CN102703595B - 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-flux detection method of the controlled extension of a kind of Base selection of the present invention and detection reagent thereof belong to high-throughput DNA sequencing field in biotechnology, particularly use s-generation high throughput sequencing technologies to carry out the detection method of STR sequence.
Background technology
S-generation high-throughput DNA sequencing: carrying out and completing of the Human Genome Project and various Model organism genome plans, has produced tremendous influence to contemporary biological study and medical research.People can be familiar with from gene level the difference of biological phenomena, and disease occurs, the rule of development, and the interaction of medicine and life entity.With regard to gene sequencing, the emphasis of genome times afterwards comprehensively is measured and has been transferred to the comparison of hereditary difference between individual hereditary difference and species on genomic dna sequence level of a certain species by the whole genome sequence of single species.At present, aspect the mutational site of the new functional gene of searching 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 of first human genome sequencing 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 is still limited to DNA sequencing technology.For this reason, U.S. Venter foundation proposed the goal in research of 1000 dollars of mankind's genome sequencings in 2003.At the beginning of 2004, the U.S. drops into the research that huge fund is supported DNA sequencing new technology in state-run commune hospital.Their target is in recent years, to develop mankind's complete genome DNA sequencing technologies of 100,000 dollars, and final attenuating is 1,000 dollars.
Since 454 Life Sciences companies in 2005 are since (within 2007, the said firm is formally purchased by Roche) released 454 FLX tetra-sodiums order-checkings platforms, once releasing the Applied BioSystem(ABI of 3730xl DNA sequencer) this family just starts to have shaken in occupation of the leading position of the company of order-checking market lion's share always, 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), another is exactly the Solexa genome analysis platform of American I llumina company release in 2006.For this reason, 2007 NianABI companies have released the SOLiD sequenator of independent research.These three order-checking platforms are the representative of current high-flux sequence platform.
The common feature of these platforms is high sequencing throughput, and with respect to the kapillary order-checking of tradition order-checking 96 road, high-flux sequence is once tested and can be read 400,000 to 4,000,000 sequences.Read length according to platform difference from 25bp to 450bp, different order-checking platforms once experiment in, can read 1G to 14G not grade base number, huge like this order-checking ability is that traditional sequenator can not be compared.
DNA fingerprint identification and STR detection technique: the geneticist Jefferys of Britain University of Leicester in 1984 and co-worker thereof first by separated people source minisatellite DNA as gene probe, the meaning is it is equally that everyone is peculiar with people's fingerprint.The image of DNA fingerprint is a series of stripeds in X-ray film, the spitting image of the barcode on commodity.DNA fingerprinting, started the diversified means of detection DNA polymorphism (biological Different Individual or different population exist difference on DNA structure), as RFLP(restriction fragment length polymorphism) analysiss, tandem repetitive sequence analysis, RAPD(randomly amplified polymorphic DNA) analysis etc.Various analytical procedures all be take the polymorphism of DNA and are basis, generation has height individual specificity's DNA fingerprinting, because DNA fingerprinting has the variability of height and stable heredity, and still by simple Mendelian's mode heredity, become the most attractive genetic marker at present.It has following feature:
1. the specificity of height: research shows, the probability that two random individuals have a same DNA figure only 3 * 10
-11; If compared with two kinds of probes simultaneously, two identical probability of individuality are less than 5 * 10
-19.Whole world population approximately 5,000,000,000,5 * 10
9.Therefore, enzygotic twins children only, otherwise may have hardly two people's the figure of DNA fingerprint identical.2. stable heredity: DNA is people's genetic material, it is characterized in that by direct heredity.Analyze to find, in DNA fingerprinting, almost each band line can find in one of its parents' collection of illustrative plates, thisly with line, meets classical mendelian inheritance, and both sides' feature on average transmits 50% to filial generation.3. somatocyte stability: the different tissues of same person is as in full accord in the DNA fingerprint figure that blood, muscle, seminal fluid etc. produce.
Within 1985, first doctor Jefferys is applied to forensic identification by DNA fingerprint technology.Within 1989, this technology obtains US Congress's approval as formal court exhibits means.China police utilize DNA fingerprint technology to track down thousands of routine difficult cases.DNA fingerprint technology has advantages of that many traditional legal medical expert's inspection methods do not possess, and in its seminal stain from 4 years, bloodstain sample, still can extract DNA and perform an analysis; If use Mitochondrial DNA inspection, the time also will extend.In addition the evaluation of mummy in thousand, in Russian revolutionary, put to death the remains of tsar Ni Gula period, and fatal crass's late United States Secretary of Commerce Blang and the evaluation of entourage's remains thereof in a mishap in area, front south recently, all adopted DNA fingerprint technology.
