity information acquisitions.
2. the present invention has introduced a pair of extension initial primers and has stopped primer with extending in the process of sequential detection, has significantly reduced the base number of sites that needs detection.Simultaneously, owing to adopted the method for controlled extension, change traditional order-checking pattern one by one into the great-jump-forward base selectivity detecting pattern that is fit to the STR detection more to each site.Like this, the length of detection can be very long, and need not the locus sequence fragmentization, reduced the complexity that detects.
3. the detection method of the present invention's employing is adaptable across the commercial order-checking of existing s-generation instrument; Even some third generations order-checking instruments (like PacBio RS etc.); And it is not high to the performance requriements of sequenator, and general low and middle-end s-generation order-checking instrument can be accomplished testing process.And of the present invention having wide range of applications can apply to many Biological Detection fields such as sex detection, identification, paternity test, prudence detection.
Description of drawings
Fig. 1 is a locus synoptic diagram at STR sequence place among the present invention.The preceding primer position of amplification in the 1 expression locus among the figure; Extend the position that stops primer hybridization on the 2 expression locus, the core sequence zone of 3 expression locus, AGAA is the repeating unit of core sequence; The hybridization position of primer before 4 expressions are extended, the hybridization position of 5 expression amplification back primers.
Fig. 2 is one group of STR sequence place locus of the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity of the present invention, is respectively D7S820, CSF1PO, D3S1358 and D13S317 locus.Wherein has identical single nucleotide base site in their the core sequence repeating unit; This base of G just; In the follow-up detection, select the nucleotide monomer dCTP of special modification can the STR sequence that these four locus amplify be detected simultaneously.
Fig. 3 is the chip direct Detection Method synoptic diagram of the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity of the present invention.The left side is a detection chip among the figure; 1 expression is the sequence of the preceding primer position of amplification on certain bar STR sequence among the figure; Its 5 ' end is modified,, formed the linking group shown in 6 among the figure with the chemical group generation ligation on the chip reaction surface shown in 7 among the figure.The termination primer is extended in 2 expressions among the figure, and its 3 ' end sealing can't continue to extend.The core sequence repeat region of 3 expression STR sequences among the figure, what need detection is exactly the multiplicity of core sequence in this zone.Initial primers is extended in 4 expressions among the figure, and the starting point of detection is exactly its 3 ' end.Through constantly repeating extension, fluoroscopic examination, fluorescence shearing and recover to extend active step, detect the core sequence multiplicity in 3 zones in publishing picture at its 3 ' end.5 expressions is the sequence of primer position, amplification back on the STR sequence among the figure.
Fig. 4 is the segmental magnetic bead detection method of the introducing nucleic acid identification synoptic diagram of the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity of the present invention.The left side is a detection chip among the figure, and the sequence of the preceding primer position of amplification on 1 certain bar STR sequence of expression is modified its 5 ' end among the figure, with the chemical group generation ligation on the magnetic bead surfaces shown in 9 among the figure, forms the linking group shown in 6 among the figure.Again magnetic bead evenly is taped against in the runner of chip, selects proper reaction conditions, magnetic bead is connected among the figure on the chip reaction surface shown in 7.The termination primer is extended in 2 expressions among the figure, and its 3 ' end sealing can't continue to extend.The core sequence repeat region of 3 expression STR sequences among the figure, what need detection is exactly the multiplicity of core sequence in this zone.Initial primers is extended in 4 expressions among the figure, and the starting point of detection is exactly its 3 ' end.Through constantly repeating extension, fluoroscopic examination, fluorescence shearing and recover to extend active step, detect the core sequence multiplicity in 3 zones in publishing picture at its 3 ' end.8 expressions is the nucleic acid identification fragment of introducing among the figure, and it comprises sample information and locus kind of information under the locus, can before the core sequence multiplicity detects, carry out reading of nucleic acid identification frag info.5 expressions is the sequence of primer position, amplification back on the STR sequence among the figure.
