CN108690843A - Amplimer group, banking process and SNP classifying methods - Google Patents
Amplimer group, banking process and SNP classifying methods Download PDFInfo
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- CN108690843A CN108690843A CN201710232840.4A CN201710232840A CN108690843A CN 108690843 A CN108690843 A CN 108690843A CN 201710232840 A CN201710232840 A CN 201710232840A CN 108690843 A CN108690843 A CN 108690843A
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Abstract
The present invention relates to molecular biology fields, provide a kind of amplimer group, it is used to prepare amplified production, the amplimer group includes the first primer and the second primer, and the first primer is from 3 ' ends to 5 ' ends successively including amplimer sequence and anchor primer sequence;The amplimer sequence is for expanding the nucleic acid fragment containing SNP site to be measured;In amplified production, the anchor primer sequence is used as sequencing primer, and the anchor primer sequence and its anchor station are located at the same side of the SNP site to be measured.The present invention also provides a kind of banking process and SNP classifying methods based on above-mentioned amplimer group.Library molecule prepared by amplimer group using the present invention, contain sequencing primer thereon, without additionally adding sequencing primer into system during SNP partings, avoid when multiple samples are sequenced due to sequencing primer is different and generations interferes with each other, improve and efficiency and accuracy be sequenced.
Description
Technical field
The present invention relates to molecular biology fields, more specifically to a kind of amplimer group, banking process and SNP
Classifying method.
Background technology
Single nucleotide polymorphism(Single Nucleotide Polymorphism, SNP)Refer to single core on genome
There are the variations such as conversion, transversion, insertion, missing on thuja acid position, there are many quantity, rich polymorphism.SNP is considered as heredity
Mark, many phenotypic differences of human body, all may be related with SNP to neurological susceptibility of drug or disease etc., therefore the parting pair of SNP
The treatment and medication of many diseases have positive meaning.
For genetic test, two generation high throughput sequencing technologies because its accurate, sensitive characteristic, application range constantly expand,
Life science and each different aspect of medical research are had been directed to, SNP is carried out using two generation high throughput sequencing technologies
The detection in site and current one of research hotspot.But based on the SNP classifying methods of two generation high throughput sequencing technologies, when
It needs that a variety of sequencing primers are added into sequencing system, different sequencing primers when sequencing sample is detected to multiple simultaneously
Between easy to produce and interfere with each other so that multidigit point is sequenced accuracy and reduces, from the accuracy rate that may be decreased SNP partings and detect.
Therefore, it is necessary to a kind of new amplimer group, banking process and SNP classifying methods, can avoid in same system
In simultaneously when being detected to multiple SNP sites to be measured, the phenomenon that being interfered with each other between different sequencing primers.
Invention content
The purpose of the present invention is to provide a kind of amplimer group, banking process and SNP classifying methods, it is intended to solve existing
When technology simultaneously detects multiple SNP sites in same system, the problem of being interfered with each other between different sequencing primers.
An object of the present invention is to provide a kind of amplimer groups, are used to prepare amplified production, the amplimer
Group includes the first primer and the second primer, and the first primer includes that amplimer sequence and anchoring are drawn successively from 3 ' ends to 5 ' ends
Object sequence;The amplimer sequence is for expanding the nucleic acid fragment containing SNP site to be measured;In amplified production, the anchoring
Primer sequence is used as sequencing primer, and the anchor primer sequence and its anchor station are located at the same side of the SNP site to be measured.
Preferably, contain cleavage site on second primer;The cleavage site is uracil or restriction enzyme
Cleavage site.
Preferably, the amplimer sequence length is 15 to 25bp;The anchor primer sequence length is 15 to 20bp.
Preferably, the annealing temperature of the amplimer sequence anchoring is 57 to 63 DEG C;The anchor primer sequence anchoring
Annealing temperature be 42 to 50 DEG C.
Second object of the present invention is to propose a kind of banking process, include the following steps:
A, PCR amplification is carried out to the nucleic acid fragment containing SNP site to be measured using amplimer group, obtains amplified production;The expansion
It includes the first primer and the second primer to increase object group, and the first primer is from 3 ' ends to 5 ' ends successively including amplimer sequence and anchor
Determine primer sequence;The amplimer sequence is for expanding the nucleic acid fragment containing SNP site to be measured;It is described in amplified production
Anchor primer sequence is used as sequencing primer, and the anchor primer sequence and its anchor station are located at the same of the SNP site to be measured
Side;
B, in jointing on the opposite end at end where amplified production sequence containing anchor primer, library molecule is obtained.
