CN106929507A

CN106929507A - Primer sets, anchor primer, kit, library construction and gene order surveying method

Info

Publication number: CN106929507A
Application number: CN201511007119.2A
Authority: CN
Inventors: 盛司潼; 钟茂春
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-12-30
Filing date: 2015-12-30
Publication date: 2017-07-07
Also published as: WO2017113655A1

Abstract

The present invention relates to biology field, there is provided a kind of primer sets, anchor primer, library constructing method and gene order surveying method.The primer sets include the first primer pair, and the sense primer in first primer pair is made up of the first general area and the first complementary region；First complementary region is connected with the 3 ' ends in the described first general area, first complementary region and First ray complete complementary；The First ray be SNP site to be measured one section of sequence in the sequence, the 3 ' ends in SNP site to be measured, 5 ' ends of the First ray are away from SNP site to be measured 1 to 7bp；The first general area is not complementary with the second sequence；Second sequence is one section of sequence being connected with 3 ' ends of First ray in sequence where SNP site to be measured.The present invention is avoided that because sequencing result is inaccurate caused by the particularity of sequence near SNP site to be measured or the appearance of failure phenomenon is sequenced.

Description

Primer sets, anchor primer, kit, library construction and gene order surveying method

Technical field

The present invention relates to biology field, more specifically to a kind of primer sets, anchor primer, library constructing method and gene order surveying method.

Background technology

Second generation high throughput sequencing technologies include connection PCR sequencing PCR and synthesis sequencing.Wherein, the connection PCR sequencing PCR is to be attached between nucleic acid fragment the fidelity during reacting based on ligase to realize, is template, anchor primer with nucleic acid fragment to be sequenced（Also known as sequencing primer, it is complementary with chain where nucleic acid fragment to be sequenced）And oligonucleotide probe（Fluorescence labeling is carried on the ad-hoc location of the probe）Reaction is attached, by detecting the fluorescence labeling on connection product so that it is determined that carrying the information of the corresponding sequence of ad-hoc location of fluorescence labeling on oligonucleotide probe.The synthesis sequencing is that the fidelity based on polymerase during nucleic acid chains are extended is realized, it is template with nucleic acid fragment to be sequenced, anchor primer complementation is bound on nucleic acid fragment to be sequenced, and the sequence information of relevant position on nucleic acid fragment to be sequenced is determined by detecting the signal produced during extension.Second generation high throughput sequencing technologies are detected because its high flux and low cost currently used for SNP partings.

A kind of SNP classifying methods of utilization second generation high throughput sequencing technologies of the prior art, comprise the following steps：A, the nucleotide sequence that acquisition SNP site to be measured is expanded by primer；B, the product based on step A build sequencing library；C, anchor primer is anchored on sequencing library molecule, the sequence information of SNP site to be measured is detected by high throughput sequencing technologies.In order to accelerate the detection of SNP site to be measured, the period of sequencing is reduced, improve SNP site detection efficiency, typically by anchor primer design in the vicinity of SNP site to be measured.But this method for designing, often because the particularity of sequence where SNP site, for example nearby there is special construction in sequence to the SNP site --- repetitive sequence, hair clip situation etc., cause the anchor primer for designing according to the method described above, precalculated position cannot be accurately anchored on, causes sequencing result inaccurate or sequencing failure；So that the method cannot large-scale promotion.

Therefore a kind of new primer sets applied widely, anchor primer, kit, library construction and gene order surveying method are needed.

The content of the invention

It is an object of the invention to provide a kind of new primer sets applied widely, anchor primer, kit, library construction and gene order surveying method, it is intended to solve the technical problem of the non-specific grappling of anchor primer in existing high flux gene sequencing technology.

In order to realize goal of the invention, the invention provides a kind of primer sets, including the first primer pair, the sense primer in first primer pair is made up of the first general area and the first complementary region；First complementary region is connected with the 3 ' ends in the described first general area, first complementary region and First ray complete complementary；The First ray be SNP site to be measured one section of sequence in the sequence, the 3 ' ends in SNP site to be measured, 5 ' ends of the First ray are away from SNP site to be measured 1 to 7bp；The first general area is not complementary with the second sequence；Second sequence is one section of sequence being connected with 3 ' ends of First ray in sequence where SNP site to be measured.

