CN108179199A - Kit and its primer special combination based on two generation sequencing technologies detection X-STR locus - Google Patents
Kit and its primer special combination based on two generation sequencing technologies detection X-STR locus Download PDFInfo
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Abstract
The invention discloses the kit based on two generation sequencing technologies detection X str locus seats and its primer special combinations.Primer combination provided by the invention, is made of 32 kinds of DNA moleculars shown in sequence in sequence table 1 to sequence 32.It is demonstrated experimentally that using the STR partings of 16 X str locus seats in the genomic DNA of the kit detection women mouth epithelial cells provided by the invention based on two generation sequencing technologies detection X str locus seats, genotyping result is accurate.Kit provided by the invention has important application value.
Description
Technical field
The present invention relates to medical jurisprudence technical fields, and in particular to the examination based on two generation sequencing technologies detection X-STR locus
Agent box and its primer special combination.
Background technology
In modern medical jurisprudence, DNA material evidences play an important role, and will become more to weigh with the development of technology
It will.Currently, DNA material evidences mainly utilize Capillary Electrophoresis (capillary electrophoresis, CE) for str locus
Parting in terms of seat carries out length polymorphism detects.But CE can only carry out parting, nucleotide quantity phase to the length of locus
Deng all allele be considered as same allele, be easy to cause the equipotential base that some sequences are different but length is identical
Because being identified as same allele, and sequence difference existing for shoulder portion can not also be detected and be utilized, these are all serious
Influence the identification of Different Individual in case.In recent years, two generations sequencing (Next generation sequencing, NGS) technology
Gradually it is applied to medical jurisprudence STR parting researchs.Compared to CE partings, NGS technologies have many advantages in STR analyses：1st, energy
Enough obtain the base sequence of complete STR, the difference between the str locus seat of Different Individual is very clear；2nd, it can all cover
Each base of lid str locus seat and the high accuracy that depth is sequenced and ensures data for passing through output；3rd, sample input quantity is low,
It is more advantageous for the parting of the sample of trace sample and high degradation；4th, there is opening, ability of discovery and the new letter of searching
It is even better in breath ability, new parting and new mutation are meaned for str locus seat；5th, it is at low cost, by each
Sample sample addition label once can carry out parallel sequencing to multiple samples simultaneously, not only saved the time but also reduce sequencing into
This.
The X chromosome Short tandem repeatSTR (X-chromosome short tandem repeats, X-STR) of the mankind refers to
It is present in the Short tandem repeatSTR segment in non-recombinant area on X chromosome.Locus has the feature of sex-linked inheritance, and hereditary feature was both
Different from autosome, also different from Y chromosome, sex-kink feature is shown as, is had in certain Relationship iden- tifications important
Reference value.Sample is mixed for some men and women, only there are one allele due to male X-STR, it is impossible to be completely covered
The X-STR allele peak of women, better effect can be obtained than euchromosome STR parting by carrying out X-STR partings.For father and daughter
Relationship and the identification with father's sisterhood, X-STR partings have special value, if the X-STR phenotypes of two identified people are different,
It can exclude affiliation.Meanwhile X-STR also has one for certain affiliations, such as the identification of grandparent and grandchild female, aunt niece etc.
Fixed effect.
The commercialization CE kits of current existing X chromosome have Investigator Argus X-12Kit
(Fa.Qiagen) and Mentype1Argus X-8PCR amplification kit (Biotype AG, Dresden,
Germany) etc., but since its length polymorphism can only be measured, there is certain limitation.Illumina companies are also proposed packet
The two generation sequencing kits containing X-STR, i.e. ForenSeqTMDNA Signature Prep Kit, but wherein contain only 7
X-STR locus (DXS10135, DXS8378, DXS7132, DXS10074, DXS10103, HPRTB and DXS7423), and should
The sequencing data that the mating data analysis program of kit can only be directed to intrinsic locus in kit is analyzed, and is had certain
Limitation.In view of the particularity of X-STR and the limitation of CE, develop the detection X-STR locus based on two generation sequencing technologies
Kit have important value.
Invention content
The purpose of the present invention is carry out X-STR partings.