Fig. 5 is the subregion among the real-time detected image result of STR sequence high throughput testing method of the controlled extension of a kind of base selectivity of the present invention.Among the figure on a certain detection site of each some position (comprising bright spot and dim spot) representative the STR sequence take turns the fluorescence situation in the detection at this.STR sequence on wherein brighter this reaction site of some bit representation still can detect fluorescent signal in epicycle, and the counting of core sequence multiplicity is proceeded.Wherein darker some bit representation wherein finishes that short STR sequence count because this STR sequence place locus comprises two kinds of allelotrope, and the integral fluorescence intensity level descends; Long that then continuing counting, still can detect a certain amount of fluorescence.Fluorescent signal situation of these some positions judged that this some position is the failpoint position when remaining non-luminous position then need begin according to detection, or shorter owing to this STR sequence nucleus, the available point position of having accomplished core sequence multiplicity counting.
Embodiment
Embodiment 1:Utilize the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity the STR sequence of Amelogenin locus to be carried out the high-throughput sex detection of multiple sample:
Utilize the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity, the detection step is:
(1) the choosing and increasing of locus: with the one couple of PCR amplimer of Amelogenin locus; Dna profiling with sample to be tested adds in the amplification system; The control proper reaction conditions is carried out the PCR reaction, obtains the STR sequence amplification product of Amelogenin locus.Wherein, be modified with attachable group on one the 5 ' end in a pair of amplimer.Different samples need independently increase in the amplification system.
(2) preparation in STR sequence library on the detection chip:, the Amelogenin locus amplified production system of different samples is put in the separated region on the detection chip reaction basal plane through the mode of point sample.Be modified with another kind of linking group on the chip reaction basal plane, the control proper reaction conditions, making has in the amplified production system that STR sequence strand of linking group to combine with linking group on the reaction basal plane, accomplishes fixing of STR sequence.
(3) detection of STR core sequence multiplicity:
A) on chip, carrying out the STR sequence holds the extension of sealing to stop the hybridization of primer with the corresponding initial primers and 3 ' of extending.Wherein, extend the 5 ' end that stops primer and do not do any modification.
B). do not do any modification owing to extend the 5 ' end that stops primer, need to use the kelnow polysaccharase that does not have 5 ' 5 prime excision enzyme activity to carry out extension.Because the Amelogenin locus of selecting has A (VITAMIN B4) base of different numbers in the sample of different sexes, so, can be A (VITAMIN B4) site as detecting nucleotide site.Therefore; Nucleotide is monomeric in the extension monomer reagent is combined as without the common nucleotide monomer of modifying of dATP, dGTP, dCTP and the dTTP nucleotide monomer of following special modification: be modified with the fluorescence that can shear on the monomer; And monomer 3 ' the terminal modified group that controlled extension is arranged; Under the certain reaction condition, monomeric 3 ' end can recover to extend active.According to the regulation of experimental standard, add corresponding other reaction reagents, and control proper reaction conditions well, carry out extension.
C). the fluorescent signal of all detection site on detection and the record by imaging chip.
D). fluorescent signal carries out fluorescence cleavage reaction and the extension active reaction that recovers monomer 3 ' end after detecting and write down and accomplishing successively.
E). repeat b, c, d step, the fluorescent signal of all detection site disappears on chip.
(4) analysis of fluorescent signal: analyze the fluorescence occurrence number and the strength change laws of respectively taking turns each fluorescence site in the fluoroscopic image, confirm whether the core sequence repeat number of STR sequence on the corresponding chip detection site, fluorescence site and the locus at place thereof have two different allelotrope.When the Amelogenin locus being carried out the sex detection; If the phenomenon that fluorescent signal weakens has appearred in certain reflecting point position; Then represent to contain two STR sequences that length is different in this site corresponding sample amplification system, also just represent this sample to come from the male sex; If fluorescent signal does not weaken, but directly disappear, the STR sequence of having only unique length in this site corresponding sample amplification system is described, sample derives from the women.
Embodiment 2:Utilize the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity that the STR sequence of D3S1358, TH01, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, TPOX, SE33, D8S1179, D21S11, vWA, 16 locus of FGA is carried out the identification detection that sample is separated, the direct preparation method of chip is adopted in the library preparation:
Utilize the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity, the detection step is:
(1) the choosing and increasing of locus: with the corresponding one couple of PCR amplimer of above-mentioned 16 locus; Dna profiling with sample to be tested adds in the amplification system; The control proper reaction conditions is carried out the PCR reaction, obtains the STR sequence amplification product of locus.Different samples need independently increase in the amplification system.