Preferably, the first complementary region sequence length is between 6 to 12bp.

Preferably, the described first general region sequence length is between 6 to 16bp.

Preferably, 5 ' ends of the First ray are away from SNP site to be measured 1 to 3bp.

Preferably, the primer sets also include the second primer pair, and first primer pair and the second primer pair are respectively the inner primer pair and outer primer pair expanded to sequence where SNP site to be measured.

Preferably, the anti-sense primer in first primer pair is made up of the second general area and the second complementary region；Second complementary region is connected with the 3 ' ends in the described second general area；Second complementary region be SNP site to be measured one section of sequence in the sequence, the 5 ' ends in SNP site to be measured；The second general area is different from the 3rd sequence；3rd sequence is one section of sequence being connected with 5 ' ends of the second complementary region in sequence where SNP site to be measured.

Preferably, U is contained in the described first general area.

In order to preferably realize the purpose of the present invention, present invention also offers a kind of anchor primer, the anchor primer and the 4th sequence complete complementary are matched；4th sequence is single stranded nucleic acid molecule, be the first primer pair in any of the above-described kind of primer sets amplified production in one section of sequence；4th sequence includes First ray and the first normalization area, and the first normalization area is connected with 3 ' ends of First ray, and 5 ' ends of the 4th sequence are away from SNP site to be measured 1 to 7bp；Or the 4th sequence includes the first complementary region and the second normalization area, the second normalization area is connected with 5 ' ends of the first complementary region, and 3 ' ends of the 4th sequence are away from SNP site to be measured 1 to 7bp.

Preferably, the 4th sequence includes First ray and the first normalization area, and the first normalization area is connected with 3 ' ends of First ray, and 5 ' ends of the First ray are away from SNP site to be measured 1 to 3bp；Or

4th sequence includes the first complementary region and the second normalization area, and the second normalization area is connected with 5 ' ends of the first complementary region, and 3 ' ends of the 4th sequence are away from SNP site to be measured 1 to 3bp.

Preferably, the length in the first normalization area is between 8 to 14bp.

In order to preferably realize the purpose of the present invention, present invention also offers a kind of kit, including any one above-mentioned primer sets.

Preferably, the kit also includes any one above-mentioned anchor primer.

In order to preferably realize the purpose of the present invention, present invention also offers another kit, including any one above-mentioned anchor primer.

Preferably, the kit also includes any one above-mentioned primer sets.

In order to preferably realize the purpose of the present invention, present invention also offers a kind of library constructing method, comprise the following steps：

A, using any one above-mentioned primer sets, sample to be tested is entered performing PCR amplification, obtain the amplified production containing SNP site to be measured；

B, will be connected with joint containing the amplified production of SNP site to be measured, formed and treat sequencing library molecule.

Preferably, the step B is：Amplified production containing SNP site to be measured is directly connected with joint, sequencing library molecule is treated in formation, and is fixed on solid phase carrier by the way that microballoon is addressable.

It is furthermore preferred that the step B is comprised the following steps：

B1, will directly be connected with the joint being fixed on microballoon containing the amplified production of SNP site to be measured, formed to be fixed on microballoon and treats sequencing library molecule；

B2, it is fixed on step B1 thus obtained microspheres are addressable on solid phase carrier.

It is furthermore preferred that the coupled reaction in the step B1 is carried out in cutting-coupled reaction system, the cutting-coupled reaction system includes：Ligase, clastogen, the first joint being fixed on microballoon and connection buffer solution；Contain U in the first general area；The clastogen is used for specificity cutting U, and the amplified production containing SNP site to be measured is formed the first cohesive end；First joint is nucleic acid molecules, contains the second cohesive end matched with the first cohesive end complete complementary.

It is furthermore preferred that containing PEG during buffer solution is connected described in step B1.