The present invention protects a kind of primer to combine first, and primer combination can be by primer 1, primer 2, primer 3, primer 4, primer
5th, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14, primer 15, primer 16,
Primer 17, primer 18, primer 19, primer 20, primer 21, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27,
Primer 28, primer 29, primer 30, primer 31 and primer 32 form.
The primer 1 can be following A1) or A2)：
A1) the single strand dna shown in the sequence 1 in sequence table；
A2) sequence 1 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 1 with phase
The DNA molecular of congenerous.
The primer 2 can be following A3) or A4)：
A3) the single strand dna shown in the sequence 2 in sequence table；
A4) sequence 2 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 2 with phase
The DNA molecular of congenerous.
The primer 3 can be following A5) or A6)：
A5) the single strand dna shown in the sequence 3 in sequence table；
A6) sequence 3 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 3 with phase
The DNA molecular of congenerous.
The primer 4 can be following A7) or A8)：
A7) the single strand dna shown in the sequence 4 in sequence table；
A8) sequence 4 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 4 with phase
The DNA molecular of congenerous.
The primer 5 can be following A9) or A10)：
A9) the single strand dna shown in the sequence 5 in sequence table；
A10 sequence 5) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 5
The DNA molecular of identical function.
The primer 6 can be following A11) or A12)：
A11) the single strand dna shown in the sequence 6 in sequence table；
A12 sequence 6) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 6
The DNA molecular of identical function.
The primer 7 can be following A13) or A14)：
A13) the single strand dna shown in the sequence 7 in sequence table；
A14 sequence 7) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 7
The DNA molecular of identical function.
The primer 8 can be following A15) or A16)：
A15) the single strand dna shown in the sequence 8 in sequence table；
A16 sequence 8) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 8
The DNA molecular of identical function.
The primer 9 can be following A17) or A18)：
A17) the single strand dna shown in the sequence 9 in sequence table；
A18 sequence 9) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 9
The DNA molecular of identical function.
The primer 10 can be following A19) or A20)：
A19) the single strand dna shown in the sequence 10 in sequence table；
A20 sequence 10) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 10
There is the DNA molecular of identical function.
The primer 11 can be following B1) or B2)：
B1) the single strand dna shown in the sequence 11 in sequence table；
B2 sequence 11) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 11
The DNA molecular of identical function.
The primer 12 can be following B3) or B4)：
B3) the single strand dna shown in the sequence 12 in sequence table；
B4 sequence 12) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 12
The DNA molecular of identical function.
The primer 13 can be following B5) or B6)：
B5) the single strand dna shown in the sequence 13 in sequence table；
B6 sequence 13) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 13
The DNA molecular of identical function.
The primer 14 can be following B7) or B8)：
B7) the single strand dna shown in the sequence 14 in sequence table；
B8 sequence 14) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 14
The DNA molecular of identical function.
The primer 15 can be following B9) or B10)：
B9) the single strand dna shown in the sequence 15 in sequence table；
B10 sequence 15) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 15
There is the DNA molecular of identical function.
The primer 16 can be following B11) or B12)：
B11) the single strand dna shown in the sequence 16 in sequence table；
B12 sequence 16) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 16
There is the DNA molecular of identical function.
The primer 17 can be following B13) or B14)：
B13) the single strand dna shown in the sequence 17 in sequence table；
B14 sequence 17) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 17
There is the DNA molecular of identical function.
The primer 18 can be following B15) or B16)：
B15) the single strand dna shown in the sequence 18 in sequence table；
B16 sequence 18) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 18
There is the DNA molecular of identical function.
The primer 19 can be following B17) or B18)：
B17) the single strand dna shown in the sequence 19 in sequence table；
B18 sequence 19) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 19
There is the DNA molecular of identical function.
The primer 20 can be following B19) or B20)：
B19) the single strand dna shown in the sequence 20 in sequence table；
B20 sequence 20) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 20
There is the DNA molecular of identical function.
The primer 21 can be following C1) or C2)：
C1) the single strand dna shown in the sequence 21 in sequence table；
C2 sequence 21) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 21
The DNA molecular of identical function.
The primer 22 can be following C3) or C4)：
C3) the single strand dna shown in the sequence 22 in sequence table；
C4 sequence 22) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 22
The DNA molecular of identical function.