Preferably, the distance between the anchor station of the anchor primer sequence and SNP site to be measured are 1 to 5bp.
Preferably, uracil is contained at 5 ' ends of the first primer, and the step B is further comprising the steps of:
B0, amplified production is cut using the enzyme of specificity cutting uracil, obtains the first cleaved products.
Preferably, cleavage site is contained on second primer, the cleavage site is uracil or restriction enzyme
Cleavage site;The step B includes the following steps:
B1, the first cleaved products are cut, obtains the second cleaved products, the is formed through digestion in second cleaved products
One cohesive end;
B2, under the action of ligase, second cleaved products jointing at the first cohesive end obtains library point
Son;Contain the second cohesive end matched with the first cohesive end complete complementary on the connector.
Third object of the present invention is to provide a kind of SNP classifying methods, including to pressing any of the above-described kind of banking process
The step of library molecule obtained is sequenced.
Preferably, when detection is when sequencing sample has multiple, different according to sample to be sequenced build library respectively, obtain more
Kind library molecule, then will be sequenced after the mixing of a variety of library molecules.
The amplimer group of the present invention is waited for by being expanded to the nucleic acid fragment containing SNP site to be measured to obtain to contain
Survey the amplified production of SNP site and sequencing primer.During SNP site to be measured is sequenced, it is connected to template to be measured
Sequencing primer on chain is anchored on the anchor station on template strand to be measured, free terminal the rising as sequencing reaction of sequencing primer
Point, to detect the genotype of SNP site to be measured.The present invention is avoided without additionally adding sequencing primer into sequencing system
To it is multiple when sample is sequenced and is carried out at the same time sequencing due to sequencing primer is different and generation interferes with each other, improve the standard of sequencing
True property;In addition, the distance between the anchor station of sequencing primer and SNP site to be measured are 1 to 5bp, fewer number only need to be carried out
Sequencing steps detection can be completed, substantially reduce sequencing the time, and make the detection to SNP site to be measured not by sequencing instrument
Long limitation is read, sequencing accuracy is improved.
Description of the drawings
Fig. 1 is the structural schematic diagram of the first primer in first embodiment of the invention.
Fig. 2 is the amplification schematic diagram in first embodiment of the invention.
Fig. 3 be in third embodiment of the invention using reverse strand as sequencing template chain when sequencing primer anchoring schematic diagram.
Fig. 4 be in third embodiment of the invention using positive chain as sequencing template chain when sequencing primer anchoring schematic diagram.
Fig. 5 is the Page gel electrophoresis figures of the amplified production of each reaction system in fourth embodiment of the invention.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.
The present invention proposes first embodiment, and as shown in Figure 1, 2, a kind of amplimer group is used to prepare amplified production 300,
Including the first primer 110 and the second primer 120, the first primer 110 is from 3 ' ends to 5 ' ends successively including amplimer sequence
111 and anchor primer sequence 112;The amplimer sequence 111 is for expanding the nucleic acid fragment 200 containing SNP site to be measured;
In amplified production 300, the anchor primer sequence 112 is used as sequencing primer, the anchor primer sequence 112 and its anchor station
311 are located at the same side of the SNP site 312 to be measured.
It should be noted that the first primer 110 of the present invention can be sense primer, can also be downstream primer.This reality
It applies in example, as shown in Fig. 2, the first primer 110 is downstream primer, is used for the reverse strand 220 of amplification of nucleic acid segment 200.Expand in PCR
In increasing process, the amplimer sequence 111 in the first primer 110 is incorporated in reverse strand 220, and the second primer 120 is incorporated in just
To on chain, amplified production 300 is obtained by PCR amplification.Contain SNP site to be measured on the positive chain 310 of amplified production 300 simultaneously
312 and anchor primer sequence 112, the anchor primer sequence 112 and its anchor station 311 be located at the SNP site 312 to be measured
The same side;Correspondingly, also containing the pairing with 112 complementary pairing of anchor primer sequence in the reverse strand 320 of amplified production 300
Sequence 113, matched sequence 113 and its anchor station 321 are located at the same side of reverse strand SNP site 322 to be measured.
Amplified production is prepared using the amplimer group of the present embodiment, anchor primer sequence is contained on obtained amplified production
Row, in the step of subsequently SNP site to be measured is sequenced, anchor primer sequence can be used as sequencing primer, without volume
Outer addition sequencing primer, simplifies subsequent sequencing steps, avoids when being carried out at the same time multiple sample sequencings due to sequencing primer
It is different and generation to interfere with each other, improve sequencing efficiency and accuracy.
