CN103898226A - Mitochondria SNP (single nucleotide polymorphism) fluorescence-labeling multiple amplification kit and application thereof - Google Patents
Mitochondria SNP (single nucleotide polymorphism) fluorescence-labeling multiple amplification kit and application thereof Download PDFInfo
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Abstract
The invention relates to a mitochondria SNP (single nucleotide polymorphism) fluorescence-labeling multiple amplification kit capable of detecting 61 SNP loci at the same time. According to a mitochondria phylogenetic tree, based on genetic characteristics of Chinese populatio, 61 SNP loci which are high in polymorphism, low in mutation rate and strong in parting capacity are selected, wherein the 61 SNP lotuses comprise 54 loci in a coding region, 7 loci in a control region; the kit can be used for detecting the 61 SNP loci by virtue of specific primers. The kit which is used as a mitochondria detecting system is capable of carrying out amplification in three tubes at the same time and performing electrophoresis at the same time, so that haplotype diversity reaches 98.6%, and therefore, the mitochondria SNP fluorescence-labeling multiple amplification kit can be used as a kit for identifying the same maternal line, after autosome STR and Y chromosome STR.
Description
Technical field
The invention belongs in vitro diagnostic techniques field, what embody a concentrated reflection of is third generation genetic marker SNP(single nucleotide polymorphism, single nucleotide polymorphism) and the combined utilization of detection technique of fluorescence, can utilize the DNA profiling mtDNA Haplogroup of definite sample fast of trace relatively, be applicable to the maternal evaluation of Mitochondrial DNA.
Background technology
On medical jurisprudence, be all to detect euchromosome STR genetic marker for the test kit of individual recognition at present, there is not association owing to not existing between them between the allelotrope of linkage relationship and different genetic markers, so product that when individual's identification, autosomal total matching probability is the matching probability of each genetic marker, therefore while carrying out individual recognition, do not need ad infinitum to increase genetic marker, the genetic marker of some amount just can meet the requirement of Forensic detection.And the mitochondrial inheritance mark that is applied to medicolegal practice is haplotype heredity, whole Mitochondrial Genome Overview is equivalent to a genetic marker, therefore the individual recognition capability of inheritance of mitochondrion DNA mark can not be calculated by product, improve mitochondrial individual recognition capability, only way finds and applies more polymorphic genetic marker exactly to cover more single times of realm type in mtDNA.
At present, mainly contain SnaPshot detection method, probe hybridization development process, direct sequencing etc. for mtDNA SNP detection method, SnaPshot detection technique refers to based on Cambridge standard sequence, 3 primers of each SNP design, wherein 2 is the amplification of object fragment, length is in 200-500bp left and right, and Tm value is in 60 degree left and right.Article 3, primer be to extend ddNTP, design is in the upstream in SNP site or reverse downstream.First enrichment obtains object fragment, removes unnecessary dNTP through enzymic digestion, after primer and other single stranded DNA products, carries out single base extension again, and reaction product is again carried out capillary electrophoresis and detected the SNP information that obtains site after enzyme is processed.This method design of primers is simple, synthetic easy low price.But this method also has obvious limitation: the digestive ferment that partial purification is used is expensive; Complex operation step, workload is large; It is low that PBR exists the site of unknown mutation to be detected as power, is in particular in that mitochondria control position point detects success ratio low.
On medical jurisprudence, the research of plastosome SNP is mainly adopted to direct sequencing at present, the main mtDNA control region (HV I HeHV II district) of detecting, this section of sequence is in the about 1122bp of mtDNA copy-controlled areas length, due to its coded protein not, therefore selective pressure is little during evolution, determines that it compares other regions and have more much higher state property.Obviously can obtain a large amount of information to this section of hypervariable region sequence order-checking, but because this region is originally as mutantional hotspot district, have genetic instability, so specificity is still not aobvious.Research finds to increase the specific pleomorphism site of coding region, can effectively expand doubly group's sensing range of plastosome list, can significantly improve mitochondrial same maternal identification capacity.Complete sequence order-checking is the best way in theory, but carries out the order-checking of mtDNA complete sequence, and workload is large, and technical requirements is higher, is difficult to promote, and is not suitable for launching large-scale application in existing legal medical expert laboratory.
The patent of Mitochondrial DNA research almost concentrates in the discussion of mitochondrial disease, and seldom relating to aspect maternal evaluation, what only one piece " a kind of method and application of detection line plastochondria gene haplotype monoid " adopted is the method for oligonucleotide probe, real-time quantitative detection method has highly sensitive, specificity is good, but exist flux low, the shortcomings such as primer probe design complexity, are not suitable for carrying out large sample detection simultaneously.
Summary of the invention
The appearance of plastosome SNP fluorescence labeling composite amplification test kit, for efficient rapid detection single nucleotide mutation provides approach.By setting up the pattern of mtDNA hypervariable region and coding region joint-detection, can set up a kind of quick, efficient, reliable mtDNA haplotype somatotype strategy and detection method.Identical with existing STR test kit working method, meet current legal medical expert's lab assistant state of the art, and the detection of applicable large sample, have great application prospect.
The object of this invention is to provide a kind of plastosome SNP fluorescence labeling composite amplification test kit, adopt four look fluorescent mark technology, specificity fluorescent primer amplification detects in conjunction with capillary electrophoresis, take FAM, HEX, TAMRA, SIZ fluorescent signal as detection signal.By hereditary sequenator, the collection of fluorescent signal is detected to specific mtDNA sudden change, therefore the present invention is increased to 61 sites on the basis in 35 SNP sites (patent publication No.: CN103290108A) of applying for before, can effectively cover more single times of realm type.