In order to preferably realize the purpose of the present invention, present invention also offers a kind of gene order surveying method, comprise the following steps：

B, will be connected with joint containing the amplified production of SNP site to be measured, formed and treat sequencing library molecule；

C, using any one above-mentioned anchor primer, treat sequencing library molecule and be sequenced, obtain the sequence information of SNP site to be measured.

Preferably, the sequence measurement in the step C is connection PCR sequencing PCR；Also include step D between step B, C, using any of the above-described kind of anchor primer, the oligonucleotide probe marked using unstressed configuration is replaced the oligonucleotide probe for having fluorescence labeling, treats sequencing library molecule and once connected sequencing reaction.

Preferably, the step D is comprised the following steps：

D1, the anchor primer is anchored on and is treated on sequencing library molecule；

D2, the oligonucleotide probe for adding unstressed configuration mark, ligase and corresponding buffer solution, are attached reaction；

D3, denaturation removal connection product.

As from the foregoing, the present invention carries out particular design by the amplimer to SNP site to be measured, so that including the first general area near SNP site to be measured in the product expanded by the amplimer, reduce the complexity of different SNP site near zones to be measured, it is then based on the sequence between the first general area and SNP site to be measured, corresponding anchor primer can be designed, anchor primer is set accurately to be anchored on target location, primer sets of the invention, anchor primer, kit, library construction and gene order surveying method are applied to the detection of various different SNP sites, it is particularly suited for being detected while many SNP sites, can be on the premise of SNP site detection efficiency be ensured, avoid because sequencing result is inaccurate caused by the particularity of sequence near SNP site to be measured or the appearance of failure phenomenon is sequenced.

Brief description of the drawings

Fig. 1 is the relation schematic diagram between sequence where sense primer and SNP site to be measured in the first exemplary embodiments of the invention in first primer pair.

Fig. 2 is the relation schematic diagram between sequence where anti-sense primer and SNP site to be measured in one embodiment of the invention in the first primer pair.

Fig. 3 is the relation schematic diagram between the anchor primer in the second exemplary embodiments of the invention and the first primer pair amplifies product.

Fig. 4 is the structural representation of the first joint in the first specific embodiment of the invention.

Fig. 5 is the sequence label and samples sources and the corresponding relation figure of SNP site of the first joint in the first specific embodiment of the invention.

Fig. 6 is experimental group sequencing flow chart in the first specific embodiment of the invention.

Fig. 7 is the sequencing flow chart of the first contrast experiment in the first specific embodiment of the invention.

Fig. 8 is the preliminary experimental results compares figure of sample 2 in the first specific embodiment of the invention.

Fig. 9 is the sequencing and image collecting result in rs671 and rs1801253 sites in the 3rd contrast experiment of the invention.

Figure 10 is the sequencing and image collecting result in rs671 and rs1801253 sites in the first specific embodiment of the invention.

Figure 11 is the sequencing and image collecting result in rs1799853 sites in the 3rd contrast experiment of the invention.

Figure 12 is the sequencing and image collecting result in rs1799853 sites in the first specific embodiment of the invention.

Specific embodiment

In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with drawings and Examples, the present invention will be described in further detail.

The present invention proposes the first exemplary embodiments, a kind of primer sets, including the first primer pair, as shown in figure 1, the sense primer in first primer pair is made up of the first general area and the first complementary region；First complementary region is connected with the 3 ' ends in the described first general area, first complementary region and First ray complete complementary；The First ray be SNP site to be measured one section of sequence in the sequence, the 3 ' ends in SNP site to be measured, 5 ' ends of the First ray are away from SNP site to be measured 1 to 7bp；The first general area is not complementary with the second sequence；Second sequence is one section of sequence being connected with 3 ' ends of First ray in sequence where SNP site to be measured.