Preferably, the amplimer sequence length is 15 to 25bp;The anchor primer sequence length is 15 to 20bp.
PCR amplification is carried out using the amplimer of this programme, while with higher amplification efficiency, the sequencing primer introduced exists
Can be preferably anchored on its anchor station in follow-up sequencing steps so that its in follow-up sequencing steps, have compared with
High sequencing efficiency.
Preferably, the annealing temperature of the amplimer sequence anchoring is 57 to 63 DEG C;The anchor primer sequence is being surveyed
The annealing temperature being anchored in program process is 42 to 50 DEG C.The amplimer group of this programme, can be by will be in PCR reaction process
Annealing temperature is controlled in 57 to 63 DEG C of ranges, and the anchor primer sequence in PCR reaction process is avoided to be anchored on sequencing template chain.
It should be noted that the amplified production 300 prepared using the amplimer group of the present embodiment, 310 He of positive chain
Reverse strand 320 may be used as the sequencing template chain in follow-up sequencing procedure.When using positive chain 310 as sequencing template chain,
Anchor primer sequence 112 is anchored at its anchor station 311, in follow-up sequencing procedure, is needed by anchor primer sequence
112 5 ' ends carry out phosphorylation, are then sequenced using connection PCR sequencing PCR in 5 ' ends of anchor primer sequence 112.When
When with reverse strand 320 being template strand, the survey with the matched sequence 113 of 112 complementary pairing of anchor primer sequence as reverse strand 310
Sequence primer is anchored at matched sequence primer anchor station 321, can be the 3 ' of matched sequence 113 in follow-up sequencing procedure
End is sequenced SNP site 322 to be measured using connection PCR sequencing PCR or synthesis sequencing.
Preferably, the first cleavage site is contained at 5 ' ends of the first primer, and first cleavage site is uracil.It adopts
Prepare amplified production with the amplimer group of this programme, in the follow-up process, using specificity cutting uracil enzyme to its into
5 ' ends of row cutting, positive chain can produce phosphate group, connect and survey when being conducive to using positive chain as sequencing template chain
Sequence probe.
Preferably, the enzyme of the specificity cutting uracil includes but not limited to USER enzymes, RNase H or UDG enzymes.
Preferably, as shown in Fig. 2, containing the second cleavage site 121 on second primer 120;Second cleavage
Point 121 is uracil or restriction enzyme cleavage sites.Using the amplimer group of this programme to the core containing SNP site to be measured
Acid fragment is expanded, and the second cleavage site 121 is contained in the reverse strand 320 of obtained amplified production 300.Using corresponding spy
The enzyme or restriction enzyme of opposite sex cutting uracil cut amplified production 300 prepared by this programme, can generate viscous
Property end.During being conducive to continue library after, by being connected at cohesive end and cohesive end complete complementary pairing
Connector, to improve joint efficiency.
Preferably, the distance between the anchor station 311 of the anchor primer sequence and SNP site 312 to be measured be 1 to
5bp, the technical solution of the present embodiment so that in follow-up sequencing procedure, the sequencing steps that need to only carry out fewer number can be complete
At detection, the sequencing time is substantially reduced, and so that the detection to SNP site to be measured is not read length by sequencing instrument and is limited, is improved
Sequencing accuracy.
The present invention proposes that second embodiment, a kind of banking process include the following steps:
A, PCR amplification is carried out to the nucleic acid fragment containing SNP site to be measured using amplimer group, obtains amplified production;The expansion
It includes the first primer and the second primer to increase primer sets, the first primer from 3 ' ends to 5 ' ends include successively amplimer sequence and
Anchor primer sequence;The amplimer sequence is for expanding the nucleic acid fragment containing SNP site to be measured;In amplified production, institute
It states anchor primer sequence and is used as sequencing primer, the anchor primer sequence and its anchor station are located at the SNP site to be measured
The same side;
B, in jointing on the opposite end at end where amplified production sequence containing anchor primer, library molecule is obtained.
The library molecule prepared using this programme, mould to be sequenced is introduced to by pcr amplification reaction by anchor primer sequence
On plate chain, in follow-up sequencing procedure, anchor primer sequence can be used as sequencing primer, without additionally adding sequencing primer,
Subsequent sequencing steps are simplified, the phase generated when being carried out at the same time sequencing to multiple samples since sequencing primer is different is avoided
Mutually interference, improves the accuracy of sequencing.