The primer 23 can be following C5) or C6)：
C5) the single strand dna shown in the sequence 23 in sequence table；
C6 sequence 23) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 23
The DNA molecular of identical function.
The primer 24 can be following C7) or C8)：
C7) the single strand dna shown in the sequence 24 in sequence table；
C8 sequence 24) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 24
The DNA molecular of identical function.
The primer 25 can be following C9) or C10)：
C9) the single strand dna shown in the sequence 25 in sequence table；
C10 sequence 25) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 25
There is the DNA molecular of identical function.
The primer 26 can be following C11) or C12)：
C11) the single strand dna shown in the sequence 26 in sequence table；
C12 sequence 26) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 26
There is the DNA molecular of identical function.
The primer 27 can be following C13) or C14)：
C13) the single strand dna shown in the sequence 27 in sequence table；
C14 sequence 27) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 27
There is the DNA molecular of identical function.
The primer 28 can be following C15) or C16)：
C15) the single strand dna shown in the sequence 28 in sequence table；
C16 sequence 28) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 28
There is the DNA molecular of identical function.
The primer 29 can be following C17) or C18)：
C17) the single strand dna shown in the sequence 29 in sequence table；
C18 sequence 29) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 29
There is the DNA molecular of identical function.
The primer 30 can be following C19) or C20)：
C19) the single strand dna shown in the sequence 30 in sequence table；
C20 sequence 30) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 30
There is the DNA molecular of identical function.
The primer 31 can be following D1) or D2)：
D1) the single strand dna shown in the sequence 31 in sequence table；
D2 sequence 31) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 31
The DNA molecular of identical function.
The primer 32 can be following D3) or D4)：
D3) the single strand dna shown in the sequence 32 in sequence table；
D4 sequence 32) is passed through into the substitution of one or several nucleotide and/or lacks and ors add and has with sequence 32
The DNA molecular of identical function.
It is the primer 1, the primer 2, the primer 3, the primer 4, the primer 5, described in primer combination
Primer 6, the primer 7, the primer 8, the primer 9, the primer 10, the primer 11, the primer 12, the primer
13rd, the primer 14, the primer 15, the primer 16, the primer 17, the primer 18, the primer 19, the primer
20th, the primer 21, the primer 22, the primer 23, the primer 24, the primer 25, the primer 26, the primer
27th, the molar ratio of the primer 28, the primer 29, the primer 30, the primer 31 and the primer 32 concretely 1：
1：1：1：1：1：1：1：2：2：2：2：1：1：2：2：1：1：2： 2：1：1：1：1：1：1：1：1：1：1：1：1.
The present invention also protects a kind of composite amplification system based on X-STR locus, it may include any of the above-described primer
Combination.
The primer 1, the primer 2, the primer 3, the primer 4, the primer 5, the primer 6, the primer
7th, the primer 8, the primer 13, the primer 14, the primer 17, the primer 18, the primer 21, the primer
22nd, the primer 23, the primer 24, the primer 25, the primer 26, the primer 27, the primer 28, described draw
The concentration of object 29, the primer 30, the primer 31 and the primer 32 in the composite amplification system concretely 0.3 μ
M。
It is the primer 9, the primer 10, the primer 11, the primer 12, the primer 15, the primer 16, described
Concretely 0.6 μM of the concentration of primer 19 and the primer 20 in the composite amplification system.
The composite amplification system may also include the reagent carried out needed for pcr amplification reaction；It is described " it is anti-to carry out PCR amplification
Answer required reagent " including the primer needed for pcr amplification reaction.
Any of the above-described composite amplification system may include the aqueous solution and human genome that Master Mix, primer are combined
DNA (as template).
Any of the above-described composite amplification system can be 20 μ L, the water including 10 μ L Master Mix, the combination of 4 μ L primers
Solution and 1ng human genome DNAs (as template).
Any of the above-described composite amplification system can be 20 μ L, can specifically be combined by 10 μ L Master Mix, 4 μ L primers
Aqueous solution, 2.5 a concentration of 0.425ng/mL of μ L women mouth epithelial cells genomic DNA aqueous solution and 3.5 μ L
ddH2O is formed.