In addition, it is used to individual differentiate, determine 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 species taxonomy, can be used for distinguishing different plant species, also there are the potentiality of distinguishing same species different lines.In the assignment of genes gene mapping of crop and breeding, also there is application very widely.
STR (short tandem repeat, STR) claim again simple repeated sequence (simple sequence repeat, SSR) or microsatellite DNA (microsatellite DNA), the core sequence of 2～6bp, consist of, multiplicity is conventionally at 15～30 times.STR is extensively present in eukaryotic gene group.Because the multiplicity of STR core sequence exists interindividual variation, there is height polymorphism, thereby be used as in the Identification of Species that a kind of genetic marker is widely used in plant, animal.In human genome, STR is dispersed in people's whole genome, and average every 15～20kb just exists a str locus seat, accounts for 3% of human genome.STR mark also has rich polymorphism, is easy to the features such as detection, therefore being widely used in the 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, is also 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 difference of the interior allelotrope length of locus and the locus of amplification, adopts high-resolution capillary electrophoresis technique to carry out separation.Conventionally, for a primer in each locus, carry out fluorescent mark, the different different fluorescence of locus primer mark, in conjunction with the mobility of amplified fragments and the color of fluorescence, applying gene sequenator and adopt corresponding analysis software can automatization to detect all information of numerous STR locus of composite amplification.Can find out, it is basis that current detection means be take the capillary electrophoresis that is similar to first-generation order-checking, and the flux of detection is less, can not meet the requirement simultaneously Massive Sample being detected simultaneously.
Summary of the invention
The object of the invention is to provide for above-mentioned weak point STR sequence high-flux detection method and the detection reagent thereof of the controlled extension of a kind of Base selection, is a kind of high-throughput, low cost, quick STR sequence detecting method and detection reagent thereof based on s-generation DNA sequencing technology.The present invention proposes the synthetic sequencing technologies of the controlled extension of a kind of Base selection, increased substantially the pattern detection flux of STR sequence, within the identical time, relatively existing detection means, the sample number detecting can reach the lifting of several orders of magnitude, correspondingly, the testing cost of single sample has also obtained significantly reducing.New detection method by design corresponding to the STR sequence of s-generation sequencing technologies, realization is incorporated into the high-throughput feature of s-generation order-checking in the detection technique of STR sequence, thereby makes STR sequential detection technology become possibility in the widespread use of the major areas such as identification, legal medical expert's detection, cracking of cases and individual people's gene identity information acquisition.
The STR sequence high-flux detection method of the controlled extension of a kind of Base selection of the present invention and detection reagent thereof are to take following technical scheme to realize
:
The step of the STR sequence high-flux detection method of the controlled extension of a kind of Base selection is:
(1) the choosing and increasing of locus: according to different detection needs, choose the STR sequence of respective numbers, as detected object; By round pcr, the above-mentioned STR sequence of choosing is increased out from the corresponding gene seat of human DNA sample.
The locus at described 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, one or more in the locus relevant to identification such as D22-GATA198B05.
Chosen after the STR sequence that needs to detect, according to the sequence information of core sequence repetition unit in STR sequence, selected to detect accordingly base site and design the detection of packets scheme that polygene seat detects simultaneously.
(2) preparation in STR sequence library in detection chip: the same STR single stranded sequence copy that only has unique sample in 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, indirectly first single stranded sequence copy is fixed on to the surface of microballoon, and then the microballoon of having fixed single stranded sequence copy is fixed on to the surface of planar substrates.
Described small spheroid can be selected magnetic bead or non magnetic microballon, 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 the sequencing library adopting separately according to the high-flux sequence instrument for detection of chip to prepare type.
(3) detection of STR core sequence multiplicity: before extension carries out, first will read out sample information and locus information under the STR sequence in each reaction site on chip; By the method for hybridization, the extension of extending initial primers and 3 ' end sealing is stopped to primer and be combined on the single stranded sequence of reaction site in detection chip; Add by 3 kinds with need to detect base mismatch right nucleotide monomer and another kind of with need to detect base pairing, with the fluorophor that can shear, 3 ' end, be the detection reagent that the nucleotide monomer of controlled sealing and archaeal dna polymerase etc. form, carry out extension; The fluorescent signal of dot matrix on detection microarray after an above-mentioned extension completes, then shears fluorescence and recovers the extension activity that monomer 3 ' is held; Then continue to add the above-mentioned detection reagent being formed by four kinds of nucleotide monomers and archaeal dna polymerase etc., carry out next round extension, until all reaction site on chip are all examined, do not measure fluorescent signal.
While reading the affiliated information of STR sequence, need to select different sample identification and read method according to the separation whether on chip with sample.If chip has the sample property separated, need to be with combining the affiliated locus of fluorescent method distinguishing sequence during detection, detected result need to obtain according to the numbering inquiry of sample areas on chip the information of sample.If chip does not have the sample property separated, need to introduce the information that nucleic acid identification fragment records sample information and affiliated locus, during detection, need to carry out the process that nucleic acid identification fragment is read.