(2) preparation in STR sequence library on the detection chip: but respectively that 16 STR sequences are corresponding, as to have a linking group side amplimer through the mode of point sample, is put in the separated region on the detection chip reaction basal plane.Be modified with another kind of linking group on the chip reaction basal plane, the control proper reaction conditions makes that the linking group on the primer combines with linking group on the reaction basal plane, accomplishes fixing of primer.Wherein a side amplimer should be noted that in the STR sequence strand at its place when selecting; Whether have in the core sequence of identical detection nucleotide site: D3S1358, TH01, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, TPOX, these 12 locus of SE33 and all comprise G (guanine) base detection site, therefore can detect on the same group.And the core sequence of D8S1179, D21S11, vWA, these 4 locus of FGA comprises is C (cytosine(Cyt)) base detection site, can't directly directly detect simultaneously with 12 top locus.But because two strands in the dna double chain satisfy the principle of complementary pairing, so core sequential detection site is G (guanine) site on the antisense strand that amplifies of D8S1179, D21S11, vWA, FGA locus.Like this; When primer is selected; The antisense strand primer of the positive-sense strand primer of selection D3S1358, TH01, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, TPOX, SE33 locus and D8S1179, D21S11, vWA, FGA locus can detect the STR sequence of these 16 locus on the same group.
After the fixedly completion of chip and primer, 16 locus mixing amplification systems of different samples respectively in the cut zone of point samples to the chip, are carried out hybridization, let the primer on the chip capture respective sample locus in the amplification system.With the sample point sample to sequence testing chip the time, need to the sample point sample to the zone carry out record, guarantee that the separated region on the chip is corresponding one by one with sample number.The control proper reaction conditions is carried out amplified reaction once more, and locus to be detected is increased on the reaction surface of chip.Like this, just accomplished the preparation in STR sequence library on the detection chip.
(3) detection of STR core sequence multiplicity:
A). in 16 STR sequences choosing, select two sections short specific sequence fragments, and with four look fluorescence on its complementary short sequence fragment difference mark, at twice with the STR sequence hybridization.16 kinds of fluorescence combined result that utilization obtains are distinguished in the chip kind of locus on all reaction site.Concrete steps are: at first complementary short sequence fragment of article one and chip are carried out hybridization, detect the fluorescence intensity signals of four look fluorescence channels after hybridization is accomplished, excise fluorescence then.With complementary short sequence of second and chip hybridization, after accomplishing, hybridization detects the fluorescence intensity signals of four look fluorescence channels again.At last, through sex change, remove the short sequence fragment on the STR sequence.
B). on chip, carry out 16 STR sequences and extend the hybridization that initial primers and 3 ' is held the extension termination primer of sealing separately accordingly.Wherein, extend the 5 ' end that stops primer and do not do any modification.
C). do not do any modification owing to extend the 5 ' end that stops primer, need to use the kelnow polysaccharase that does not have 5 ' 5 prime excision enzyme activity to carry out extension.Because 16 STR sequences selecting have same detection nucleotide site G (guanine) site.Therefore; Nucleotide is monomeric in the extension monomer reagent is combined as without the common nucleotide monomer of modifying of dATP, dGTP, dTTP and the dCTP nucleotide monomer of following special modification: be modified with the fluorescence that can shear on the monomer; And monomer 3 ' the terminal modified group that controlled extension is arranged; Under the certain reaction condition, monomeric 3 ' end can recover to extend active.According to the regulation of experimental standard, add corresponding other reaction reagents, and control proper reaction conditions well, carry out extension.
D). the fluorescent signal of all detection site on detection and the record by imaging chip.
E). fluorescent signal carries out fluorescence cleavage reaction and the extension active reaction that recovers monomer 3 ' end after detecting and write down and accomplishing successively.
F). repeat c, d, e step, the fluorescent signal of all detection site disappears on chip.