Any of the above-described Master Mix concretely Material Evidence Identification Center, Ministry of Public Security's products.
The present invention also protects a kind of kit, can contain any of the above-described primer combination or any of the above-described described compound
Amplification system；The purposes of the kit can be following (h1) or (h2) or (h3) or (h4)：(h1) X-STR partings；(h2)
SNP partings；(h3) Indel partings；(h4) genetic marker is detected.
The present invention also protects the preparation method of any of the above-described composite amplification system or the kit；The preparation method
The step of may include individually packing each primer in any of the above-described primer combination.
The present invention also protects any of the above-described primer combination or any of the above-described composite amplification system in reagent preparation
Application in box；The purposes of the kit can be following (h1) or (h2) or (h3) or (h4)：(h1) X-STR partings；(h2)
SNP partings；(h3) Indel partings；(h4) genetic marker is detected.
The present invention also protects any of the above-described primer combination or any of the above-described composite amplification system in detection X-STR
Application in parting and/or SNP partings and/or Indel partings and/or detection genetic marker.
The present invention also protects a kind of method for the genotype for detecting sample to be tested X-STR locus, specifically may include as follows
Step：
(1) using the genomic DNA of sample to be tested as template, PCR amplification is carried out using any of the above-described primer combination, is obtained
To pcr amplification product；
(2) pcr amplification product is subjected to the sequencing of two generations, the genotype of X-STR locus is judged according to sequencing result.
In the above method, the X-STR locus can be DXS6804, DXS10079, GATA31E08, GATA172D05,
DXS7423、DXS7424、DXS8378、DXS9895、DXS7132、DXS7133、DXS6789、DXS101、DXS6800、
16 in GATA165B12, DXS9902 and DXS6799, it is 15 arbitrary, 14 arbitrary, 13 arbitrary, 12 arbitrary, arbitrary
11, it is 10 arbitrary, 9 arbitrary, 8 arbitrary, 7 arbitrary, 6 arbitrary, 5 arbitrary, 4 arbitrary, 3 arbitrary, arbitrary 2
It is a or 1 arbitrary.
In the above method, the sample to be tested can be human oral epithelial cells.
Compared to the ForenSeq of illumina companiesTMDNA Signature Prep Kit, it is provided by the invention to be based on
Comprising 16 X-STR locus in the kit of the detection X-STR locus of two generation sequencing technologies, more new heredity letters are provided
Breath.And the maximum amplicon length of all X-STR locus is both less than 300bp in composite amplification system provided by the invention,
Be conducive to the analysis of degradation sample.It is demonstrated experimentally that it is based on two generation sequencing technologies detection X-STR locus using provided by the invention
Kit detection women mouth epithelial cells genomic DNA in 16 X-STR locus STR partings, genotyping result is accurate
Really.Kit provided by the invention has important application value.
Description of the drawings
Fig. 1 is the length of pcr amplification product of the corresponding primer of each X-STR locus in hg19 mankind's reference gene group.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is conventional method unless otherwise specified.Experiment material used in following embodiments is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative experiment in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Agencourt AMPure XP 5mL Kit are the product of Beckman Coulter companies, and catalog number is
A63880.2000 quantitative instruments of NanoDrop are the product of Thermo Fisher Scientific companies.Bis- generations of Miseq FGx
Sequenator is the product of Illumina companies.TruSeq DNA PCR-Free HT Library Prep Kit are Illumina
The product of company, catalog number FC-121-3003.KAPA Library Quantification Kit are KAPA companies
Product, catalog number KK4824.
Embodiment 1, based on two generation sequencing technologies detection X-STR locus kit preparation
First, the preparation of primer combination
Primer combination is made of 32 primers, for detecting 16 X-STR locus.The title of each X-STR locus,
It is detailed to expand the information such as the range of the corresponding Primer of X-STR locus, the nucleotide sequence of primer and allele genotype
It is shown in Table 1.In hg19 mankind's reference gene group, (information of hg19 human genomes is shown in net to the corresponding primer of each X-STR locus
Location http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/hg19.2bi t) in PCR expand
The length and nucleotide sequence for increasing production object refer to table 2.