The method that combination fluorescence is distinguished the affiliated locus of STR sequence is: the specific sequence fragment of selecting suitable quantity, modified several fluorescence, the different fluorescence results that produce during by these specific sequence fragments and STR sequence hybridization, distinguish the STR sequence type connecting in each reaction site of chip.
The object of selectivity extension method is to skip the base site without detecting.If current extension site is not the base site that needs detection, 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: by recording fluorescence occurrence number and the fluorescence intensity that on chip, each reaction site of STR microarray detects, determine the repeat number of the STR core sequence of locus to be detected; By to same reaction site the fluorescence intensity in each extension result analyze, judge whether the STR sequence place locus connecting in this reaction site has two different allelotrope, and analyze two allelotrope STR sequences core sequence multiplicity separately.Final detected result is whether STR sequence place locus has two not isoalleles, and the core sequence multiplicity of STR sequence, comprises unique or two kinds of different core sequence multiplicity.
The detected result finally obtaining can apply to different detection fields, as: the sex-screening that the independent detected result of Amelogenin locus be can be used for to unknown sample; Detected result to Amelogenin, D18S1364, D12S391, D13S325, D6S1043, D2S1772, D11S2368, D8S1132, these 10 locus of D7S3048, 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, these 20 locus of D6S1043, D2S1338, can be for carrying out individual identification in DNA fingerprint detection technique, thus be further applied to the major areas such as crime Database, legal medical expert's detection, cracking of cases and individual people's gene identity information acquisition.
The detection reagent of the STR sequence high-flux detection method of the described controlled extension of a kind of Base selection comprises the biochemical reagents that need to use when various biochemical reactions carry out in extension 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 pack and contained four kinds of different nucleotide monomer dNTP, wherein N is in A, T, C, tetra-kinds of bases of G; Three kinds of monomers unpaired with detecting base site in four kinds of nucleotide monomers are common nucleotide monomer, remaining a kind of monomer with detecting the pairing of base site is through following modification: on monomer mark the fluorescence that can shear, and the 3 ' terminal modified chemical group that has controlled extension of monomer; The mixed system of four kinds of nucleotide monomers is as extending monomer reagent, and encapsulation is preserved separately; Whether 5 ' the end that when archaeal dna polymerase using during detection need to be according to experimental design, order-checking stops primer is modified with blocking group, selects the polysaccharase of respective type.Described extension primer reagent, reaction buffer, fluorescence are sheared reagent, are extended and recover reagent, sex change liquid reagent, all select biochemical reagents conventional in biological experiment.Before detection reaction, the ratio that enough corresponding detection reagent are required according to experimental standard and order join in reaction system.
Beneficial effect of the present invention: compared with prior art, tool has the following advantages in the present invention:
1. great advantage of the present invention is to have invented the synthetic sequencing technologies of the controlled extension of a kind of Base selection, and be used in the detection of STR sequence, STR sequential detection and s-generation high throughput sequencing technologies are adapted, greatly improve each detectable sample size, thereby greatly reduced the cost of detection and the time of expending.What during existing technology for detection, adopt 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 is based on s-generation high throughput sequencing technologies, can detect tens thousand of hundreds thousand of samples even at every turn, improved at least 3 orders of magnitude.The raising that detects flux can greatly promote STR sequential detection technology in the application of the major areas such as identification, crime Database, legal medical expert's detection, cracking of cases and individual people's gene identity information acquisition.
2. the present invention, in the process of sequential detection, has introduced a pair of extension initial primers and has extended stopping primer, has greatly reduced the base number of sites that needs detection.Meanwhile, owing to having adopted the method for controlled extension, the great-jump-forward Base selection detecting pattern that traditional Mode change that checks order one by one to each site is detected for more applicable STR.Like this, the length of detection can be very long, and without by locus sequence fragment, reduced the complexity detecting.
3. the detection method that the present invention adopts is widely used in the commercial order-checking of existing s-generation instrument, some third generations order-checking instruments (as PacBio RS etc.) even, and it is not high to the performance requriements of sequenator, general low and middle-end s-generation order-checking instrument can complete testing process.And of the present invention having wide range of applications, can apply to many Biological Detection fields such as sex-screening, identification, paternity test, Forensic detection.
Accompanying drawing explanation
Fig. 1 is a locus schematic diagram at STR sequence place in the present invention.In figure, 1 represents the front primer of amplification position in locus, 2 represent to extend the position that stops primer hybridization on locus, and 3 represent the core sequence region of locus, the repeating unit that AGAA is core sequence, 4 represent to extend the hybridization position of front primer, the hybridization position of primer after 5 expression amplifications.