Preferably, the amplimer sequence length is 15 to 25bp;The anchor primer sequence length is 15 to 20bp.
The banking process of this programme, while with preferable amplification efficiency, the sequencing primer being introduced into is in follow-up sequencing steps
Can preferably be anchored on its anchor station, to make library molecule its in follow-up sequencing steps, there is preferable sequencing
Efficiency.
Preferably, the annealing temperature of the amplimer sequence anchoring is 57 to 63 DEG C;The anchor primer sequence is being surveyed
The annealing temperature being anchored in program process is 42 to 50 DEG C;The annealing temperature of the PCR amplification is 57 to 63 DEG C.This programme can exempt from
Anchor primer sequence is anchored on sequencing template chain in PCR reaction process.
Preferably, the distance between the anchor station of the anchor primer sequence and SNP site to be measured are 1 to 5bp, this reality
Apply the technical solution of example so that in follow-up sequencing procedure, need to only pass through connection sequencing or the synthesis order-checking of fewer number, you can
The genotype of SNP site to be measured is detected.
Preferably, the first cleavage site is contained at 5 ' ends of the first primer, and the cleavage site is uracil;The step
Rapid B is further comprising the steps of:
B0, amplified production is cut using the enzyme of specificity cutting uracil, obtains the first cleaved products.
Preferably, the enzyme of the specificity cutting uracil is USER enzymes, RNase H or UDG enzymes.
5 ' ends of library molecule prepared by this programme, positive chain form phosphate group.It is used in follow-up sequencing procedure
When positive chain is as sequencing template chain, convenient for connecting sequencing probe in 5 ' ends.
Preferably, the second cleavage site is contained on second primer, second cleavage site is uracil or limitation
Property inscribe cleavage sites;The step B includes the following steps:
B1, the first cleaved products are cut, obtains the second cleaved products, the is formed through digestion in second cleaved products
One cohesive end;
B2, under the action of ligase, second cleaved products jointing at the first cohesive end obtains library point
Son;Contain the second cohesive end matched with the first cohesive end complete complementary on the connector.
The library molecule prepared using this programme can by the connection between the first cohesive end and the first cohesive end
So that the joint efficiency higher between connector and amplified production, can further increase and build library efficiency.
Further, the cleavage reaction of the step B1 and B2 and connection reaction can be in same cutting-linked systems
It carries out.Compared with stepwise reaction, this programme, which simplifies, builds library step, improves and builds library efficiency.
When second cleavage site is restriction enzyme cleavage sites, restriction enzyme is used in the step B1
The first amplified production of cleavage.Points of the step B0 and step B1 without priority.
Preferably, second cleavage site is uracil, cuts the enzyme of uracil in the step B1 using specificity
Cut the first amplified production.Compared with said program, B0 synchronous can be carried out with B1 in this programme, it is only necessary to be added into amplified production
The enzyme for entering specificity cutting uracil simplifies reaction system to reduce reagent type.
The enzyme of the specificity cutting uracil includes but not limited to USER enzymes, RNase H or UDG enzymes.
It should be noted that when being free of uracil in the first primer, directly amplified production is carried out in the step B1
Cutting.
The ligase can realize that DNA fragmentation is connected without specifically limited, it is preferred that the ligase is T4
DNA ligase can not only connect cohesive end, but also can connect flat end.
Preferably, contain biotin labeling, the text on the opposite end at end where the second cohesive end on the connector
Library molecule can be fixed on by the biotin labeling on connector on the magnetic bead containing Avidin.
Preferably, including sequence label on the connector, the library molecule prepared through this embodiment contains label thereon
Sequence can be by detecting the label sequence on library molecule when detecting multiple samples to be tested simultaneously in subsequent sequencing procedure
Row carry out the differentiation between sample.
Preferably, the sequence label length is the arbitrary base sequence of 4bp, the sequence label structure letter of the present embodiment
It is single, it is easy detection.
The present invention proposes 3rd embodiment, a kind of SNP classifying methods, including is made to pressing banking process in second embodiment
Library molecule the step of being sequenced.
The SNP classifying methods of the present embodiment include SNP site to be measured and sequencing primer on sequencing template chain, are treating
It surveys during SNP site is sequenced, sequencing primer is anchored on the template strand where itself, without additionally adding survey
Sequence primer simplifies subsequent sequencing steps, avoid to it is multiple wait be sequenced sample be carried out at the same time sequencing when due to sequencing primer
It is different and generation mutual, improve the accuracy of sequencing.