It should be noted that the primer sets can be used to expand sequence where SNP site to be measured, gained amplified production can be used to build the sequencing library containing SNP site to be measured, and then carry out the high-flux sequence detection of SNP site to be measured.The present invention is by the particular design to the sense primer in the first primer pair so that in the amplified production based on the first primer pair, and a sequence containing the first general area is increased near SNP site to be measured.The sequence in the first general area neither sequence in the template molecule of sample to be tested near SNP site to be measured, nor the complementary series in the template molecule of sample to be tested near SNP site to be measured.The first general area causes that one end of the amplified production for SNP site to be measured increased a normalization sequence.The first general area is not complementary with the second sequence, and without repetitive sequence, itself will not form hair clip.This programme is particularly suited for the complicated sample to be tested of sequential structure near the SNP site to be measured on the template molecule in sample to be tested, the sequential structure near SNP site to be measured can effectively be simplified, reduce the design difficulty of anchor primer, can effectively ensure that anchor primer is accurately anchored on target location, avoid because sequencing result is inaccurate caused by the particularity of sequence near SNP site to be measured or the appearance of failure phenomenon is sequenced, it is adaptable to the detection of all types of SNP sites to be measured.For different SNP sites to be measured, the first general region sequence can be identical.

In addition, 5 ' ends of First ray are away from SNP site to be measured 1 to 7bp, so that based on the first primer pair amplifies product gained when sequencing library molecule carries out high flux gene sequencing, the distance between the end of anchor primer and SNP site to be measured are between 1 to 7bp, when using connecting PCR sequencing PCR and when T4 ligases are sequenced, the connection only by the probe of an anchor primer and ad-hoc location containing fluorescence labeling can be ensured, it is capable of achieving to treat the detection for surveying SNP site, improves the efficiency of high flux gene sequencing.

In one embodiment of the invention, 5 ' ends of the First ray are away from SNP site to be measured 1 to 3bp.

This programme can effectively control the spacing between SNP site to be measured and the first universal sequence so that the complementary series of the first universal sequence containing one section of sufficient length or the first universal sequence in anchor primer；When this detects multiple SNP sites to be measured at the same time, both ensured that each anchor primer can be accurately anchored on respective target location, cause that each anchor primer combines situation with target location again more consistent, normalization, reduce because each anchor primer is combined the difference of situation with target location, so as to reduce the difference of each SNP site detection signal to be measured, the accuracy of sequencing is improved.

For the primer pair of utility first expand the purity of products therefrom, it is to avoid non-specific amplification, the present invention proposes an embodiment on the basis of the first exemplary embodiments, and the first complementary region sequence length is between 6 to 12bp.

This programme ensure that the specific subset of sense primer in the first primer pair and the template sequence containing SNP site to be measured, it is to avoid non-specific amplification, in turn ensure that different SNP sites to be measured nearby have enough discriminations between sequence.It is furthermore preferred that the first complementary region sequence length is between 7 to 10bp.

The sequence complexity of different SNP site near zones to be measured is reduced to be further ensured that, the present invention proposes another embodiment on the basis of above-described embodiment, and the first general region sequence length is between 6 to 16bp.

In this programme, first universal sequence is largely avoided because sequence is long or too short caused first universal sequence of sequence is internally formed secondary structure, or first possibility for forming secondary structure between universal sequence and other sequences, reduce the design difficulty of anchor primer.

On the basis of any of the above-described embodiment, the present invention proposes another embodiment, and the primer sets also include the second primer pair, and first primer pair and the second primer pair are respectively the inner primer pair and outer primer pair expanded to sequence where SNP site to be measured.

In this programme, for SNP site to be measured, using inner primer pair and outer primer to carrying out nested PCR amplification to sequence where SNP site to be measured, so as to improve the purity of target molecule in the first primer pair amplifies product.

In order to further reduce using the architectural difference between above-mentioned primer sets amplification products therefrom, based on any of the above-described embodiment, the present invention proposes another specific embodiment, as shown in Fig. 2 the anti-sense primer in first primer pair is made up of the second general area and the second complementary region；Second complementary region is connected with the 3 ' ends in the described second general area；Second complementary region be SNP site to be measured one section of sequence in the sequence, the 5 ' ends in SNP site to be measured；The second general area is different from the 3rd sequence；3rd sequence is one section of sequence being connected with 5 ' ends of the second complementary region in sequence where SNP site to be measured.