Fig. 2 is one group of STR sequence place locus of the STR sequence high-flux detection method of the controlled extension of a kind of Base selection of the present invention, is respectively D7S820, CSF1PO, D3S1358 and D13S317 locus.Wherein in their core sequence repeating unit, there is identical single nucleotide base site, this base of G namely, in follow-up detection, select the STR sequence that the nucleotide monomer dCTP of special modification can amplify these four locus to detect simultaneously.
Fig. 3 is the chip direct Detection Method schematic diagram of the STR sequence high-flux detection method of the controlled extension of a kind of Base selection of the present invention.In figure, left side is detection chip, in figure, 1 expression is the sequence of the front primer of amplification position in certain STR sequence, its 5 ' end is modified, with the chemical group generation ligation on the chip reaction surface shown in 7 in figure, formed the linking group shown in 6 in figure.In figure, 2 represent to extend termination primer, and its 3 ' end sealing, cannot continue to extend.The core sequence repeat region of 3 expression STR sequences in figure, what need detection is exactly the multiplicity of core sequence in this region.In figure, 4 represent to extend initial primers, and the starting point of detection is exactly its 3 ' end.By constantly repeating extension, fluoroscopic examination, fluorescence shearing and recover to extend active step at its 3 ' end, detect the core sequence multiplicity in 3 regions in publishing picture.In figure, 5 expressions is the sequence of the rear primer of amplification position in STR sequence.
Fig. 4 is the magnetic bead detection method schematic diagram of introducing nucleic acid identification fragment of the STR sequence high-flux detection method of the controlled extension of a kind of Base selection of the present invention.In figure, left side is detection chip, and in figure, the sequence of the front primer of amplification position in certain STR sequence of 1 expression, is modified its 5 ' end, with the chemical group generation ligation in the magnetic bead surfaces shown in 9 in figure, forms the linking group shown in 6 in figure.Again magnetic bead is evenly taped against in the runner of chip, selects suitable reaction conditions, magnetic bead is connected in figure on the chip reaction surface shown in 7.In figure, 2 represent to extend termination primer, and its 3 ' end sealing, cannot continue to extend.The core sequence repeat region of 3 expression STR sequences in figure, what need detection is exactly the multiplicity of core sequence in this region.In figure, 4 represent to extend initial primers, and the starting point of detection is exactly its 3 ' end.By constantly repeating extension, fluoroscopic examination, fluorescence shearing and recover to extend active step at its 3 ' end, detect the core sequence multiplicity in 3 regions in publishing picture.In figure, 8 expressions is the nucleic acid identification fragment of introducing, and it comprises sample information and locus kind of information under locus, can before core sequence multiplicity detects, carry out reading of nucleic acid identification frag info.In figure, 5 expressions is the sequence of the rear primer of amplification position in STR sequence.
Fig. 5 is the subregion in the real-time detected image result of STR sequence high-flux detection method of the controlled extension of a kind of Base selection of the present invention.In figure, each some position (comprising bright spot and dim spot) represents that in a certain detection site, STR sequence is taken turns the fluorescence situation in detection at this.Wherein the STR sequence in 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, because this STR sequence place locus comprises two kinds of allelotrope, wherein finishes that shorter STR sequence count, and integral fluorescence intensity level declines; Long that, continuing counting, still can detect a certain amount of fluorescence.When remaining non-luminous position needs to start according to detection, fluorescent signal situations of these some positions judge that this some position is failpoint position, still because this STR sequence nucleus is shorter, have completed the available point position of core sequence multiplicity counting.
Embodiment
embodiment 1:utilize the STR sequence high-flux detection method of the controlled extension of a kind of Base selection the STR sequence of Amelogenin locus to be carried out to the high-throughput sex detection of multiple sample:
The STR sequence high-flux detection method that utilizes the controlled extension of a kind of Base selection, detecting step is:
(1) the choosing and increasing of locus: by the one couple of PCR amplimer of Amelogenin locus, add in amplification system with together with the DNA profiling of sample to be tested, control suitable reaction conditions, carry out PCR reaction, obtain the STR sequence amplification product of Amelogenin locus.Wherein, in pair for amplification primer, on one 5 ' end, be modified with attachable group.Different samples need to independently increase in amplification system.
(2) preparation in STR sequence library in detection chip: by the mode of point sample, the Amelogenin locus amplified production system of different samples is put in the separated region on detection chip reaction basal plane.On chip reaction basal plane, be modified with another kind of linking group, control suitable reaction conditions, make to have in amplified production system that STR sequence strand of linking group to combine with the linking group on reaction basal plane, complete the fixing of STR sequence.