In the present embodiment, the anchor primer sequence may be designed as being located at anti-in the one section of sequence in SNP site 3 ' to be measured end
To sequence.Sequencing primer on library molecule prepared by this programme can match with the point sequence complete complementary on template strand to be measured
It is right, to improve sequencing efficiency.
Preferably, the sequencing approach can be second generation high throughput gene sequencing technology, including but not limited to connection sequencing
Method or synthesis sequencing.
It should be noted that in the present embodiment, it is high-temperature denatured to library molecule progress, obtain two kinds of positive chain and reverse strand
Sequencing template chain.As shown in figure 3, using reverse strand 320 as sequencing template chain, it is complementary with anchor primer sequence in sequencing procedure
The matched sequence 113 of pairing is anchored at matched sequence anchor region 321, sequencing of the matched sequence 113 as sequencing template chain
Primer, may be used connection PCR sequencing PCR or SNP site to be measured is sequenced in synthesis sequencing.
As shown in figure 4, when using positive chain 310 as sequencing template chain, anchor primer sequence 112 is anchored on anchor primer
At anchor region 311, in the present embodiment, since 5 ' ends of anchor primer sequence 112 are free of phosphate group, need anti-in sequencing
Before should starting, phosphate group is formed in 5 ' ends of anchor primer sequence.Preferably, 5 ' ends of the first primer are contained
Cleavage site, the cleavage site can be uracil or restriction enzyme cleavage sites, and the present embodiment is uracil, is being surveyed
Further include the steps that using USER cleavages template strand to be measured before sequence reaction starts, to obtain 5 ' ends of phosphorous acid groups
End, may be used connection PCR sequencing PCR, sequencing probe be connected in 5 ' ends, to which SNP site be sequenced.
Preferably, in the SNP classifying methods, the annealing temperature of sequencing primer anchoring is 42 to 50 DEG C.This programme avoids
Interference, improves the anchoring efficiencies of sequencing primer.
The present invention can carry out SNP partings to a variety of library molecules simultaneously.SNP points are carried out to a variety of library molecules when simultaneously
When type, the SNP genotyping results for distinguishing different library molecules are needed.
Preferably, the SNP classifying methods further include by the addressable step being fixed on solid phase carrier of library molecule.
Addressable fixation refers to the fixation that can determine location information, so that it is determined that the SNP parting knots of different library molecules
Fruit.
Further, the library molecule can be fixed on by direct or indirect mode on solid phase carrier.
In one embodiment of the invention, library molecule is fixed on by connector on slide and is sequenced.
In another embodiment of the present invention, by library molecule by the biotin labeling on connector, surface is fixed on containing affine
On the magnetic bead of element, then magnetic bead is fixed on slide again and is sequenced.
Preferably, include sequence label on the connector, in subsequent sequencing procedure, when simultaneously to multiple samples to be tested
When being sequenced, the differentiation between sample can be carried out by the sequence label on library molecule.
Preferably, the sequence label length is the arbitrary base sequence of 4bp, the sequence label structure letter of the present embodiment
It is single, it is easy detection.
The present invention proposes fourth embodiment, a kind of SNP classifying methods, using the poba gene group DNA of two people as template,
SNP site rs1799853, rs1057910 of CYP2C9 genes in each template, the SNP site of VKORC1 genes are detected respectively
SNP site rs4244285, rs4986893 of rs9923231, CYP2C19 gene.Reaction system is established according to the following steps:
A1, it is directed to above-mentioned two sample, each sample prepares 5 first round nested PCR amplifications totally 10 reaction systems respectively:
1.0 μ L of 50ng/ μ L poba gene groups DNA;2×long Taq Mix 10.0μL;10 μM of 0.4 μ L of the first sense primer;10μ
The 0.4 μ L of the first downstream primer of M;Each 4 μ L of dNTP of 2.5mM;Add deionized water to 50 μ L;Mixing simultaneously centrifuges.In the following conditions
Lower carry out PCR amplification:Continue 4 minutes under the conditions of 94 DEG C;It is for 20 seconds under the conditions of 94 DEG C, it is for 20 seconds under the conditions of 49 DEG C, 72 DEG C
Under the conditions of continue 1 minute, altogether 30 cycle;Continue 3 minutes under the conditions of 72 DEG C;After the completion of amplified reaction, the first amplification is obtained
Product.Wherein, the first rs1799853 sense primers that the first rs1799853 amplified productions are used to prepare in two samples are SEQ
ID NO:1, the first rs1799853 downstream primers are SEQ ID NO:2;It is used to prepare the of the first rs1057910 amplified productions
One rs1057910 sense primers are SEQ ID NO:3, the first rs1057910 downstream primers are SEQ ID NO:4;It is used to prepare
First rs9923231 sense primers of the first rs9923231 amplified productions are SEQ ID NO:5, the first downstreams rs9923231 are drawn
Object is SEQ ID NO:6;The first rs4244285 sense primers for being used to prepare the first rs4244285 amplified productions are SEQ ID
NO:7, the first rs4244285 downstream primers are SEQ ID NO:8;It is used to prepare the first of the first rs4986893 amplified productions
Rs4986893 sense primers are SEQ ID NO:9;First rs4986893 downstream primers are SEQ ID NO:10;
A2, using the first amplified production of above-mentioned two sample as template, it is each prepare 5 second wheel nested PCR amplifications totally 10 it is anti-
Answer system:The 10.0 μ L of the first amplified production of 100 times of dilution;2×long Taq Mix 10.0μL;Draw 10 μM of the second upstream
0.4 μ L of object;10 μM of 0.4 μ L of the second downstream primer;Each 4 μ L of dNTP of 2.5mM;Add deionized water to 50 μ L;Mixing simultaneously centrifuges.