It should be noted that the sequence in the second general area is neither sequence in the template molecule of sample to be tested near SNP site to be measured, nor the complementary series in the template molecule of sample to be tested near SNP site to be measured.Without repetitive sequence in the second general area, itself will not form hair clip.The first primer pair amplifies products therefrom based on this programme, respectively has a universal sequence at SNP site two ends to be measured, and for different SNP sites to be measured, the first general area and the second general region sequence can all sames；So as to reduce the sequence complexity of different SNP site near zones to be measured.Also cause that the amplification efficiency for each SNP site to be measured is more consistent simultaneously.

Based on any of the above-described embodiment, in order that the amplified production that must be based on the acquisition of the first primer pair can faster complete the structure of sequencing library, the present invention proposes an embodiment, and containing in the first general area can broken site.It is described can broken site can be cut agent specificity cutting so that based on the first primer pair obtain amplified production formed the first cohesive end.

In this programme, based on the first general area in can broken site, after amplified production is cut out the first cohesive end, can be directly connected to containing the linkers with the second cohesive end of the first cohesive end complementary pairing, improve joint efficiency.

Preferably, it is described can broken site be ribonucleotide, RNA sequence or restriction enzyme digestion sites.Accordingly, the clastogen is RNase H, RNase H or restriction enzyme.

When it is described can broken site be U when, the clastogen is preferably USER enzymes.

Preferably, it is described can broken site and the distance of chain 5 ' end where it be 1 to 6 base.Now, the first cohesive end is higher with the complementary pairing coupled reaction efficiency of the second cohesive end of joint, more preferably 4 or 5bp, the efficiency highest of now coupled reaction.

In order to preferably realize the purpose of the present invention, the present invention proposes the second exemplary embodiments, as shown in figure 3, a kind of anchor primer, the anchor primer and the 4th sequence complete complementary are matched；4th sequence is single stranded nucleic acid molecule, be the first primer pair in any of the above-described kind of primer sets amplified production in one section of sequence；As shown in Figure 3 a, the 4th sequence includes First ray and the first normalization area, and the first normalization area is connected with 3 ' ends of First ray, and 5 ' ends of the 4th sequence are away from SNP site to be measured 1 to 7bp；Or as shown in Figure 3 b, the 4th sequence includes the first complementary region and the second normalization area, the second normalization area is connected with 5 ' ends of the first complementary region, and 3 ' ends of the 4th sequence are away from SNP site to be measured 1 to 7bp.

It should be noted that it is described first normalization area be the first general area complementary series in be connected one section of sequence with 3 ' ends of First ray；The second normalization area is one section of sequence being connected with 5 ' ends of the first complementary region in the first general area.Anchor primer in this programme is based on the sequencing library of the amplified production structure of the first primer pair of primer sets in any of the above-described scheme and designs, for different SNP sites to be measured, these anchor primers contain a common first normalization sequence or the second normalization sequence, they accurately can be anchored on target location, avoid because sequencing result is inaccurate caused by the particularity of sequence near SNP site to be measured or the appearance of failure phenomenon is sequenced, cause that each anchor primer combines situation with target location again more consistent, normalization, reduce because each anchor primer is combined the difference of situation with target location, so as to reduce the difference of each SNP site detection signal to be measured, improve the accuracy of sequencing.

Based on above-described embodiment, the present invention proposes another embodiment, and the 4th sequence includes First ray and the first normalization area, and the first normalization area is connected with 3 ' ends of First ray, and 5 ' ends of the First ray are away from SNP site to be measured 1 to 3bp；Or the 4th sequence includes the first complementary region and the second normalization area, the second normalization area is connected with 5 ' ends of the first complementary region, and 3 ' ends of the 4th sequence are away from SNP site to be measured 1 to 3bp.