(3) detection of STR core sequence multiplicity:
A) on chip, carrying out STR sequence holds the extension of sealing to stop the hybridization of primer with the corresponding initial primers and 3 ' of extending.Wherein, the 5 ' end that extends termination primer is not done any modification.
B). owing to extending 5 ' end of termination primer, do not do any modification, need to use the kelnow polysaccharase without 5 ' 5 prime excision enzyme activity to carry out extension.Because the Amelogenin locus of selecting has the A(VITAMIN B4 of different numbers in the sample of different sexes) base, so, can be using A(VITAMIN B4) site is as detecting nucleotide site.Therefore, extend the dTTP nucleotide monomer that is combined as not modified dATP, dGTP, the common nucleotide monomer of dCTP and following special modification of nucleotide monomer in monomer reagent: on monomer, be modified with the fluorescence that can shear, and the terminal modified group that has controlled extension of monomer 3 ', under certain reaction conditions, 3 ' end of monomer can recover to extend active.According to the regulation of experimental standard, add corresponding other reaction reagents, and control suitable reaction conditions well, carry out extension.
C). the fluorescent signal of all detection site on detection record by imaging chip.
D). after fluorescent signal detection and record complete, the extension active reaction of carrying out successively fluorescence cleavage reaction and recovering monomer 3 ' end.
E). repeat b, c, d step, until the fluorescent signal of all detection site disappears on chip.
(4) analysis of fluorescent signal: analyze fluorescence occurrence number and the strength change laws respectively take turns each fluorescence site in fluoroscopic image, determine on chip detection site corresponding to fluorescence site whether the core sequence repeat number of STR sequence and the locus at place thereof have two different allelotrope.When Amelogenin locus is carried out to sex-screening, if there is the phenomenon that fluorescent signal weakens in certain reflecting point position, represent to contain two STR sequences that length is different in sample amplification system corresponding to this site, also just represent that this sample comes from the male sex; If fluorescent signal does not weaken, but directly disappear, the STR sequence of only having unique length in sample amplification system corresponding to this site is described, sample derives from women.
embodiment 2:utilize the STR sequence high-flux detection method of the controlled extension of a kind of Base selection 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 to be carried out to the identification detection of sample separation, library preparation adopts the direct preparation method of chip:
The STR sequence high-flux detection method that utilizes the controlled extension of a kind of Base selection, detecting step is:
(1) the choosing and increasing of locus: by the corresponding one couple of PCR amplimer of above-mentioned 16 locus, add in amplification system with together with the DNA profiling of sample to be tested, control suitable reaction conditions, carry out PCR reaction, obtain the STR sequence amplification product of locus.Different samples need to independently increase in amplification system.
(2) preparation in STR sequence library in detection chip: respectively 16 STR sequences are corresponding, with a side amplimer that can linking group, by the mode of point sample, point is in the separated region on detection chip reaction basal plane.On chip reaction basal plane, be modified with another kind of linking group, control suitable reaction conditions, the linking group on primer is combined with the linking group on reaction basal plane, complete the fixing of primer.Wherein a side amplimer should be noted that in the STR sequence strand at its place when selecting, whether there is identical detection nucleotide site: in the core sequence of D3S1358, TH01, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, TPOX, these 12 locus of SE33, all comprise G(guanine) base detection site, therefore can detect on the same group.And the core sequence of D8S1179, D21S11, these 4 locus of vWA, FGA comprises, be C(cytosine(Cyt)) base detection site cannot directly directly detect with 12 locus above simultaneously.But because two strands in DNA double chain meet the principle of complementary pairing, so on the antisense strand that amplifies of D8S1179, D21S11, vWA, FGA locus, core sequential detection site is G(guanine) site.Like this, when Primer selection, 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 on the same group to the STR sequence of these 16 locus.
After fixedly the completing of chip and primer, by 16 locus mixing amplification systems of different samples respectively point samples in the cut zone on chip, carry out hybridization, allow the primer on chip capture respective sample locus in amplification system.By sample point sample on sequence testing chip time, need to sample point sample to region carry out record, guarantee that the separated region on chip is corresponding one by one with sample number.Control suitable reaction conditions, again carry out amplified reaction, locus to be detected is increased on the reaction surface of chip.Like this, just completed the preparation in STR sequence library in detection chip.
(3) detection of STR core sequence multiplicity:
A). in 16 STR sequences choosing, select two sections of short specific sequence fragments, and by four look fluorescence on its complementary short sequence fragment difference mark, at twice with STR sequence hybridization.16 kinds of fluorescence combined result that utilization obtains, distinguish in chip the kind of locus in all reaction site.Concrete steps are: first the complementary short sequence fragment of article one and chip are carried out to hybridization, hybridized the fluorescence intensity signals of rear detection four look fluorescence channels, then excise fluorescence.By the complementary short sequence of second and chip hybridization, hybridized the fluorescence intensity signals of rear detection four look fluorescence channels again.Finally, by sex change, remove the short sequence fragment in STR sequence.