Technical scheme is:
A kind of plastosome SNP fluorescence labeling composite amplification test kit, include the primer for following 61 the SNP sites of increasing: G10398A, Norm9bpDel, C10873T, A3010G, A709G, C7196A, C12705T, C3970T, G13104A, G10310A, C5178A, G13928C, G6446A, C8414T, C8794T, C8793T, C16126T, G16129A, A15043G, C16311T, A8701G, A8697G, C4883T, C10400T, (CA) n, G1719A, C14668T, C12811T, C, T9824A, C9698T, A9123G, C7028T, G11719A, G8584A, A11251G, G8020A, G5460A, G2706A, C11215T, T4216C, G12372A, C16362T, C1541T, C8684T, G9477A, A4491G, G1811A, G16316A, A16319G, G9545A, C11944T, C152T, G14569A, C8964T, G10397A, G3348A, G4833A, G7600A, G5417A, C5442T, C15784T.
Further, above-mentioned primer is preferably as follows:
A709G, SEQ ID NO.1-3; A3010G, SEQ ID NO.4-6; C5178A, SEQ ID NO.7-9; Norm9bpDel, SEQ ID NO.10-11; G10398A, SEQ ID NO.12-14; C10873T, SEQ ID NO.15-17; C12705T, SEQ IDNO.18-20; G13928C, SEQ ID NO.21-23; C16311T, SEQ ID NO.24-26; A15043G, SEQ IDNO.27-29; C3970T, SEQ ID NO.30-32; C8414T, SEQ ID NO.33-35; G6446A, SEQ ID NO.36-38; C7196A, SEQ ID NO.39-41; G10310A, SEQ ID NO.42-44; G13104A, SEQ ID NO.45-47; C8793T and C8794T, SEQ ID NO.48-51; C16126T and G16129A, SEQ ID NO.52-55; C4883T, SEQ IDNO.56-58; C10400T, SEQ ID NO.59-61; (CA) n, SEQ ID NO.62-63; G11719A, SEQ ID NO.64-66; G1719A and C1541T, SEQ ID NO.67-71; G8584A and A8701G and A8697G, SEQ ID NO.72-77; A9123G, SEQ ID NO.78-80; C7028T, SEQ ID NO.81-83; C14668T, SEQ ID NO.84-86; G2706A, SEQ ID NO.87-89; G12372A, SEQ ID NO.90-92; T4216C, SEQ ID NO.93-95; A11251G and C11215T, SEQ ID NO.96-100; G5460A, SEQ ID NO.101-103; C12811T, SEQ ID NO.104-106; G8020A, SEQ ID NO.107-109; C, T9824A, SEQ ID NO.110-112; C9698T, SEQ ID NO.113-115; C16362T, SEQ ID NO.116-118; C152T, SEQ ID NO.119-121; G1811A, SEQ ID NO.122-124; A4491G, SEQ ID NO.125-127; G4833A, SEQ ID NO.128-130; G5417A and C5442T, SEQ IDNO.131-135; G7600A, SEQ ID NO.136-138; C8684T, SEQ ID NO.139-141; C8964T, SEQ IDNO.142-144; G9477A and G9545A, SEQ ID NO.145-149; G10397A, SEQ ID NO.150-152; G3348A, SEQ ID NO.153-155; C11944T, SEQ ID NO.156-158; G14569A, SEQ ID NO.159-161; C15784T, SEQ ID NO.162-164; G16316A and A16319G, SEQ ID NO.165-168.
In above primer, C8793T and C8794T, C16126T and G16129A, G16316A and A16319G, G11719A and G1719A, G8584A and A8701G and A8697G, A11251G and C11215T, C, T9824A and C9698T, G5417A and C5442T, G9477A and G9545A site share same forward primer.
Further, above primer is packet marking, and G10398A, Norm9bpDel, C10873T, A3010G, A709G, C7196A, C12705T, C3970T, G13104A, G10310A, C5178A, G13928C, G6446A, C8414T, C8794T, C8793T, C16126T, G16129A, A15043G, primer corresponding to C16311T site are first group; A8701G, A8697G, C4883T, C10400T, (CA) n, G1719A, C14668T, C12811T, C, T9824A, C9698T, A9123G, C7028T, G11719A, G8584A, A11251G, G8020A, G5460A, G2706A, C11215T, T4216C, G12372A, C16362T, primer corresponding to C1541T site are second group; C8684T, G9477A, A4491G, G1811A, G16316A, A16319G, G9545A, C11944T, C152T, G14569A, C8964T, G10397A, G3348A, G4833A, G7600A, G5417A, C5442T, primer corresponding to C15784T site are the 3rd group.Above-described " first ", " second " and " the 3rd " do not form the restriction to precedence.
Wherein, 9824 sites have the situation that base is C, T or A, are described as in the present invention C, T9824A.
Further, the forward primer in above primer is fluorescently-labeled.
Further, in above primer the forward primer of first group by FAM mark; The forward primer of second group is by HEX mark; The forward primer of the 3rd group is by TAMRA mark.
The present invention also provides the using method of mentioned reagent box, and concrete technical scheme is:
A using method for plastosome SNP fluorescence labeling composite amplification test kit, comprises the steps:
S1: extract Mitochondrial DNA from biological material, as mt DNA profiling; Described biological material can be hair, stub, tooth, cast-off cells, bone, blood, seminal stain, saliva etc.; The method of above DNA extraction method DNA can be that paramagnetic particle method, Chelex-100 extraction method, silicon fiml method etc. are diluted to suitable concn after simply quantitatively;
S2: preparation PCR reaction solution, add reaction buffer, Taq enzyme, composite primer, water, DNA profiling, adopts three step TRAP, and sample is increased;
S3: amplified production is carried out to electrophoresis, and software analysis electrophoresis result is also carried out Haplogroup somatotype.When electrophoresis, need in race model hole, add in deionized formamide, molecular weight and mark; Interior target effect is the interior mark indication for electrophoresis process.Can also add allelic ladder, be used for comparing definite genotype with unknown sample.
The application of above test kit in case investigation, paternity test and corpse source are assert.