In this programme, the distance for being used for one end and SNP site to be measured for extending on anchor primer is 1 to 3bp, when being sequenced using connection sequencing technologies, the connection only by the probe of an anchor primer and ad-hoc location containing fluorescence labeling can be ensured, realize the detection to SNP site to be measured, the efficiency of high flux gene sequencing is improved, and 1 to 3bp is in the fidelity range of T4 ligases（1 to 7bp）In in high-fidelity scope, the accuracy in detection of SNP site to be measured can be effectively improved；When being sequenced using synthesis sequencing technologies, 4th sequence includes the first complementary region and the second normalization area, the second normalization area is connected with 5 ' ends of the first complementary region, 3 ' ends of the 4th sequence are away from SNP site 1bp to be measured, now only need to extend 1 bp, carry out 1 reaction of circulation, you can realize the detection to SNP site to be measured, detection efficiency is high.

In one embodiment of the invention, the first complementary region sequence length is between 6 to 12bp.This programme further ensures the specificity that anchor primer is combined with the target location near SNP site to be measured.It is furthermore preferred that the first complementary region sequence length is between 7 to 10bp.

In one embodiment of the invention, the length in the first normalization area is between 8 to 14bp.This programme ensure that the uniformity of the anchor primer and the joint efficiency of target location for different SNP site designs to be measured.

In order to preferably realize the purpose of the present invention, the present invention proposes the 3rd exemplary embodiments, a kind of kit, including any one above-mentioned primer sets.

Kit in this programme can be amplification kit, library construction Kit or sequencing kit.When the kit is amplification kit, its other reagent needed for may also include PCR amplifications；When the kit is library construction Kit, it may also include other reagents needed for library construction process, the reagent needed for may also comprise PCR amplifications；When the kit is sequencing kit, it may include other reagents needed for sequencing procedure, the reagent needed for may also comprise PCR amplifications, may also comprise the reagent needed for library construction process.

In one embodiment of the invention, the kit also includes any one above-mentioned anchor primer.

In order to preferably realize the purpose of the present invention, the present invention proposes the 4th exemplary embodiments, a kind of kit, including any one above-mentioned anchor primer.

Kit in this programme is generally sequencing kit, and it may include other reagents needed for sequencing procedure, the reagent needed for may also comprise PCR amplifications, may also comprise the reagent needed for library construction process.

In one embodiment of the invention, the kit also includes any one above-mentioned primer sets.

In order to preferably realize the purpose of the present invention, the present invention proposes the 5th exemplary embodiments, and a kind of library constructing method is comprised the following steps：

It should be noted that being obtained after the amplified production that sequencing library molecule is gained after entering performing PCR amplification to sample to be tested based on any one above-mentioned primer sets in this programme, therefore, a sequence containing the first general area is increased near SNP site to be measured.The first general area is not complementary with the second sequence, and without repetitive sequence, itself will not form hair clip.Therefore, it is possible to effectively simplify the sequential structure near SNP site to be measured, reduce the design difficulty of anchor primer, it is particularly suited for the complicated sample to be tested of sequential structure near the SNP site to be measured on the template molecule in sample to be tested, avoid because sequencing result is inaccurate caused by the particularity of sequence near SNP site to be measured or the appearance of failure phenomenon is sequenced, it is adaptable to the detection of all types of SNP sites to be measured.For different SNP sites to be measured, the first general region sequence can be identical.

In addition, in for step B, amplified production and the connection of joint containing SNP site to be measured will below be further elaborated from many aspects.

In one embodiment of the invention, only have one end in the amplified production containing SNP site to be measured to be connected with joint, and then sequencing library molecule is treated in formation.The connection end can be the either end of the amplified production containing SNP site to be measured.

In another embodiment of the present invention, the two ends of the amplified production containing SNP site to be measured are connected with joint, and then sequencing library molecule is treated in formation.

It should be noted that the sequence of the joint of two ends connection may be the same or different.

Preferably, the sequence of the joint of two ends connection is different, sequencing library molecule so can be treated based on the two different joints carries out amplification checking, whether verification library molecular structure is correct, may be based on the two different joints and treat sequencing library molecule carrying out unimolecule amplification, then second generation high flux gene sequencing is carried out to unimolecule amplified production, so as to obtain the sequence information of SNP site to be measured.