B). on chip, carry out 16 STR sequences and the hybridization that extends accordingly separately the extension termination primer of initial primers and 3 ' end sealing.Wherein, the 5 ' end that extends termination primer is not done any modification.
C). owing to extending 5 ' end of termination primer, do not do any modification, need to use the kelnow polysaccharase without 5 ' 5 prime excision enzyme activity to carry out extension.Because 16 STR sequences selecting have same detection nucleotide site G(guanine) site.Therefore, extend the dCTP nucleotide monomer that is combined as not modified dATP, dGTP, the common nucleotide monomer of dTTP and following special modification of nucleotide monomer in monomer reagent: on monomer, be modified with the fluorescence that can shear, and the terminal modified group that has controlled extension of monomer 3 ', under certain reaction conditions, 3 ' end of monomer can recover to extend active.According to the regulation of experimental standard, add corresponding other reaction reagents, and control suitable reaction conditions well, carry out extension.
D). the fluorescent signal of all detection site on detection record by imaging chip.
E). after fluorescent signal detection and record complete, the extension active reaction of carrying out successively fluorescence cleavage reaction and recovering monomer 3 ' end.
F). repeat c, d, e step, until the fluorescent signal of all detection site disappears on chip.
(4) analysis of fluorescent signal: analyze fluorescence occurrence number and the strength change laws respectively take turns each fluorescence site in fluoroscopic image, determine on chip detection site corresponding to fluorescence site whether the core sequence repeat number of STR sequence and the locus at place thereof have two different allelotrope.As: the D18S51 locus of certain sample in detected result, front 13, take turns in fluoroscopic examination, all there is fluorescent signal, 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, takes turns in fluoroscopic examination front 9, all occurs compared with hyperfluorescenceZeng Yongminggaoyingguang signal, 10 to 15, takes turns in detection, and fluorescence signal intensity is decayed but do not disappeared, and 16 take turns in detection afterwards, fluorescent signal completely dissolve.The D8S1179 locus of therefore reaching a conclusion as 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, in can be used for the identification of individual of sample to detect.
Use present method to detect standard samples, the detected result of detected result and kit test method in correspondence with each other.
embodiment 3:utilize the STR sequence high-flux detection method of the controlled extension of a kind of Base selection to carry out the detection of mixing sample to F13A1, F13B, FES/FPS, HPRTB, LPL, D22S1045, DYS19, DYS385 a/b, DYS388, DYS389 I/II, DYS390, DYS391, DYS392, DYS393, DYS434, these 17 locus of DYS437, Y-GATA-H4, library preparation adopts magnetic bead preparation method simultaneously:
The STR sequence high-flux detection method that utilizes the controlled extension of a kind of Base selection, detecting step is:
(1) the choosing and increasing of locus: owing to being mixing sample system, need to introducing nucleic acid identification fragment and distinguish different samples and different locus.First on the corresponding one couple of PCR amplimer of above-mentioned 17 locus, introduce corresponding nucleic acid identification fragment, by having introduced together with the amplimer of nucleic acid identification fragment and the DNA profiling of sample to be tested, add in amplification system again, control suitable reaction conditions, carry out PCR reaction, obtain the STR sequence of locus containing the amplified production of nucleic acid identification fragment.Different samples need to independently increase in amplification system.After having increased, each amplification system is distributed into two parts of equivalent.
(2) preparation in STR sequence library in detection chip: respectively 17 STR sequences are corresponding, with a side amplimer that can linking group, splashing into finishing has in the turbid liquid of magnetic bead of another kind of linking group, control suitable reaction conditions, make linking group on primer and magnetic bead surfaces linking group combine, complete primer fixing on magnetic bead, formed the magnetic bead that is fixed with different primers sequence on 17 kinds of surfaces.
Because 17 locus detection base site is separately different, therefore said gene seat need to be divided two groups detect.Due to the complementary pairing principle of base, can be G(guanine by detecting base site) site and C(cytosine(Cyt)) site be classified as one group, in like manner, detecting base site is A(VITAMIN B4) site and T(thymus pyrimidine) site be classified as another group.Like this, detection C(cytosine(Cyt)) above-mentioned 17 locus in site are: F13A1, DYS385 a/b, DYS389 I/II, DYS390, DYS391, DYS434, these 7 locus of DYS437, that wherein amplimer is selected antisense strand primer is F13A1 and DYS385 a/b.Detection T(thymus pyrimidine) above-mentioned 17 locus in site are: F13B, FES/FPS, HPRTB, LPL, D22S1045, DYS19, DYS388, DYS392, these 10 locus of DYS393, Y-GATA-H4, that wherein amplimer is selected antisense strand primer is D22S1045, DYS388 and DYS392.