Beneficial effect
1, Mitochondrial DNA is divided into control region and coding region, wherein the HVI of control region and HVII region mutation rate are higher, the part mainly detecting for current legal medical expert's plastosome, but there are some researches show, the random coupling probability of HV I is 2%, HV II is 4%, only rely on control region to be sometimes difficult to two irrelevant individualities to make a distinction, therefore the present invention has rationally avoided this shortcoming choosing of site, 54 coding regions and site, 7 control regions joint-detection are adopted, the feature of high site of control region mutation rate and site, coding region genetic stability is combined, greatly increase the distinguishing ability of test kit, optimize the multiplex PCR system in composite amplification SNP site, and DNA composite amplification is combined with capillary electrophoresis technique, greatly improve check speed and efficiency, surmount the detection speed of existing detection method completely, can become the main detection method of case check and database establishment.
2, the inventive method is reasonable in design, has set up a kind of novel mtDNA Haplogroup classifying method of fluorescent dye primer specific amplification in conjunction with capillary electrophoresis technique that utilize;
3, sample strong adaptability, for the little sample of some DNA extraction amounts, as bone, tooth and hair, this test kit still can detect preferably.Even when sample extraordinary obsolescence or severely degrade, the mt DNA in tenuigenin remains reliable detection material, and it is many that this has benefited from cell mt DNA copy number, and be wrapped in by double-layer cell wall, is difficult for degraded;
4, this test kit is controlled in 325bp the detection clip size of SNP, makes to be detected as power and greatly improves.Tradition STR detects failed situation still can successful somatotype;
5, apply plastosome SNP fluorescence detection reagent kit of the present invention and carry out maternal evaluation, more traditional sequence measurement cost is low, easy and simple to handle, data analysis is simple, can in 3～4 hours, draw experimental result, therefore can in extensive corpse source identification, paternity test, human origin and Study on Evolution, be used widely.
Accompanying drawing explanation
Fig. 1 is the position view of plastosome SNP61 site layout viewing and interior mark siz-500 clip size;
Fig. 2 is the corresponding design of primers principle schematic in SNP site and contiguous site;
Fig. 3 a is that heterogeneous site kit detects collection of illustrative plates;
Fig. 3 b is heterogeneous site kit order-checking collection of illustrative plates;
Fig. 4 a is that sister I sisterhood is identified collection of illustrative plates;
Fig. 4 b is that sister II sisterhood is identified collection of illustrative plates;
Fig. 5 a is that standard substance 9947A detects collection of illustrative plates;
Fig. 5 b is the detection collection of illustrative plates through the DNA profiling of artificial reconstructed and the complementation of 9947A somatotype.
Embodiment
By reference to the accompanying drawings the present invention is described in further detail below by embodiment.But it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, (for example show with reference to J. Pehanorm Brooker etc. according to the described technology of the document in this area or condition, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
The foundation of the choosing of embodiment 1 plastosome SNP of the present invention site, grouping, design of primers and reaction system
1, SNP site screening
Reference line plastochondria phylogenetic tree (mtDNA tree Build16 (19Feb2014)), in conjunction with Chinese population inheritance of mitochondrion DNA feature, filters out the characteristic site of main chondriogen Haplogroup and hypotype thereof; Polymorphism investigation and literature survey, database analysis according to Chinese population mtDNA Haplogroup somatotype tree joint line plastochondria in Chinese population finally filter out 61 mtDNA characteristic sites, all there is higher GD value, contained the main mtDNA Haplogroup of Chinese population and Main Subtype thereof.Wherein 73.7% site GD value is positioned at more than 0.2, and maximum value has reached 0.5.Guarantee the distinguishing ability (selected 61 sites of the present invention and GD value see attached list 3) of human mtdna checking system.
Gene diversity (GD), by formula GD=N(1-∑ P
i 2)/(N-1) calculated, and n is Sample size, P
ifor gene frequency.
The allelotrope in all 61 sites that table 1 the present invention relates to and site GD value
| SNP site | Allelotrope | GD value | SNP site | Allelotrope | GD value |
| 10398 | G,A | 0.50 | 11719 | G,A | 0.22 |
| 9bp | DEL,NORM | 0.30 | 8584 | G,A | 0.24 |
| 10873 | T,C | 0.45 | 11251 | A,G | 0.09 |
| 3010 | G,A | 0.32 | 8020 | G,A | 0.28 |
| 709 | G,A | 0.27 | 5460 | G,A | 0.22 |
| 7196 | C,A | 0.03 | 2706 | G,A | 0.19 |
| 12705 | C,T | 0.50 | 11215 | C,T | 0.23 |
| 3970 | C,T | 0.21 | 4216 | T,C | 0.09 |
| 13104 | G,A | 0.12 | 12372 | G,A | 0.14 |
| 10310 | G,A | 0.13 | 16362 | C,T | 0.36 |
| 5178 | C,A | 0.20 | 1541 | C,T | 0.30 |
| 13928 | G,C | 0.21 | 9698 | C,T | 0.17 |
| 6446 | G,A | 0.25 | 8684 | T,C | 0.23 |
| 8414 | C,T | 0.18 | 9477 | G,A | 0.23 |
| 8794 | C,T | 0.34 | 4491 | A,G | 0.14 |
| 8793 | C,T | 0.30 | 1811 | G,A | 0.16 |
| 15043 | A,G | 0.41 | 16319 | G,A | 0.18 |
| 16311 | C,T | 0.30 | 16316 | G,A | 0.19 |
| 16129 | A,G | 0.27 | 9545 | G,A | 0.40 |
| 16126 | C,T | 0.25 | 11944 | C,T | 0.12 |
| 8701 | A,G | 0.45 | 152 | C,T | 0.43 |
| 8697 | A,G | 0.50 | 14569 | G,A | 0.14 |
| 4883 | C,T | 0.20 | 8964 | C,T | 0.16 |
| 10400 | C,T | 0.39 | 10397 | G,A | 0.44 |
| |
3,4,5,6,7,8 | 0.51 | 3348 | G,A | 0.04 |
| 1719 | G,A | 0.34 | 4833 | G,A | 0.11 |
| 14668 | C,T | 0.18 | 7600 | G,A | 0.12 |
| 12811 | C,T | 0.42 | 5417 | G,A | 0.14 |
| 9824 | T,C,A | 0.35 | 5442 | C,T | 0.18 |
| 9123 | A,G | 0.11 | 15784 | C,T | 0.24 |
| 7028 | CT | 0.19 |
Note: Norm refers to the normal allelotrope of sequence length, and Del refers to lack the allelotrope of 9bp base.