Table 1
Table 2
2nd, the preparation of the kit of the detection X-STR locus based on two generation sequencing technologies
The kit of detection X-STR locus based on two generation sequencing technologies includes primer mixture.Primer mixture by
32 primers prepared by step 1 mix.
Embodiment 2, based on two generation sequencing technologies detection X-STR locus kit application
First, DNA sample prepares
The genomic DNA of a women mouth epithelial cells sample is extracted, is then diluted with ultra-pure water, is obtained a concentration of
The genomic DNA aqueous solution of the women mouth epithelial cells of 0.425ng/ μ L.
2nd, prepared by library
1st, PCR amplification
The genomic DNA aqueous solution of the women mouth epithelial cells obtained using step 1 is template, with 1 step 2 of embodiment
The primer mixture of preparation is primer, carries out PCR amplification, obtains pcr amplification product.
Reaction system is 20 μ L, is mixed by 10 μ L Master Mix (product of Material Evidence Identification Center, Ministry of Public Security), 4 μ L primers
Close the genomic DNA aqueous solution and 3.5 μ L ddH of object, 2.5 μ L women's mouth epithelial cells2O is formed.In the reaction system, draw
Object 1, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 13, primer 14, primer 17, primer 18, draws primer 2
Object 21, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27, primer 28, primer 29, primer 30,31 and of primer
The concentration of primer 32 is 0.3 μM, primer 9, primer 10, primer 11, primer 12, primer 15, primer 16, primer 19 and primer
20 concentration is 0.6 μM.
Response procedures：95℃11min；94 DEG C of 30s, 60 DEG C of 2min, 72 DEG C of 1min, 28 cycles；60℃60min； 4℃
It preserves.
2nd, it purifies and quantitative
(1) pcr amplification product is taken, is purified according to the specification step of Agencourt AMPure XP 5mL Kit.
(2) after completing step (1), pcr amplification product using 2000 quantitative instruments of NanoDrop is quantified, is obtained
PCR purified products.
3rd, prepared by library
PCR purified products are taken, are operated according to the specification of TruSeq DNA PCR-Free HT Library Prep Kit
Step carries out end reparation successively, product purification is repaired in end, the purifying for connecting A-tail, connection Adapter and connection product,
Then it is quantified according to the specification step of KAPA Library Quantification Kit progress library and library standardizes,
Library is completed to prepare.
3rd, loading is tested
The library that step 2 is taken to prepare, utilizes sequencing reagent Miseq Reagent Nano Kit v2 (illumina companies
Product) be sequenced in Miseq FGx bis- generations sequenators.
Experimental result is shown in Table 3.The result shows that the genomic DNA of the women mouth epithelial cells sample has obtained completely
STR partings are fully able to the requirement for meeting legal medical expert STR inspections.
Table 3
Embodiment 3, based on two generation sequencing technologies detection X-STR locus kit Accuracy Verification
First, DNA sample prepares
The genomic DNA of a women mouth epithelial cells sample is extracted, is then diluted with ultra-pure water, is obtained a concentration of
The genomic DNA aqueous solution of the women mouth epithelial cells of 0.425ng/ μ L.
2nd, capillary electrophoresis detection
The genomic DNA of women mouth epithelial cells that 1ng step 1 obtains is taken, according to the DNATyper X19 (Ministry of Public Security
The product of material evidence evaluating center) specification step carry out capillary electrophoresis detection, obtain the allele genotype of locus.
Genotyping result refers to the row of table 4 the 2nd.
3rd, two generation sequencing technologies detect
The genomic DNA of women mouth epithelial cells that method detecting step one according to embodiment 2 obtains, obtains gene
The allele genotype of seat.Genotyping result refers to the row of table 4 the 3rd.
The result shows that embodiment 1 prepare based on two generation sequencing technologies detection X-STR locus kit with
The locus that DNATyper X19 are overlapped, the parting knot in the genomic DNA of the women mouth epithelial cells of step 1 acquisition
Fruit is completely the same.
Table 4
Note：" -- " represents to be not present.