The magnetic bead that is fixed with different primers sequence 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, control suitable reaction conditions, again carry out amplified reaction, will divide containing 17 locus STR sequences to be detected of nucleic acid identification fragment the surface of 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, be again fixed reaction, magnetic bead is fixed on the reaction basal plane of chip, so far, the library based on magnetic bead is prepared complete.
(3) detection of STR core sequence multiplicity:
A). according to the read step of nucleic acid identification fragment, the STR sequence on each magnetic bead of chip is carried out to the order-checking of nucleic acid identification fragment, and contrast coding schedule, read type information and the affiliated sample information of each magnetic bead locus gene seat.
B). in chip runner, carry out respectively two groups of STR sequences and the hybridization that extends accordingly separately the extension termination primer of initial primers and 3 ' end sealing.Wherein, extend to stop 5 ' of primer and terminal modifiedly have a circumscribed blocking group.
C). owing to extending 5 ' of termination primer, hold protected radical protection, can use common Taq polysaccharase to carry out extension.Because 17 STR sequences have two different detection nucleotide site C(cytosine(Cyt)s) site 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, be paved with the nucleotide monomer that extends monomer reagent in the runner at magnetic bead place of fixing upper F13A1, DYS385 a/b, DYS389 I/II, DYS390, DYS391, DYS434, DYS437 locus STR sequence and be combined as not modified dATP, dCTP, the common nucleotide monomer of dTTP and be modified with fluorescence, the 3 ' the terminal modified dGTP nucleotide monomer that has controlled extension group that can shear; Because detection site is T(thymus pyrimidine) site, be paved with the nucleotide monomer that extends monomer reagent in the runner at magnetic bead place of fixing upper F13B, FES/FPS, HPRTB, LPL, D22S1045, DYS19, DYS388, DYS392, DYS393, Y-GATA-H4 locus STR sequence and be combined as not modified dGTP, dCTP, the common nucleotide monomer of dTTP and be modified with fluorescence, the 3 ' the terminal modified dATP nucleotide monomer that has controlled extension group that can shear.According to the regulation of experimental standard, add corresponding other reaction reagents, and control suitable reaction conditions well, carry out extension.
D). the fluorescent signal of all detection site on detection record by imaging chip.
E). after fluorescent signal detection and record complete, the extension active reaction of carrying out successively fluorescence cleavage reaction and recovering monomer 3 ' end.
F). repeat c, d, e step, until the fluorescent signal of all detection site disappears on chip.
(4) analysis of fluorescent signal: analyze fluorescence occurrence number and the strength change laws respectively take turns each fluorescence site in fluoroscopic image, determine on chip magnetic bead site corresponding to fluorescence site whether the core sequence repeat number of STR sequence and the locus at place thereof have two different allelotrope.The result of specific experiment result and embodiment 2 is similar, no longer repeats to introduce.
Claims (6)
1. a STR sequence high-flux detection method for the controlled extension of Base selection, is characterized in that comprising following operation:
(1) the choosing and increasing of locus: according to different detection needs, choose the STR sequence of respective numbers, as detected object; By round pcr, the above-mentioned STR sequence of choosing is increased out from the corresponding gene seat of human DNA sample;
The locus at the described STR sequence place as detected object 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, these detect one or more in relevant locus to identification D22-GATA198B05,
(2) preparation in STR sequence library in detection chip: the same STR single stranded sequence copy that only has unique sample in 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, indirectly first single stranded sequence copy is fixed on to micro-spherular surface, and then the small spheroid of having fixed single stranded sequence copy is fixed on to the surface of planar substrates;
(3) detection of STR core sequence multiplicity: before extension carries out, first to read out sample information and locus information under the STR sequence in each reaction site on chip, by the method for hybridization, the extension of extending initial primers and 3 ' end sealing is stopped to primer and be combined on the single stranded sequence of reaction site in detection chip; Add the detection reagent being formed by extension monomer reagent, archaeal dna polymerase, extension primer reagent, reaction buffer, fluorescence shearing reagent, extension recovery reagent, sex change liquid, carry out extension; The fluorescent signal of dot matrix on detection microarray after an above-mentioned extension completes, then shears fluorescence and recovers the extension activity that monomer 3 ' is held; Then continue to add the above-mentioned detection reagent being formed by four kinds of nucleotide monomers and archaeal dna polymerase etc., carry out next round extension, until all reaction site on chip are all examined and do not measured fluorescent signal;
Wherein, while reading sample information under the STR sequence in each reaction site on chip and locus information, need to select different sample identification and read method according to the separation whether on chip with sample; If chip has the sample property separated, need to be with combining the affiliated locus of fluorescent method distinguishing sequence during detection, detected result need to obtain according to the numbering inquiry of sample areas on chip the information of sample; If chip does not have the sample property separated, need to introduce the information that nucleic acid identification fragment records sample information and affiliated locus, during detection, need to carry out the process that nucleic acid identification fragment is read; Locus under described combination fluorescent method distinguishing sequence, its method is for selecting the specific sequence fragment of suitable quantity, modified several fluorescence, the different fluorescence results that produce during by these specific sequence fragments and STR sequence hybridization, distinguish the STR sequence type connecting in each reaction site of chip;
What described extension adopted is the method that selectivity is extended, if current extension site is not the base site that needs detection, 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: by recording fluorescence occurrence number and the fluorescence intensity that on chip, each reaction site of STR microarray detects, determine the repeat number of the STR core sequence of locus to be detected; By to same reaction site the fluorescence intensity in each extension result analyze, judge whether the STR sequence place locus connecting in this reaction site has two different allelotrope, and analyze two allelotrope STR sequences core sequence multiplicity separately.