2, design of primers
After having determined SNP site, according to mutational site design specific detection primer (test kit characteristic site primer sequence is in table 2).By artificial mutation is set, the artificial mutation optimum position of every primer is carried out groping one by one, and constructed plasmid corresponding to each SNP site and carry out primer test and screening, finally retain the primer of the high and high specificity of efficiency, be used for assembly composite amplification system.
Each design of primers principle as shown in Figure 2, except indivedual contiguous sites are as (C8793T and C8794T), (C16126T and G16129A), (G11719A and G1719A), (G8584A and A8701G and A8697G), (A11251G and C11215T), (C, T9824A and C9698T), (G5417A and C5442T), outside employing shared labeled primer in (G9477A and G9545A) site detects simultaneously, all there is each self-corresponding labeled primer in all the other sites, the primer in each SNP site is according to revised Cambridge reference sequences (revised Cambridge Reference Sequence, rCRS) NC_012920.1 designs, wherein 5 ' of F-primer end carries out mark with corresponding fluorescein, 3 ' end of two other non-marked primer is to equipotential gene order specific recognition, article two, wherein arbitrary the 5 ' end of nonstandard primer introduced 3～4 and the unpaired Nucleotide of template, general " TATA " base of adding, minimum on the impact of primer TM value.Because the length difference of two non-marked primers causes amplified production length difference, electrophoretic migration speed difference, finally reaches the object of detection.
The polymorphism that due to 9bp disappearance and (CA) n repeats both shows as length polymorphism, only need to design respectively a F-primer and non-marked primer, just can vary in size and be added on differentiation according to product.
For most site, each site comprises two non-marked primers and a fluorescent dye primer, 3 ' of above-mentioned two primers hold last base pair allelotrope template to carry out specific recognition, in order further to improve specific amplification, introducing artificial mispairing apart from 3 ' 2～7 base places of end, artificial mispairing position is also different apart from the specificity of different primers from 3 ' end, find through repeatedly groping, for the higher FAM of fluorescence efficiency, the fluorescein-labeled primer of HEX, when artificial mispairing from 3 ' end when the 2nd base place primer mispairing specificity best, for the fluorescein-labeled primer of the poor TAMRA of fluorescence efficiency, artificial mispairing is placed in from the 3rd base place effect of 3 ' end better.Follow following principle as for the base of introducing what type: if 3 ' end is strong mispairing (T-C, A-G pairing), should introduce weak mispairing (A-C, G-T pairing); If 3 ' end is moderate mispairing (A-A, G-G, C-C, T-T pairing), just introduce moderate mispairing; If 3 ' end is weak mispairing, should introduce strong mispairing.In addition, in order to improve allelic resolution, two non-marked primers are by holding plug-in 3～4 bases to make amplified production have difference in length 5 ', and the allelotrope in each site can reach by amplified production length difference the object of differentiation.Consider the needs of follow-up primer composite amplification, all between 62～64 ℃ (TM value calculating method is nearest neighbor algorithm (the nearestneighbor method)) of the primer Tm of this design value.
The primer that each site of table 2 is corresponding
Owing to there are differences between every primer efficiency, dosage difference when making every primer will reach same amplification peak value in system, the proper operation concentration of each primer adopts primer concentration grads experiment to grope, and has finally obtained the working concentration of each primer, as shown in table 3.
In composite primer, the working concentration of every primer is to test acquisition by primer concentration grads, every primer concentration does concentration gradient from 0.01 μ M to 0.2 μ M, gradient is spaced apart 0.02 μ M, do single larger primer concentration scope of acquisition that expands and then organize further fine setting primer concentration of multiple expansion primer repetition measurement examination, most important index is to occur non-specific peak; According to detected result, select the peak height RFU primer concentration between 2000～2500 as preferably.
The final concentration in amplification system of the primer in the each site of table 3 test kit
3, the SNP assembled scheme of fluorescence labeling composite amplification system design
In test kit provided by the invention, the corresponding upstream primer (F-primer) in each SNP site can carry out mark by isotropic substance, fluorescein, relatively finds that fluorescein-labelled effect is better.Corresponding downstream primer (R-primer) is the primer not being labeled.
The present invention has carried out discriminating, has selected fluorescence dye, has selected blue, green, black, four kinds of fluorescent markers of orange, has built four look fluorescence assembled schemes.
Determining on the basis of 4 look fluorescence assembled schemes, by experiment repeatedly in a large number, follow low fluorescein-labelled as TAMRA of use fluorescence efficiency that primer efficiency is high, high fluorescein-labelled as FAM of the inefficient use fluorescence efficiency of primer, the principle that be labeled as HEX of primer efficiency in holding.Find out the fluorescent mark type in 61 SNP Sites Combination modes and each site, 61 SNP sites are divided into 3 groups, in molecular weight, mark carries out mark with orange fluorescence dye SIZ.Being combined as of final definite fluorochrome label: wherein the F-primer in G10398A, Norm9bpDel, C10873T, A3010G, A709G, C7196A, C12705T, C3970T, G13104A, G10310A, C5178A, G13928C, G6446A, C8414T, C8794T, C8793T, C16126T, G16129A, these 20 sites of A15043G, C16311T is by FAM mark.A8701G, A8697G, C4883T, C10400T, (CA) n, G1719A, C14668T, C12811T, C, the F-primer in T9824A, C9698T, A9123G, C7028T, G11719A, G8584A, A11251G, G8020A, G5460A, G2706A, C11215T, T4216C, G12372A, C16362T, these 23 sites of C1541T is by HEX mark.The F-primer in C8684T, G9477A, A4491G, G1811A, G16316A, A16319G, G9545A, C11944T, C152T, G14569A, C8964T, G10397A, G3348A, G4833A, G7600A, G5417A, these 18 sites of C5442T, C15784T is by TAMRA mark.By this packet mode site of can as much as possiblely arranging on the one hand, cover more Haplogroup; Can make on the other hand the non-object band producing because interacting between primer outside software monitoring range, in actual use, the primer of FAM color marker and corresponding nonstandard primer thereof are formulated as to FAM look composite primer; In like manner prepare HEX look composite primer and TAMRA look composite primer.Interior mark is selected fluorescent orange mark, fluorescent marker is SIZ (marking in the siz500 fluorescent mark of preparation method with reference to our company's independent research disclosing in patent CN101307226, altogether 13 fluorescent mark fragment: 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 500bp).This SNP Sites Combination mode makes only to need 4 kinds of fluorescence of mark just can realize these 61 SNP sites and detects analysis simultaneously.Four look layout viewings are shown in Fig. 1.