<110>Material Evidence Identification Center, Ministry of Public Security
<120>The kit of detection X-STR locus based on two generation sequencing technologies and its primer special combination
<160> 32
<170> PatentIn version 3.5
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<210> 8
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
tgaaagcccg gattcaaa 18
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 9
tagcgcctgg cacatagtag 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 10
agatttcctc cccatccatc 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 11
ggaacacgca catttgagaa 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 12
ctgggaaaca caggaagacc 20
<210> 13
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 13
cgacaagagc gaaactcca 19
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 14
tccatcctgg gacagttcac 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 15
tggctctgct caaggaatta 20
<210> 16
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 16
ttgggtgggg acacagag 18
<210> 17
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 17
ccagagaaac agaaccaata gga 23
<210> 18
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 18
tcccctctca tctatctgac tg 22
<210> 19
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 19
ttttaggtgt agcttcctta gatgg 25
<210> 20
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 20
catcttccaa gaatcagaag tctc 24
<210> 21
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 21
acagaaccaa taggagatag atgg 24
<210> 22
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 22
cctcgtgatc atgtaagttg g 21
<210> 23
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 23
tcagtccaaa tatctccctt ca 22
<210> 24
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 24
gcgcatgtat cccagaactt a 21
<210> 25
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 25
gggctggttc ccctgata 18
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 26
tgggaccttg tgattgtgtg 20
<210> 27
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 27
gccaagtatt ggactaaact gtacc 25
<210> 28
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 28
agttgactgt gattcctggt tt 22
<210> 29
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 29
ctgggtgaag agaagcagga 20
<210> 30
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 30
cattcatatc aggagtatgg gatca 25
<210> 31
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 31
gaagacattt tcgagctcat tc 22
<210> 32
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 32
ctacttgcac atgggatgga 20
Claims (10)
1. primer combines, by primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer
10th, primer 11, primer 12, primer 13, primer 14, primer 15, primer 16, primer 17, primer 18, primer 19, primer 20, primer
21st, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27, primer 28, primer 29, primer 30, primer 31 and draw
Object 32 forms；
The primer 1 is following A1) or A2)：
A1) the single strand dna shown in the sequence 1 in sequence table；
A2) sequence 1 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 1 with identical work(
The DNA molecular of energy；
The primer 2 is following A3) or A4)：
A3) the single strand dna shown in the sequence 2 in sequence table；
A4) sequence 2 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 2 with identical work(
The DNA molecular of energy；
The primer 3 is following A5) or A6)：
A5) the single strand dna shown in the sequence 3 in sequence table；
A6) sequence 3 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 3 with identical work(
The DNA molecular of energy；
The primer 4 is following A7) or A8)：
A7) the single strand dna shown in the sequence 4 in sequence table；
A8) sequence 4 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 4 with identical work(
The DNA molecular of energy；
The primer 5 is following A9) or A10)：
A9) the single strand dna shown in the sequence 5 in sequence table；
A10) sequence 5 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 5 with identical
The DNA molecular of function；
The primer 6 is following A11) or A12)：
A11) the single strand dna shown in the sequence 6 in sequence table；
A12) sequence 6 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 6 with identical
The DNA molecular of function；
The primer 7 is following A13) or A14)：
A13) the single strand dna shown in the sequence 7 in sequence table；
A14) sequence 7 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 7 with identical
The DNA molecular of function；
The primer 8 is following A15) or A16)：
A15) the single strand dna shown in the sequence 8 in sequence table；
A16) sequence 8 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 8 with identical
The DNA molecular of function；
The primer 9 is following A17) or A18)：
A17) the single strand dna shown in the sequence 9 in sequence table；
A18) sequence 9 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 9 with identical
The DNA molecular of function；
The primer 10 is following A19) or A20)：
A19) the single strand dna shown in the sequence 10 in sequence table；
A20) sequence 10 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 10 with phase
The DNA molecular of congenerous；
The primer 11 is following B1) or B2)：
B1) the single strand dna shown in the sequence 11 in sequence table；
B2) sequence 11 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 11 with identical
The DNA molecular of function；
The primer 12 is following B3) or B4)：
B3) the single strand dna shown in the sequence 12 in sequence table；