PCR amplification is carried out under the following conditions:Continue 4 minutes under the conditions of 94 DEG C;It is for 20 seconds under the conditions of 94 DEG C, continue under the conditions of 57 DEG C
For 20 seconds under the conditions of 20 seconds, 72 DEG C, 30 recycle altogether;Continue 3 minutes under the conditions of 72 DEG C;After the completion of amplified reaction, obtain
Second amplified production.Wherein, the 2nd rs1799853 sense primers for being used to prepare the 2nd rs1799853 amplified productions are SEQ
ID NO:11, the 2nd rs1799853 downstream primers are SEQ ID NO:12;It is used to prepare the 2nd rs1057910 amplified productions
2nd rs1057910 sense primers are SEQ ID NO:13, the 2nd rs1057910 downstream primers are SEQ ID NO:14;For
The 2nd rs9923231 sense primers for preparing the 2nd rs9923231 amplified productions are SEQ ID NO:15, the 2nd rs9923231
Downstream primer is SEQ ID NO:16;The 2nd rs4244285 sense primers for being used to prepare the 2nd rs4244285 amplified productions are
SEQ ID NO:17, the 2nd rs4244285 downstream primers are SEQ ID NO:18;It is used to prepare the 2nd rs4986893 amplification productions
2nd rs4986893 sense primers of object are SEQ ID NO:19, the 2nd rs4986893 downstream primers are SEQ ID NO:20.
It after the completion of second wheel nested PCR amplification, is detected through Page gel electrophoresis, as shown in figure 5, swimming lane 0 is that molecule is big in figure
Tick marks object, swimming lane 1 to 5 are respectively the 2nd rs1799853 amplified productions of sample one, the 2nd rs1057910 amplified productions, the
Two rs9923231 amplified productions, the 2nd rs4244285 amplified productions, the 2nd rs4986893 amplified productions.
The 2nd rs1799853 amplified productions of two samples are can be seen that in 104bp from Page gel electrophoresis testing results
There is target molecule band at place;2nd rs1057910 amplified productions have target molecule band at 85bp;Second
Rs9923231 amplified productions have target molecule band at 80bp;2nd rs4244285 amplified productions have mesh at 90bp
Mark molecular band;2nd rs4986893 amplified productions have target molecule band at 83bp, this is consistent completely with expection.This
Illustrate that amplimer group using the present invention can prepare the amplified production containing sequencing primer.