(4) analysis of fluorescent signal: analyze the fluorescence occurrence number and the strength change laws of respectively taking turns each fluorescence site in the fluoroscopic image, confirm whether the core sequence repeat number of STR sequence on the corresponding chip detection site, fluorescence site and the locus at place thereof have two different allelotrope.As: the D18S51 locus of certain sample in the detected result, take turns in the fluoroscopic examination preceding 13, fluorescent signal all occurs, and take turns backward the 14th, no longer detect fluorescent signal.Therefore two allelotrope of D18S51 locus that draw this sample are identical, have 13 core sequence repeating units.The D8S1179 locus of another sample is taken turns in the fluoroscopic examination preceding 9, all occurs than the hyperfluorescence signal, takes turns in the detection 10 to 15, and fluorescence signal intensity is decayed but disappeared, and 16 take turns in the detection fluorescent signal completely dissolve afterwards.The D8S1179 locus of therefore reaching a conclusion to this sample has two different allelotrope, and its core sequence repeating unit is respectively 9 and 15.The detected result of above-mentioned 16 locus of same sample promptly can be used in the individual identification detection of sample.
Utilization present method detects standard samples, and the detected result of detected result and kit test method in correspondence with each other.
Embodiment 3:Utilize the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity that F13A1, F13B, FES/FPS, HPRTB, LPL, D22S1045, DYS19, DYS385 a/b, DYS388, DYS389 I/II, DYS390, DYS391, DYS392, DYS393, DYS434, DYS437, these 17 locus of Y-GATA-H4 are carried out the detection of mixing sample simultaneously, the magnetic bead preparation method is adopted in the library preparation:
Utilize the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity, the detection step is:
(1) the choosing and increasing of locus:, need to introduce the nucleic acid identification fragment and distinguish different samples and different gene seat owing to be the mixing sample system.At first on the corresponding one couple of PCR amplimer of above-mentioned 17 locus, introduce corresponding nucleic acids sign fragment; To introduce nucleic acid identification segmental amplimer again adds in the amplification system with the dna profiling of sample to be tested; The control proper reaction conditions; Carry out the PCR reaction, the STR sequence that obtains locus contains the segmental amplified production of nucleic acid identification.Different samples need independently increase in the amplification system.After amplification is accomplished, each amplification system is distributed into two parts of equivalent.
(2) preparation in STR sequence library on the detection chip: but respectively that 17 STR sequences are corresponding, as to have a linking group side amplimer; Splashing into finishing has in the turbid liquid of magnetic bead of another kind of linking group; The control proper reaction conditions; Make linking group and magnetic bead surfaces on the primer linking group combine, accomplish primer fixing on magnetic bead, formed the magnetic bead that is fixed with different primer sequences on 17 kinds of surfaces.
Because 17 locus detection base sites separately are different, therefore need divide two groups to detect the said gene seat.Because the complementary pairing principle of base, can with detect the base site be G (guanine) site and C (cytosine(Cyt)) site be classified as one group, in like manner, detection base site is another group that is classified as in A (VITAMIN B4) site and T (thymus pyrimidine) site.Like this; Above-mentioned 17 locus that detect C (cytosine(Cyt)) site are: F13A1, DYS385 a/b, DYS389 I/II, DYS390, DYS391, DYS434, these 7 locus of DYS437, that wherein amplimer is selected the antisense strand primer for use is F13A1 and DYS385 a/b.Above-mentioned 17 locus that detect T (thymus pyrimidine) site are: F13B, FES/FPS, HPRTB, LPL, D22S1045, DYS19, DYS388, DYS392, DYS393, these 10 locus of Y-GATA-H4, that wherein amplimer is selected the antisense strand primer for use is D22S1045, DYS388 and DYS392.
The magnetic bead that is fixed with different primer sequences on 17 kinds of surfaces according to above-mentioned rule of classification, is divided into two groups.Join respectively in two parts of independent sample amplification systems that the first step makes, the control proper reaction conditions is carried out amplified reaction once more, will contain the surface that segmental 17 the locus STR sequences to be detected of nucleic acid identification are divided two groups of magnetic beads that increase.Subsequently, two groups of magnetic beads are injected respectively in the different runner of sequence testing chip, after evenly spreading out, carry out fixation reaction once more, magnetic bead is fixed on the reaction basal plane of chip, so far, finish based on the preparation of the library of magnetic bead.