In test kit, also include two cover positive controls, a set of is 9947A, and another set of is and the engineered DNA sequence dna of the each site of 9947A somatotype complementation that positive control is as the positive quality control product of each experiment, for monitoring the exactness of pcr amplification system, get rid of false negative; Negative control, for controlling the pollution condition of preparation plant process, is got rid of false positive.As follows with the artificial sequence preparation method of 9947A somatotype complementation: successively to adopt rite-directed mutagenesis, merging PCR and clone technology obtains, first according to each site allelotrope information of 9947A, design another allelic rite-directed mutagenesis primer of each site, catastrophe point is placed in the middle of primer, approximately 78 ℃ of primer TM values and just reverse complemental of upstream and downstream primer, amplify like this each fragment owing to containing complementary sequence, can be directly used in the fusion product that next step merges each fragment of amplification acquisition, fusion product is proceeded in DH5 α and preserved through being cloned into pMD18-T, extract plasmid and be standard substance.
Primer in test kit of the present invention, through strict design and checking, has specific amplification and the species specificity of height.3, amplification and product detect
(1) first complete the preparation of the PCR reaction system outside removing template on ice, finally add template, positive control and negative control after the first sample to be checked of template addition sequence, the Taq enzyme in the present embodiment test kit is to adopt the warm start high temperature resistant enzyme disclosing in patent publication No. CN101050453; While extracting Mitochondrial DNA template, preferably will be by 50～100 times of the DNA dilutions of extracting, in actually operating, the mt DNA profiling that generally can get 1～2 μ l increases.Concrete dosage is as shown in table 4.
Table 4 test kit amplification system
(2) instantaneous centrifugal after above-mentioned system preparation, be placed on thermal cycler, increase according to the program shown in table 5.
Table 5 plastosome SNP detection kit amplification amplification program
(3) amplified production detects and interpretation of result
Adopt mark (AGCU Marker SIZ-500) in the DNA denaturing agent deionized formamide molecular weight supporting with test kit to form loading mixed solution for 12:0.5 mixes by volume.12.5 μ L loading mixed solutions and 1 μ L amplified production and the supporting alleles analysis standard substance of test kit are mixed, mix rear instantaneous centrifugal removal bubble.After 98 ℃ of sex change 5min, place immediately 3min on ice, carry out electrophoresis detection with genetic analyzer ABI3100, GeneMapperID v3.2 software is analyzed result.
The result increasing respectively to standard substance 9947A with through artificial reconstructed and the DNA profiling complementation of 9947A somatotype, as shown in Fig. 5 a and Fig. 5 b, can find out that the amplified production peak between each site is obvious, occurs without other non-specific amplification product.
In Fig. 5 a, the site that each of the first row detects is:
10398,9bp, 10873,3010,709,7196,12705,3970,13104,10310,5178,13928,6446,8414,8794,15043,16311, (16126 and 16129).
Each site detecting of the second row is:
8701、4883、10400、(CA)n、1719、14668、12811、9824、9123、7028、11719、8584、11251、8020、5460、2706、11215、4216、12372、16362、9698、1541。
The site that each of the third line detects is:
8684,9477,4491,1811, (16316 and 16319), 9545,11944,152,14569,8964,10397,3348,4833,7600,5417,5442,15784.
The Accuracy Verification of embodiment 2 plastosome SNP of the present invention detection kit and large sample test
Adopt primer, grouping fluorescent mark and amplification system and parameter in embodiment 1, carry out large sample plastosome SNP and detect, choose at random altogether 200 Han nationality and 200 crowd of Uygur nationality samples, sample increases with test kit after extracting, and shows: the main somatotype result D4* of Han nationality, B5 through electrophoresis detection result, D5a, and D4b2 accounts for respectively 7%, 6.5%, 4.5%, and 4.5%, D4* in the crowd of the Uygur nationality equally, T, account for respectively 7.5%, 7% with W, and 6%.Han nationality is as the offspring of ancient family of China, and its diffusion profile meets De meter Qi diffusion model.At population Onset, between it and multiple nationality, there is gene exchange; For the Uygur nationality, their somatotype has at least 12 haplotypes and European and other ethnic groups common, and this demonstrates, and the Uygur nationality has European and gook mixes blood lineage.400 increments have originally been divided into into 106 kinds of haplotypes, and resolving power has reached 98.6%.In order to verify the accuracy of test kit, random choose three samples in each site use the BigDye sequencing kit direct Sequencing of AB company, and result shows that the two is in full accord, has fully proved the accuracy of this test kit, can be used in mtDNA SNP and detect analysis.
The detection of embodiment 3 test kit provided by the present invention to mtDNA SNP site heterogeneity
The existence of mtDNA heterogeneity, has brought uncertainty to medicolegal same maternal evaluation, should in actual case, evaluate accordingly.International medicolegal genetics meeting (International Society for Forensic Genetics, ISFG) regulation, if run into a base difference of two samples appearance, and to be recognized be heterogeneous focus in this base position, can not get rid of; When running into two base positions when different, if two positions are all heterogeneous focuses, can not get rid of, otherwise can not judge; In the time occurring that three positions are different, can make eliminating conclusion.Test kit provided by the present invention, not only can realize the detection in general SNP site, and still can successfully detect in the time that site exists heterogeneity.In actual sample detects, find that in the Uygur nationality's 200 increments bases, some sample 16319 site exists heterogeneous, and adopt direct Sequencing method validation, the two result is consistent, test kit detects electrophoretogram and accompanying drawing 3 is shown in by order-checking collection of illustrative plates, wherein 3a is that heterogeneous site kit detects collection of illustrative plates, and 3b is heterogeneous site order-checking collection of illustrative plates.