B, due to containing U on the second sense primer, contain U on the second amplified production, for the second amplification
Product prepares cutting-connection totally 10 reaction systems respectively:Second amplified production, 20 μ L, T4 ligase buffer solution, 8 μ L, T4 DNA
The 2 μ L of USER enzymes of 1 μ L, 1U/ μ L of ligase, are fixed on the 1 μ L of connector of 10 μM on magnetic bead in advance(Per μ L there are about 3 ~ 4 ×
106A magnetic bead);Add deionized water to 40 μ L;It reacts 1 hour at room temperature, obtains connection product, that is, be fixed on the library point on magnetic bead
Son;Wherein, the rs1799853 connectors being connect with the 2nd rs1799853 amplified productions are by SEQ ID NO:21 and SEQ ID NO:
22 compositions;The rs1057910 connectors being connect with the 2nd rs1057910 amplified productions are by SEQ ID NO:23 and SEQ ID NO:24
Composition;The rs9923231 connectors being connect with the 2nd rs9923231 amplified productions are by SEQ ID NO:25 and SEQ ID NO:26 groups
At;The rs4244285 connectors being connect with the 2nd rs4244285 amplified productions are by SEQ ID NO:27 and SEQ ID NO:28 groups
At;The rs4986893 connectors being connect with the 2nd rs4986893 amplified productions are by SEQ ID NO:29 and SEQ ID NO:30 groups
At.Contain wherein on rs1799853 connectors and contain sequence label TGCA on sequence label ACGT, rs1057910 connector,
Contain on rs9923231 connectors and contains sequence label CATG, rs4986893 connector on sequence label GTAC, rs4244285 connector
It is upper to contain sequence label AGTC.
C, totally 10 reaction systems mix the magnetic bead in step B to two samples, separation removal supernatant;It is dense that 20 μ L are added
Degree is the NaOH of 0.1M, and it is single-stranded to make its denaturation, removal supernatant is then detached again, with 1 × TE(Containing 0.01% triton)Washing
Both sides are resuspended in 20 1 × TE of μ L and are used as sequencing template chain;By the sequencing template chain point sample being fixed on magnetic bead to different sulphur cyanogen
The load sample piece of base modification(Slide), in 37 DEG C of fixed 1h, that is, complete to be fixed on magnetic bead to wait for that the addressable of library molecule is fixed.
D, using the high-throughput gene sequencer Pstar II A sequenators of Shenzhen HYK Gene Technology Co., Ltd., and
It is sequenced using connection PCR sequencing PCR, the sequencing template chain of different samples is distinguished according to the detection of sequence label.
In the present embodiment, the genotype for obtaining the sites rs1799853 of sample one is CC, is wild type;Rs1057910
The genotype of point is AC, for the heterozygote of mutation;The genotype in the sites rs9923231 is CC, is wild type;Rs4244285
The genotype of point is GG;For wild type type;The genotype in the sites rs4986893 is GG, is wild type.
The genotype for obtaining the sites rs1799853 of sample two is CC, is wild type;The genotype in the sites rs1057910
It is wild type for AA;The genotype in the sites rs9923231 is TT, is no mutant homozygote;The genotype in the sites rs4244285 is
GG;For wild type;The genotype in the sites rs4986893 is GG, is wild type.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
SEQUENCE LISTING
<110>Guangzhou Kang Xin Rui Jiyin health Science and Technology Ltd.
<120>Amplimer group, banking process and SNP classifying methods
<130>
<160> 30
<170> PatentIn version 3.3
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ggatggaaaa cagagactta caga 24
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atatggccac ccctgaaatg t 21
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ttcatatacc cctgaattgc taca 24
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tggggacttc gaaaacatgg 20
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caccaagacg ctagacccaa 20
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tagatgtgag aaacagcatc tgga 24
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accagagctt ggcatattgt atc 23
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gcagaacaga gcttttccta tcct 24
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aagacaaata ggccgggaat gt 22
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cttagaagcc tgatctatat tggga 25
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cgguggggaa gaggagcatt gagg 24
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gtgttcaaga ggaaggggtc acccaccctt ggtttt 36
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cggutgcacg aggtccagag atac 24
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ttgaccttct ccccatgtca caggtcactg catgg 35
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cggugcgtga gccaccgcac c 21
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ggccaatggt tgttttggga agtcaagcaa gagaa 35
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cggutcccac tatcattgat tatttccc 28
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ggaacccata acaaacactt tccataaaag caaggtt 37
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cggutcagga ttgtaagcac cccct 25
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atccaggtaa ggccaagtgg tttctcagga agcaaa 36
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cctccctgca gtctctatgg gcacgtctgc tagtcgctga agtagtcggt 50
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ctacttcagc gactagcaga cgtgcccata gagactgcag gg 42
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cctccctgca gtctctatgg gctgcactgc tagtcgctga agtagtcggt 50
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ctacttcagc gactagcagt gcagcccata gagactgcag gg 42
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cctccctgca gtctctatgg gcgtacctgc tagtcgctga agtagtcggt 50
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ctacttcagc gactagcagg tacgcccata gagactgcag gg 42
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cctccctgca gtctctatgg gccatgctgc tagtcgctga agtagtcggt 50
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ctacttcagc gactagcagc atggcccata gagactgcag gg 42
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cctccctgca gtctctatgg gcagtcctgc tagtcgctga agtagtcggt 50
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<211> 42
<212> DNA
<213>Artificial sequence
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ctacttcagc gactagcagg actgcccata gagactgcag gg 42
Claims (10)
1. a kind of amplimer group, is used to prepare amplified production, which is characterized in that the amplimer group include the first primer and
Second primer, the first primer is from 3 ' ends to 5 ' ends successively including amplimer sequence and anchor primer sequence;The amplification
Primer sequence is for expanding the nucleic acid fragment containing SNP site to be measured;In amplified production, the anchor primer sequence is used as sequencing
Primer, the anchor primer sequence and its anchor station are located at the same side of the SNP site to be measured.