(3) detection of STR core sequence multiplicity:
A). according to the segmental read step of nucleic acid identification, the STR sequence on each magnetic bead of chip is carried out the segmental order-checking of nucleic acid identification, and the contrast coding schedule, the type information and the affiliated sample information of reading each magnetic bead locus gene seat.
B). in the chip runner, carry out two groups of STR sequences respectively and extend the hybridization that initial primers and 3 ' is held the extension termination primer of sealing separately accordingly.Wherein, extend to stop 5 ' of primer and terminal modified circumscribed blocking group arranged.
C). protected by blocking group owing to extend the 5 ' end that stops primer, can use common Taq polysaccharase to carry out extension.Because 17 STR sequences have two different detection nucleotide site C (cytosine(Cyt)) sites and T (thymus pyrimidine) site.Therefore; The extension monomer reagent that different runner domestic demands will add is different: because detection site is C (cytosine(Cyt)) site, is paved with the nucleotide monomer that extends monomer reagent in the runner at magnetic bead place of the fixing F13A1 of going up, DYS385 a/b, DYS389 I/II, DYS390, DYS391, DYS434, DYS437 locus STR sequence and is combined as without the common nucleotide monomer of modifying of dATP, dCTP, dTTP and is modified with fluorescence, 3 ' the terminal modified dGTP nucleotide monomer that controlled extension group is arranged that can shear; Because detection site is T (thymus pyrimidine) site, is paved with the nucleotide monomer that extends monomer reagent in the runner at magnetic bead place of the fixing F13B of going up, FES/FPS, HPRTB, LPL, D22S1045, DYS19, DYS388, DYS392, DYS393, Y-GATA-H4 locus STR sequence and is combined as without the common nucleotide monomer of modifying of dGTP, dCTP, dTTP and is modified with fluorescence, 3 ' the terminal modified dATP nucleotide monomer that controlled extension group is arranged that to shear.According to the regulation of experimental standard, add corresponding other reaction reagents, and control proper reaction conditions well, carry out extension.
D). the fluorescent signal of all detection site on detection and the record by imaging chip.
E). fluorescent signal carries out fluorescence cleavage reaction and the extension active reaction that recovers monomer 3 ' end after detecting and write down and accomplishing successively.
F). repeat c, d, e step, the fluorescent signal of all detection site disappears on chip.
(4) analysis of fluorescent signal: analyze the fluorescence occurrence number and the strength change laws of respectively taking turns each fluorescence site in the fluoroscopic image, confirm whether the core sequence repeat number of STR sequence on the corresponding chip magnetic bead site, fluorescence site and the locus at place thereof have two different allelotrope.The result of concrete experimental result and embodiment 2 is similar, no longer repeats to introduce.
Claims (10)
1. the STR sequence high throughput testing method of the controlled extension of a base selectivity is characterized in that comprising following operation:
(1) the choosing and increasing of locus:, choose the STR sequence of respective numbers, as detected object according to the different detection needs; Through round pcr, the above-mentioned STR sequence of choosing is come out from the corresponding gene seat amplification of human DNA sample;
(2) preparation in STR sequence library on the detection chip: same a kind of STR single stranded sequence copy that unique sample is only arranged on each reaction site of chip; Above-mentioned STR single stranded sequence copy can be directly fixed on the planar solid substrate surface of chip; Also can pass through small spheroid; Earlier the single stranded sequence copy is fixed on the surface of small spheroid indirectly, and then will have fixed the surface that small spheroid that single stranded sequence copies is fixed in planar substrates;
(3) detection of STR core sequence multiplicity: before extension carries out; To read out sample information and locus information under the STR sequence on each reaction site on the chip earlier; Through the method for hybridization, the extension that will extend initial primers and 3 ' end sealing stops primer and is combined on the single stranded sequence of reaction site in the detection chip; Adding is carried out extension by the detection reagent that extension monomer reagent, archaeal dna polymerase, extension primer reagent, reaction buffer, fluorescence shearing reagent, extension recovery reagent, sex change liquid constitute; After accomplishing, an above-mentioned extension detects the fluorescent signal of dot matrix on the microarray, and then that the extension of fluorescence shearing and recovery monomer 3 ' end is active; Then continue to add the above-mentioned detection reagent that constitutes by four kinds of nucleotide monomers and archaeal dna polymerase etc., carry out the next round extension, till all reaction site on the chip all can not detect fluorescent signal;
(4) analysis of fluorescent signal:, confirm the repeat number of the STR core sequence of locus to be detected through the detected fluorescence occurrence number of each reaction site and the fluorescence intensity of STR microarray on the record chip; Through the fluorescence intensity of same reaction site in each extension result analyzed; Judge whether the STR sequence place locus that connects on this reaction site has two different allelotrope, and analyze two allelotrope STR sequences core sequence multiplicity separately.