The application of embodiment 4 test kit provided by the present invention in maternal relation is identified
Test kit provided by the present invention has played booster action in a routine doubtful sister relation is identified case, has improved the confidence level of this routine maternal relation evaluation case from matrilinear inheritance angle.
1, determination step is as follows:
(1) collect the hair swatch in case to be identified: sisterhood identifies that sample is provided by certain forensic science laboratory.
(2) sample Mitochondrial DNA extracts: take DTT digestion to extract in conjunction with Chelex method.
(3) augmentation detection: the SNP that carries out Mitochondrial DNA according to test kit implementation step in embodiment 1 detects, the somatotype of sister I the results are shown in Figure 4a, the somatotype of sister II the results are shown in Figure 4b, and its comparing result sees the following form 6, and different sites are shown by bold Italic.
61 Genotypings of plastosome of table 6sister I and sister II
Result shows: there are 7 differences in 61 sites of detected result of the plastosome SNP of sister I and sister II, carry out haplotype analysis according to plastosome system growth tree and show that sister I is G type, sister II is C type, can be to get rid of their possibility from same maternal family in conjunction with these 2.
The explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of the claims in the present invention.To the above-mentioned explanation of the disclosed embodiments, make professional and technical personnel in the field can realize or use the present invention.To be apparent for those skilled in the art to the multiple modification of these embodiment, General Principle as defined herein can, in the situation that not departing from the spirit or scope of the present invention, realize in other embodiments.Therefore, the present invention will can not be restricted in these embodiment shown in this article, meet the wider scope consistent with principle disclosed herein and features of novelty but can be applied to.
SEQUENCE LISTING
<110> Shanghai Jin Bo Bioisystech Co., Ltd
Zhongde Meilian Biotech Co., Ltd. Wuxi
Shanghai City science and technology concerning criminal matters research institute
<120> plastosome SNP fluorescence labeling composite amplification test kit and application thereof
<130> none
<160> 168
<170> PatentIn version 3.5
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<212> DNA
<213> artificial sequence
<400> 115
gagacataca gaaatagtca aaccacat 28
<210> 116
<211> 25
<212> DNA
<213> artificial sequence
<400> 116
acattacagt caaatccctt cttgt 25
<210> 117
<211> 28
<212> DNA
<213> artificial sequence
<400> 117
atatcattac agtcaaatcc cttctagc 28
<210> 118
<211> 23
<212> DNA
<213> artificial sequence
<400> 118
ataccaaatg catggagagc tcc 23
<210> 119
<211> 28
<212> DNA
<213> artificial sequence
<400> 119
atattgaacg taggtgcgat aaatatta 28
<210> 120
<211> 25
<212> DNA
<213> artificial sequence
<400> 120
ttgaacgtag gtgcgataaa tactg 25
<210> 121
<211> 25
<212> DNA
<213> artificial sequence
<400> 121
ataagacatc acgatggatc acagg 25
<210> 122
<211> 29
<212> DNA
<213> artificial sequence
<400> 122
atgttagtcc ttgctatatt atgctttgc 29
<210> 123
<211> 32
<212> DNA
<213> artificial sequence
<400> 123
tataggttag tccttgctat attatgctag gt 32
<210> 124
<211> 25
<212> DNA
<213> artificial sequence
<400> 124
accttactac cagacaacct tagcc 25
<210> 125
<211> 25
<212> DNA
<213> artificial sequence
<400> 125
ctgcaaagat ggtagagtag acgat 25
<210> 126
<211> 29
<212> DNA
<213> artificial sequence
<400> 126
attcctgcaa agatggtaga gtagattac 29
<210> 127
<211> 24
<212> DNA
<213> artificial sequence
<400> 127
accccatcct aaagtaaggt cagc 24
<210> 128
<211> 21
<212> DNA
<213> artificial sequence
<400> 128
acccagaggt tacccaagtc g 21
<210> 129
<211> 24
<212> DNA
<213> artificial sequence
<400> 129
tatacccaga ggttacccaa tgca 24
<210> 130
<211> 26
<212> DNA
<213> artificial sequence
<400> 130
tatgttaggg ttgtacggta gaactg 26
<210> 131
<211> 23
<212> DNA
<213> artificial sequence
<400> 131
gggtgggttt tgtatgttca tac 23
<210> 132
<211> 26
<212> DNA
<213> artificial sequence
<400> 132
tataggtggg ttttgtatgt tctaat 26
<210> 133
<211> 21
<212> DNA
<213> artificial sequence
<400> 133
agcgatgagt gtggggagca g 21
<210> 134
<211> 23
<212> DNA
<213> artificial sequence
<400> 134
atagtgatga gtgtggggac gaa 23
<210> 135
<211> 23
<212> DNA
<213> artificial sequence
<400> 135
gcacctgaaa caagctaaca tga 23
<210> 136
<211> 27
<212> DNA
<213> artificial sequence
<400> 136
gaagtagcgt cttgtagacc tactagt 27
<210> 137
<211> 24
<212> DNA
<213> artificial sequence
<400> 137
gtagcgtctt gtagacctac tggc 24
<210> 138
<211> 24
<212> DNA
<213> artificial sequence
<400> 138
gtagaagaac cctccataaa cctg 24
<210> 139
<211> 27
<212> DNA
<213> artificial sequence
<400> 139
tacccaacaa tgactaatca aactcac 27
<210> 140
<211> 24
<212> DNA
<213> artificial sequence
<400> 140
ccaacaatga ctaatcaaac gaat 24