2. amplimer group according to claim 1, which is characterized in that contain cleavage site on second primer;Institute
It is uracil or restriction enzyme cleavage sites to state cleavage site.
3. amplimer group according to claim 1, which is characterized in that the amplimer sequence length be 15 to
25bp;The anchor primer sequence length is 15 to 20bp.
4. amplimer group according to claim 1, which is characterized in that the annealing temperature of the amplimer sequence anchoring
It is 57 to 63 DEG C;The annealing temperature of the anchor primer sequence anchoring is 42 to 50 DEG C.
5. a kind of banking process, which is characterized in that include the following steps:
A, PCR amplification is carried out to the nucleic acid fragment containing SNP site to be measured using amplimer group, obtains amplified production;The expansion
It includes the first primer and the second primer to increase object group, and the first primer is from 3 ' ends to 5 ' ends successively including amplimer sequence and anchor
Determine primer sequence;The amplimer sequence is for expanding the nucleic acid fragment containing SNP site to be measured;It is described in amplified production
Anchor primer sequence is used as sequencing primer, and the anchor primer sequence and its anchor station are located at the same of the SNP site to be measured
Side;
B, in jointing on the opposite end at end where amplified production sequence containing anchor primer, library molecule is obtained.
6. banking process according to claim 5, which is characterized in that the anchor station of the anchor primer sequence with it is to be measured
The distance between SNP site is 1 to 5bp.
7. banking process according to claim 5, which is characterized in that uracil is contained at 5 ' ends of the first primer, described
Step B is further comprising the steps of:
B0, amplified production is cut using the enzyme of specificity cutting uracil, obtains the first cleaved products.
8. banking process according to claim 5, which is characterized in that contain cleavage site on second primer, it is described
Cleavage site is uracil or restriction enzyme cleavage sites;The step B includes the following steps:
B1, the first cleaved products are cut, obtains the second cleaved products, the is formed through digestion in second cleaved products
One cohesive end;
B2, under the action of ligase, second cleaved products jointing at the first cohesive end obtains library point
Son;Contain the second cohesive end matched with the first cohesive end complete complementary on the connector.
9. a kind of SNP classifying methods, which is characterized in that include to by the banking process system described in any one of claim 5 to 8
The step of library molecule obtained is sequenced.
10. SNP classifying methods according to claim 9, which is characterized in that when detection wait be sequenced sample have multiple when,
Library is built respectively according to the difference of sample to be sequenced, and obtains a variety of library molecules, then be sequenced after a variety of library molecules are mixed.
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| CN201710232840.4A CN108690843A (en) | 2017-04-11 | 2017-04-11 | Amplimer group, banking process and SNP classifying methods |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103937896A (en) * | 2014-04-25 | 2014-07-23 | 深圳华因康基因科技有限公司 | SNP typing method and kit |
| CN104480534A (en) * | 2014-12-29 | 2015-04-01 | 深圳华因康基因科技有限公司 | Rapid library building method |
| CN105886608A (en) * | 2015-12-22 | 2016-08-24 | 武汉康昕瑞基因健康科技有限公司 | ApoE gene primer group, detection kit and detection method |
-
2017
- 2017-04-11 CN CN201710232840.4A patent/CN108690843A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103937896A (en) * | 2014-04-25 | 2014-07-23 | 深圳华因康基因科技有限公司 | SNP typing method and kit |
| CN104480534A (en) * | 2014-12-29 | 2015-04-01 | 深圳华因康基因科技有限公司 | Rapid library building method |
| CN105886608A (en) * | 2015-12-22 | 2016-08-24 | 武汉康昕瑞基因健康科技有限公司 | ApoE gene primer group, detection kit and detection method |
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Application publication date: 20181023 |