2. according to the STR sequence high-flux detection method of the controlled extension of a kind of Base selection described in claim 1, while it is characterized in that in step (1), locus is chosen, can select to detect accordingly base site and design the detection of packets scheme that polygene seat detects simultaneously according to the sequence information of core sequence repetition unit in STR sequence.
3. the STR sequence high-flux detection method of the controlled extension of a kind of Base selection according to claim 1, it is characterized in that the STR sequence copy of sample to be fixed on the chip of high-flux sequence instrument directly or indirectly when prepared by the STR sequence library described in step (2): be directly fixed as the primer of STR sequence is directly fixed on sequence testing chip by the interaction between chemical group, indirectly be fixed as a sample primer and be first fixed on small spheroid, more small spheroid is fixed on sequence testing chip.
4. according to the STR sequence high-flux detection method of the controlled extension of a kind of Base selection described in claim 1, it is characterized in that: whether the fluorescent signal described in step (4) need to be identical according to two allelotrope of each STR sequence place locus in every fluorescence intensity information of taking turns in extension result is carried out judgement sample of reaction site on chip while analyzing; If identical, obtain unique core sequence multiplicity result; If different, need analysis to draw two allelotrope STR sequences core sequence multiplicity result separately.
5. the detection reagent of the STR sequence high-flux detection method of the controlled extension of a kind of Base selection described in claim 1, it is characterized in that: the detection reagent described in 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 to use when various biochemical reactions carry out in these testing processes; Extension monomer pack has wherein contained four kinds of different nucleotide monomer dNTP, and N is in A, T, C, tetra-kinds of bases of G; Wherein three kinds of monomers unpaired with detecting base site are common nucleotide monomer, remaining a kind of monomer with detecting the pairing of base site is through following modification: on monomer mark the fluorescence that can shear, and the 3 ' terminal modified chemical group that has controlled extension of monomer; The mixed system of four kinds of nucleotide monomers is as extending monomer reagent, and encapsulation is preserved separately; Whether 5 ' the end that when archaeal dna polymerase using during detection need to be according to experimental design, order-checking stops primer is modified with blocking group, selects the polysaccharase of respective type.
6. according to the detection reagent of the STR sequence high-flux detection method of the controlled extension of a kind of Base selection described in claim 5, it is characterized in that: described extension primer reagent, reaction buffer, fluorescence are sheared reagent, extended and recover reagent, sex change liquid reagent, all select biochemical reagents conventional in biological experiment.
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| CN104152568B (en) * | 2014-08-19 | 2016-03-16 | 东南大学 | High-throughput STR sequence core repeat number detection method |
| 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 |
| CN105483262B (en) * | 2016-01-11 | 2018-08-31 | 中国科学院北京基因组研究所 | A kind of detection kit of 10 STR bit points based on high-flux sequence |
| CN106222258B (en) * | 2016-07-27 | 2018-10-16 | 中国科学院北京基因组研究所 | The detection kit of the 35 STR allele based on high-flux sequence |
| CN105969905B (en) * | 2016-07-27 | 2018-10-16 | 中国科学院北京基因组研究所 | The detection kit of the 38 STR allele based on high-flux sequence |
| CN106048053B (en) * | 2016-08-03 | 2018-08-31 | 中国科学院北京基因组研究所 | The detection kit of the 38 STR allele based on high-flux sequence |
| CN106086210B (en) * | 2016-08-03 | 2018-08-31 | 中国科学院北京基因组研究所 | The detection kit of the 40 STR allele based on high-flux sequence |
| CN106399496B (en) * | 2016-09-06 | 2019-12-20 | 承启医学（深圳）科技有限公司 | Library building kit for high-throughput detection of STR genetic markers |
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