<210> 141
<211> 25
<212> DNA
<213> artificial sequence
<400> 141
catttttaat cttagagcga aagcc 25
<210> 142
<211> 22
<212> DNA
<213> artificial sequence
<400> 142
aggttgaatg agtaggctgt tg 22
<210> 143
<211> 25
<212> DNA
<213> artificial sequence
<400> 143
atttggttga atgagtaggc ttata 25
<210> 144
<211> 24
<212> DNA
<213> artificial sequence
<400> 144
tttattgcca caactaacct cctc 24
<210> 145
<211> 24
<212> DNA
<213> artificial sequence
<400> 145
ataaaatcct gcgaagaaaa acac 24
<210> 146
<211> 27
<212> DNA
<213> artificial sequence
<400> 146
atagaaaaat cctgcgaaga aaacaat 27
<210> 147
<211> 20
<212> DNA
<213> artificial sequence
<400> 147
atgttggggg ccagtgaccc 20
<210> 148
<211> 23
<212> DNA
<213> artificial sequence
<400> 148
ataatattgg gggccagtgt cct 23
<210> 149
<211> 21
<212> DNA
<213> artificial sequence
<400> 149
caaagcacat accaaggcca c 21
<210> 150
<211> 27
<212> DNA
<213> artificial sequence
<400> 150
gagtgactac aaaaaggatt agacttg 27
<210> 151
<211> 30
<212> DNA
<213> artificial sequence
<400> 151
atagagtgac tacaaaaagg attagactca 30
<210> 152
<211> 28
<212> DNA
<213> artificial sequence
<400> 152
gagagtagct ataatgaaca gcgatagt 28
<210> 153
<211> 28
<212> DNA
<213> artificial sequence
<400> 153
attcctactc ctcattgtac ccatacta 28
<210> 154
<211> 25
<212> DNA
<213> artificial sequence
<400> 154
cctactcctc attgtaccca ttgtg 25
<210> 155
<211> 23
<212> DNA
<213> artificial sequence
<400> 155
ggggttcata gtagaagagc gat 23
<210> 156
<211> 27
<212> DNA
<213> artificial sequence
<400> 156
tcctgatcaa atatcactct cctaatc 27
<210> 157
<211> 30
<212> DNA
<213> artificial sequence
<400> 157
tatacctgat caaatatcac tctcctcctt 30
<210> 158
<211> 24
<212> DNA
<213> artificial sequence
<400> 158
agaatggggg ataggtgtat gaac 24
<210> 159
<211> 24
<212> DNA
<213> artificial sequence
<400> 159
aggggtttag tattgattgt tcgc 24
<210> 160
<211> 27
<212> DNA
<213> artificial sequence
<400> 160
tatcggggtt tagtattgat tgtgagt 27
<210> 161
<211> 24
<212> DNA
<213> artificial sequence
<400> 161
ctaaccccac taaaacactc acca 24
<210> 162
<211> 25
<212> DNA
<213> artificial sequence
<400> 162
tgctacttgt ccaatgatgg taatg 25
<210> 163
<211> 28
<212> DNA
<213> artificial sequence
<400> 163
atatgctact tgtccaatga tggtcaaa 28
<210> 164
<211> 21
<212> DNA
<213> artificial sequence
<400> 164
ggcgacccag acaattatac c 21
<210> 165
<211> 23
<212> DNA
<213> artificial sequence
<400> 165
tgctatgtac ggtaaatggc tgc 23
<210> 166
<211> 27
<212> DNA
<213> artificial sequence
<400> 166
attgtgctat gtacggtaaa tggttgt 27
<210> 167
<211> 25
<212> DNA
<213> artificial sequence
<400> 167
tatgctatgt acgataaatg gtgtc 25
<210> 168
<211> 23
<212> DNA
<213> artificial sequence
<400> 168
ccccatgctt acaagcaagt aca 23
Claims (10)
1. a plastosome SNP fluorescence labeling composite amplification test kit, is characterized in that: include the primer for following 61 the SNP sites of increasing: G10398A, Norm9bpDel, C10873T, A3010G, A709G, C7196A, C12705T, C3970T, G13104A, G10310A, C5178A, G13928C, G6446A, C8414T, C8794T, C8793T, C16126T, G16129A, A15043G, C16311T, A8701G, A8697G, C4883T, C10400T, (CA) n, G1719A, C14668T, C12811T, C, T9824A, C9698T, A9123G, C7028T, G11719A, G8584A, A11251G, G8020A, G5460A, G2706A, C11215T, T4216C, G12372A, C16362T, C1541T, C8684T, G9477A, A4491G, G1811A, G16316A, A16319G, G9545A, C11944T, C152T, G14569A, C8964T, G10397A, G3348A, G4833A, G7600A, G5417A, C5442T, C15784T.
2. plastosome SNP fluorescence labeling composite amplification test kit according to claim 1, is characterized in that, described primer is: A709G, SEQ ID NO.1-3; A3010G, SEQ ID NO.4-6; C5178A, SEQ ID NO.7-9; Norm9bpDel, SEQ ID NO.10-11; G10398A, SEQ ID NO.12-14; C10873T, SEQ ID NO.15-17; C12705T, SEQ ID NO.18-20; G13928C, SEQ ID NO.21-23; C16311T, SEQ ID NO.24-26; A15043G, SEQ ID NO.27-29; C3970T, SEQ ID NO.30-32; C8414T, SEQ ID NO.33-35; G6446A, SEQ ID NO.36-38; C7196A, SEQ ID NO.39-41; G10310A, SEQ ID NO.42-44; G13104A, SEQ ID NO.45-47; C8793T and C8794T, SEQ ID NO.48-51; C16126T and G16129A, SEQ ID NO.52-55; C4883T, SEQ ID NO.56-58; C10400T, SEQ ID NO.59-61; (CA) n, SEQ ID NO.62-63; G11719A, SEQ ID NO.64-66; G1719A and C1541T, SEQ ID NO.67-71; G8584A and A8701G and A8697G, SEQ ID NO.72-77; A9123G, SEQ ID NO.78-80; C7028T, SEQ ID NO.81-83; C14668T, SEQ ID NO.84-86; G2706A, SEQ ID NO.87-89; G12372A, SEQ ID NO.90-92; T4216C, SEQ ID NO.93-95; A11251G and C11215T, SEQ ID NO.96-100; G5460A, SEQ ID NO.101-103; C12811T, SEQ ID NO.104-106; G8020A, SEQ ID NO.107-109; C, T9824A, SEQ ID NO.110-112; C9698T, SEQ ID NO.113-115; C16362T, SEQ ID NO.116-118; C152T, SEQ ID NO.119-121; G1811A, SEQ ID NO.122-124; A4491G, SEQ ID NO.125-127; G4833A, SEQ ID NO.128-130; G5417A and C5442T, SEQ ID NO.131-135; G7600A, SEQ ID NO.136-138; C8684T, SEQ ID NO.139-141; C8964T, SEQ ID NO.142-144; G9477A and G9545A, SEQ ID NO.145-149; G10397A, SEQ ID NO.150-152; G3348A, SEQ ID NO.153-155; C11944T, SEQ ID NO.156-158; G14569A, SEQ ID NO.159-161; C15784T, SEQ ID NO.162-164; G16316A and A16319G, SEQ ID NO.165-168.