B4) sequence 12 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 12 with identical
The DNA molecular of function；
The primer 13 is following B5) or B6)：
B5) the single strand dna shown in the sequence 13 in sequence table；
B6) sequence 13 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 13 with identical
The DNA molecular of function；
The primer 14 is following B7) or B8)：
B7) the single strand dna shown in the sequence 14 in sequence table；
B8) sequence 14 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 14 with identical
The DNA molecular of function；
The primer 15 is following B9) or B10)：
B9) the single strand dna shown in the sequence 15 in sequence table；
B10) sequence 15 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 15 with phase
The DNA molecular of congenerous；
The primer 16 is following B11) or B12)：
B11) the single strand dna shown in the sequence 16 in sequence table；
B12) sequence 16 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 16 with phase
The DNA molecular of congenerous；
The primer 17 is following B13) or B14)：
B13) the single strand dna shown in the sequence 17 in sequence table；
B14) sequence 17 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 17 with phase
The DNA molecular of congenerous；
The primer 18 is following B15) or B16)：
B15) the single strand dna shown in the sequence 18 in sequence table；
B16) sequence 18 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 18 with phase
The DNA molecular of congenerous；
The primer 19 is following B17) or B18)：
B17) the single strand dna shown in the sequence 19 in sequence table；
B18) sequence 19 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 19 with phase
The DNA molecular of congenerous；
The primer 20 is following B19) or B20)：
B19) the single strand dna shown in the sequence 20 in sequence table；
B20) sequence 20 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 20 with phase
The DNA molecular of congenerous；
The primer 21 is following C1) or C2)：
C1) the single strand dna shown in the sequence 21 in sequence table；
C2) sequence 21 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 21 with identical
The DNA molecular of function；
The primer 22 is following C3) or C4)：
C3) the single strand dna shown in the sequence 22 in sequence table；
C4) sequence 22 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 22 with identical
The DNA molecular of function；
The primer 23 is following C5) or C6)：
C5) the single strand dna shown in the sequence 23 in sequence table；
C6) sequence 23 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 23 with identical
The DNA molecular of function；
The primer 24 is following C7) or C8)：
C7) the single strand dna shown in the sequence 24 in sequence table；
C8) sequence 24 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 24 with identical
The DNA molecular of function；
The primer 25 is following C9) or C10)：
C9) the single strand dna shown in the sequence 25 in sequence table；
C10) sequence 25 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 25 with phase
The DNA molecular of congenerous；
The primer 26 is following C11) or C12)：
C11) the single strand dna shown in the sequence 26 in sequence table；
C12) sequence 26 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 26 with phase
The DNA molecular of congenerous；
The primer 27 is following C13) or C14)：
C13) the single strand dna shown in the sequence 27 in sequence table；
C14) sequence 27 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 27 with phase
The DNA molecular of congenerous；
The primer 28 is following C15) or C16)：
C15) the single strand dna shown in the sequence 28 in sequence table；
C16) sequence 28 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 28 with phase
The DNA molecular of congenerous；
The primer 29 is following C17) or C18)：
C17) the single strand dna shown in the sequence 29 in sequence table；
C18) sequence 29 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 29 with phase
The DNA molecular of congenerous；
The primer 30 is following C19) or C20)：
C19) the single strand dna shown in the sequence 30 in sequence table；
C20) sequence 30 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 30 with phase
The DNA molecular of congenerous；
The primer 31 is following D1) or D2)：
D1) the single strand dna shown in the sequence 31 in sequence table；
D2) sequence 31 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 31 with identical
The DNA molecular of function；
The primer 32 is following D3) or D4)：
D3) the single strand dna shown in the sequence 32 in sequence table；
D4) sequence 32 by the substitution of one or several nucleotide and/or is lacked and ored add and with sequence 32 with identical
The DNA molecular of function.
2. primer combination as described in claim 1, it is characterised in that：It is the primer 1, the primer 2, the primer 3, described
Primer 4, the primer 5, the primer 6, the primer 7, the primer 8, the primer 9, the primer 10, the primer
11st, the primer 12, the primer 13, the primer 14, the primer 15, the primer 16, the primer 17, the primer
18th, the primer 19, the primer 20, the primer 21, the primer 22, the primer 23, the primer 24, the primer
25th, the primer 26, the primer 27, the primer 28, the primer 29, the primer 30, the primer 31 and described draw
The molar ratio of object 32 is 1：1：1：1：1：1：1：1：2：2：2：2：1：1：2：2：1：1：2：2：1：1：1：1：1：1：1：1：1：1：1：
1。
3. a kind of composite amplification system based on X-STR locus, is combined including primer described in claims 1 or 2.