2. according to the STR sequence high throughput testing method of the controlled extension of the described a kind of base selectivity of claim 1, it is characterized in that described in the step (1) that locus as the STR sequence place of detected object is one or more in ACTBP2, APOAI1, CYAR04, FABP, FOLP23, GABARB1, PLA2A1, TFIIDA, CD4, CSF1PO, F13A1, F13B, FES/FPS, FGA (FIBRA), HPRTB, LPL, Penta D, Penta E, SE33, TH01, TPOX, VWA, D1S1656, D2S441, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D14S1434, D16S539, D18S51, D19S433, D21S11, D22S1045, DYS19, DYS385 a/b, DYS388, DYS389 I/II, DYS390, DYS391, DYS392, DYS393, DYS434, DYS437, DYS438, DYS439, DYS447, DYS448, DYS456, DYS458, DYS460, DYS464, DYS635, Y-GATA-A4, Y-GATA-A7.1, Y-GATA-A7.2, Y-GATA-A10, Y-GATA-H4, Amelogenin, these locus relevant with the identification detection of D22-GATA198B05.
3. according to the STR sequence high throughput testing method of the controlled extension of the described a kind of base selectivity of claim 1; When it is characterized in that locus is chosen in the step (1), can select relevant detection base site and design the detection of packets scheme that the polygene seat detects simultaneously according to the sequence information of core sequence repetition unit in the STR sequence.
4. the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity according to claim 1; It is characterized in that need the STR sequence copy of sample being fixed on the chip of high-flux sequence instrument directly or indirectly when the STR sequence library described in the step (2) prepares: the primer that directly is fixed as the STR sequence directly is fixed on the sequence testing chip through the interaction between chemical group; Indirect securement is fixed on small spheroid on the sequence testing chip for to be fixed on the sample primer earlier on the small spheroid again.
5. the STR sequence high throughput testing method of the controlled extension of a kind of base selectivity according to claim 1; It is characterized in that described in the step (3) read the affiliated information of STR sequence the time, sample identification and read method that need be different according to the separation property selection that whether has sample on the chip; If chip has the sample property separated, need use the affiliated locus of combination fluorescent method distinguishing sequence during detection, detected result need obtain the information of sample according to the numbering inquiry of sample areas on the chip; If chip does not have the sample property separated, need to introduce the information that the nucleic acid identification fragment writes down sample information and affiliated locus, need carry out nucleic acid identification fragment process of reading during detection.
6. according to the STR sequence high throughput testing method of the controlled extension of the described a kind of base selectivity of claim 5; It is characterized in that: locus under the described combination phosphor region sub-sequence; Its method is for selecting the specific sequence fragment of suitable quantity; It is modified last some kinds of fluorescence, and the different fluorescence results that produce during with the STR sequence hybridization through these specific sequence fragments distinguish the STR sequence type that connects on each reaction site of chip.
7. according to the STR sequence high throughput testing method of the controlled extension of the described a kind of base selectivity of claim 1; It is characterized in that: what the extension described in the step (3) adopted is the method that selectivity is extended; If current extension site is not the base site that needs detection; Then extension can not ended, and proceeds; If current extension site is the base site that needs detection, extension is ended.