3. plastosome SNP fluorescence labeling composite amplification test kit according to claim 1, it is characterized in that, described primer is packet marking, and G10398A, Norm9bpDel, C10873T, A3010G, A709G, C7196A, C12705T, C3970T, G13104A, G10310A, C5178A, G13928C, G6446A, C8414T, C8794T, C8793T, C16126T, G16129A, A15043G, primer corresponding to C16311T site are first group; A8701G, A8697G, C4883T, C10400T, (CA) n, G1719A, C14668T, C12811T, C, T9824A, C9698T, A9123G, C7028T, G11719A, G8584A, A11251G, G8020A, G5460A, G2706A, C11215T, T4216C, G12372A, C16362T, primer corresponding to C1541T site are second group; C8684T, G9477A, A4491G, G1811A, G16316A, A16319G, G9545A, C11944T, C152T, G14569A, C8964T, G10397A, G3348A, G4833A, G7600A, G5417A, C5442T, primer corresponding to C15784T site are the 3rd group.
4. plastosome SNP fluorescence labeling composite amplification test kit according to claim 1, is characterized in that, the forward primer in described primer is fluorescently-labeled.
5. plastosome SNP fluorescence labeling composite amplification test kit according to claim 1, is characterized in that, in primer, the forward primer of first group is by FAM mark; The forward primer of second group is by HEX mark; The forward primer of the 3rd group is by TAMRA mark.
6. plastosome SNP fluorescence labeling composite amplification test kit according to claim 1, is characterized in that, the final concentration in amplification system of primer is:
7. a using method for plastosome SNP fluorescence labeling composite amplification test kit, is characterized in that, comprises the steps:
S1: extract Mitochondrial DNA from biological material, as mt DNA profiling;
S2: preparation PCR reaction solution, add reaction buffer, Taq enzyme, composite primer, water, DNA profiling, adopts three step TRAP, and sample is increased;
S3: amplified production is carried out to electrophoresis, and software analysis electrophoresis result is also carried out Haplogroup somatotype.
8. the using method of plastosome SNP fluorescence labeling composite amplification test kit according to claim 7, is characterized in that, described biological material can be hair, stub, tooth, cast-off cells, bone, blood, seminal stain, saliva.
9. the using method of plastosome SNP fluorescence labeling composite amplification test kit according to claim 7, is characterized in that: the program in pcr amplification is: (1) initial sex change, 95 ℃ of 3min; (2) thermal cycling, 94 ℃ of 30s, totally 10 circulations; 60 ℃ of 45s; 72 ℃ of 1min; 94 ℃ of 30s, totally 20 circulations; 58 ℃ of 45s; 72 ℃ of 1min; (3) whole extension, 72 ℃ of 10min; (4) insulation, 4 ℃.
10. the application of plastosome SNP fluorescence labeling composite amplification test kit claimed in claim 1 in case investigation, paternity test and corpse source are assert.
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| CN201410145409.2A CN103898226B (en) | 2014-04-11 | 2014-04-11 | A kind of plastosome SNP fluorescence labeling composite amplification test kit and application thereof |
| PCT/CN2014/077784 WO2015154332A1 (en) | 2014-04-11 | 2014-05-19 | Mitochondria snp fluorescence-labeling multiple amplification kit and use thereof |
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| CN201410145409.2A CN103898226B (en) | 2014-04-11 | 2014-04-11 | A kind of plastosome SNP fluorescence labeling composite amplification test kit and application thereof |
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| CN104212894A (en) * | 2014-08-25 | 2014-12-17 | 四川大学 | 26 mitochondria SNP genetic markers-based forensic medical composite detection kit |
| CN108998507A (en) * | 2018-07-24 | 2018-12-14 | 陈梦麟 | A kind of noninvasive high-flux detection method applied to crowd's complexity Relationship iden- tification |
| CN109280696A (en) * | 2017-07-21 | 2019-01-29 | 安塞斯（北京）生物技术有限公司 | The method of SNP detection technique fractionation mixing sample |
| CN109439765A (en) * | 2018-11-20 | 2019-03-08 | 江苏苏博生物医学科技南京有限公司 | Composite amplification detection kit that is a kind of while detecting 60 autosomes and Y chromosome locus |
| CN109706248A (en) * | 2018-07-06 | 2019-05-03 | 四川大学 | Forensic medicine composite detection kit and its application based on SNP-STR genetic marker |
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| CN107326075A (en) * | 2017-07-05 | 2017-11-07 | 公安部第研究所 | Target preparation method in a kind of fluorescence molecule amount |
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| CN109706248A (en) * | 2018-07-06 | 2019-05-03 | 四川大学 | Forensic medicine composite detection kit and its application based on SNP-STR genetic marker |
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| CN109439765A (en) * | 2018-11-20 | 2019-03-08 | 江苏苏博生物医学科技南京有限公司 | Composite amplification detection kit that is a kind of while detecting 60 autosomes and Y chromosome locus |
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Also Published As
| Publication number | Publication date |
|---|---|
| CN103898226B (en) | 2016-02-10 |
| WO2015154332A1 (en) | 2015-10-15 |
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