4. composite amplification system as claimed in claim 3, it is characterised in that：
The primer 1, the primer 2, the primer 3, the primer 4, the primer 5, the primer 6, the primer 7, institute
State primer 8, the primer 13, the primer 14, the primer 17, the primer 18, the primer 21, the primer 22, institute
State primer 23, the primer 24, the primer 25, the primer 26, the primer 27, the primer 28, the primer 29, institute
State a concentration of 0.3 μM of primer 30, the primer 31 and the primer 32 in the composite amplification system；
The primer 9, the primer 10, the primer 11, the primer 12, the primer 15, the primer 16, the primer
19 and a concentration of 0.6 μM in the composite amplification system of the primer 20.
5. the composite amplification system as described in claim 3 or 4, it is characterised in that：The composite amplification system, which further includes, carries out PCR
Reagent needed for amplified reaction；" the carrying out the reagent needed for pcr amplification reaction " does not include drawing needed for pcr amplification reaction
Object.
6. a kind of kit contains primer combination or any composite amplification of claim 3 to 5 described in claims 1 or 2
System；The purposes of the kit is following (h1) or (h2) or (h3) or (h4)：(h1) X-STR partings；(h2) SNP partings；
(h3) Indel partings；(h4) genetic marker is detected.
7. the preparation method of kit described in any composite amplification system of claim 3 to 5 or claim 6, including inciting somebody to action
The step of each primer in primer combination described in claims 1 or 2 is individually packed.
8. primer combination or, any composite amplification system of claim 3 to 5 described in claims 1 or 2, in reagent preparation
Application in box；The purposes of the kit is following (h1) or (h2) or (h3) or (h4)：(h1) X-STR partings；(h2)SNP
Parting；(h3) Indel partings；(h4) genetic marker is detected.
9. primer combination or, any composite amplification system of claim 3 to 5 described in claims 1 or 2, in detection X-
Application in STR partings and/or SNP partings and/or Indel partings and/or detection genetic marker.
10. a kind of method for the genotype for detecting sample to be tested X-STR locus, includes the following steps：
(1) using the genomic DNA of sample to be tested as template, PCR amplification is carried out using primer combination described in claims 1 or 2, is obtained
To pcr amplification product；
(2) pcr amplification product is subjected to the sequencing of two generations, the genotype of X-STR locus is judged according to sequencing result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810168413.9A CN108179199B (en) | 2018-02-28 | 2018-02-28 | Kit for detecting X-STR locus based on second-generation sequencing technology and special primer combination thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810168413.9A CN108179199B (en) | 2018-02-28 | 2018-02-28 | Kit for detecting X-STR locus based on second-generation sequencing technology and special primer combination thereof |
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| CN108179199B CN108179199B (en) | 2020-09-22 |
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| CN101413030A (en) * | 2008-11-25 | 2009-04-22 | 中山大学 | Fluorescence labeled X-STR locus composite amplification system and use thereof |
| CN101787396A (en) * | 2010-03-26 | 2010-07-28 | 中山大学 | X-STR locus fluorescence labeling composite amplification system and application thereof |
| CN101956004A (en) * | 2010-08-13 | 2011-01-26 | 司法部司法鉴定科学技术研究所 | Fluorescent-marked 16 X-STR loci multiplex amplification system and application thereof |
| CN102250883A (en) * | 2011-07-01 | 2011-11-23 | 中山大学 | Fluorescent labeled X-STR gene locus multiplex PCR method and application thereof |
| CN103789414A (en) * | 2014-01-06 | 2014-05-14 | 基点认知技术(北京)有限公司 | Multiplex amplification kit of 17 short tandem repeats (STR) on X chromosomes |
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| CN107099529A (en) * | 2017-07-05 | 2017-08-29 | 公安部物证鉴定中心 | The kit of detection locus based on two generation sequencing technologies and its primer special combination |
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