8. according to the STR sequence high throughput testing method of the controlled extension of the described a kind of base selectivity of claim 1, it is characterized in that: need come two allelotrope of each STR sequence place locus in the judgement sample whether identical in every fluorescence intensity information of taking turns among the extension result according to reaction site on the chip when fluorescent signal described in the step (4) is analyzed; If identical, obtain unique core sequence multiplicity result; If different, need analysis to draw two allelotrope STR sequences core sequence multiplicity result separately.
9. the detection reagent of the STR sequence high throughput testing method of the controlled extension of the described a kind of base selectivity of claim 1; It is characterized in that: the detection reagent described in the step (3) comprises extends monomer reagent, archaeal dna polymerase, extension primer reagent, reaction buffer, fluorescence shearing reagent, extension recovery reagent, sex change liquid, the biochemical reagents that need use when various biochemical reactions carry out in these testing processes; Extension monomer reagent has wherein comprised four kinds of different nucleotide monomer dNTP, and N is in A, T, C, four kinds of bases of G; Wherein three kinds is common nucleotide monomer with detecting the unpaired monomer in base site; Remaining a kind of with detect base site paired monomer through following modification: the monomer marked fluorescence that can shear, and monomeric 3 ' the terminal modified chemical group that controlled extension is arranged; The mixed system of four kinds of nucleotide monomers is as extending monomer reagent, and encapsulation is preserved separately; Whether 5 ' the end that order-checking stopped primer when the archaeal dna polymerase that uses during detection need be according to experimental design is modified with blocking group, selects the polysaccharase of respective type.
10. according to the detection reagent of the STR sequence high throughput testing method of the controlled extension of the described a kind of base selectivity of claim 9; It is characterized in that: said extension primer reagent, reaction buffer, fluorescence are sheared reagent, are extended and recover reagent, sex change liquid reagent, all select biochemical reagents commonly used in the biological experiment for use.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102943111A (en) * | 2012-11-16 | 2013-02-27 | 北京爱普益生物科技有限公司 | Application of high-pass DNA (Deoxyribonucleic Acid) sequencing method on determination of short tandem repeat gene locus in human genome and method |
| CN104152568A (en) * | 2014-08-19 | 2014-11-19 | 东南大学 | High-flux STR sequence core replication number detection method |
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| CN105483262A (en) * | 2016-01-11 | 2016-04-13 | 中国科学院北京基因组研究所 | Detection kit for ten STR loci based on high-throughput sequencing |
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| CN106399496A (en) * | 2016-09-06 | 2017-02-15 | 承启医学（深圳）科技有限公司 | Library building kit for high flux detection of STR genetic markers |
| CN107012225A (en) * | 2017-04-20 | 2017-08-04 | 司法部司法鉴定科学技术研究所 | A kind of detection kit and detection method of the str locus seat based on high-flux sequence |
| CN110265088A (en) * | 2019-06-14 | 2019-09-20 | 孙翊鸣 | A kind of method that DNA is converted into pattern |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1446921A (en) * | 2002-03-25 | 2003-10-08 | 江斌 | New technique for preparing STR allelomorphic gene stairs and kit possessing the stairs |
| CN1926247A (en) * | 2004-03-01 | 2007-03-07 | 生物概念股份有限公司 | Detection of STRP, such as fragile X syndrome |
| CN102203287A (en) * | 2008-08-26 | 2011-09-28 | 弗卢迪格姆公司 | Assay methods for increased throughput of samples and/or targets |
-
2012
- 2012-06-13 CN CN201210193643.3A patent/CN102703595B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1446921A (en) * | 2002-03-25 | 2003-10-08 | 江斌 | New technique for preparing STR allelomorphic gene stairs and kit possessing the stairs |
| CN1926247A (en) * | 2004-03-01 | 2007-03-07 | 生物概念股份有限公司 | Detection of STRP, such as fragile X syndrome |
| CN102203287A (en) * | 2008-08-26 | 2011-09-28 | 弗卢迪格姆公司 | Assay methods for increased throughput of samples and/or targets |
Non-Patent Citations (1)
| Title |
|---|
| 杨电等: "联合应用多个DNA位点进行尸源鉴定", 《法律与医学杂志》 * |
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