Based on any of the above-described kind of embodiment, the present invention proposes another embodiment, and the step B is：

Amplified production containing SNP site to be measured is directly connected with joint, sequencing library molecule is treated in formation, and is fixed on solid phase carrier by the way that microballoon is addressable.

In this programme, the amplified production containing SNP site to be measured is directly connected with joint, is effectively reduced experimental procedure, improves conventional efficient.

It should be noted that the step B there can be multiple embodiments, will below be illustrated by multiple embodiments.

In one embodiment of the invention, the step B is comprised the following steps：

B1, the just amplified production containing SNP site to be measured are directly connected with joint, and then connection product is fixed on microballoon, must be fixed on microballoon and be treated sequencing library molecule；

It should be noted that in the above two embodiments, being marked containing modification on the joint, for making it be fixed on microballoon.The modification mark can be biotin, Avidin, Streptavidin, antigen, antibody, acceptor, part, polyhistidine, nm of gold, iodacetyl, sulfydryl, amino, aldehyde radical, carboxyl, isothiocyano, silylation or acrylamide, and they specific can be combined with corresponding group or molecule.

Addressable fixation, refers to the fixation that can determine positional information.That is, that is fixed on each particular location on immobilization carrier treats that is fixed on sequencing library molecule and other particular locations treats that sequencing library molecule can be distinguished clearly.

In above-described embodiment, the joint can take various forms, including but not limited to flat end fitting, protruding terminus joint, Y connection or the joint containing loop-stem structure.

The flat end fitting refers to the double-stranded nucleic acid linker of complete complementary pairing between double-strand.Preferably, 5 ' ends of two chains of the flat end fitting are free of phosphate group, and it can avoid connection procedure center tap from the appearance for connecting phenomenon, reduce the interference to follow-up sequencing experiment, improve the accuracy rate of genetic test.

The protruding terminus joint, refers to that at least one end in double chain acid molecule carries prominent nucleotide sequence, and the double-stranded nucleic acid linker of remaining nucleotides then complete complementary.Protruding terminus joint can be for single protruding terminus, or containing two double protruding terminuses of protruding terminus, and the two protruding terminuses can be on a nucleotide chain or on different nucleotide chains.The protruding terminus joint can avoid connection procedure center tap from the appearance for connecting phenomenon, reduce the interference to follow-up sequencing experiment, improve the accuracy rate of genetic test.In the specific embodiment of the present embodiment, the joint is preferably single protruding terminus joint, and the protruding terminus is 3 ' ends of chain where it, and base is T；The joint can be directly connected to the pcr amplification product containing A tails expanded by Taq enzyme, improve joint efficiency.

The bifurcated type joint includes complementary region and crotch region, and the nucleotide complementary of complementary region double-strand is matched, and the nucleotides logarithm of pairing is not limited.Complementary region end can be flat end or protruding terminus.The bifurcated type joint can avoid connection procedure center tap from the appearance for connecting phenomenon, reduce the interference to follow-up sequencing experiment, improve the accuracy rate of SNP partings detection.In the specific embodiment of the present embodiment, the 3 ' ends that the joint is preferably complementary region are protruding terminus, and last base of protruding terminus is the T end furcations joints of T；The joint can be directly connected to the pcr amplification product containing A tails expanded by Taq enzyme, improve joint efficiency.

The joint with loop-stem structure, there is multiple embodiments.In one embodiment, the joint is single stranded nucleic acid molecule, and the single stranded nucleic acid molecule includes the first complementary pairing area, stem ring area and the second complementary pairing area successively, and the first complementary pairing area can match with the second complementary pairing area complete complementary.In another embodiment, the joint with loop-stem structure can also carry protruding terminus, and the protruding terminus can be located at 3 ' ends of single stranded nucleic acid molecule.The presence of protruding terminus 4 is prevented from joint from the generation for connecting phenomenon, reduces the interference to follow-up sequencing experiment, improves the accuracy rate of SNP partings detection.The protruding terminus is preferably T；The joint can be directly connected to the pcr amplification product containing A tails expanded by Taq enzyme, improve joint efficiency.

In above-mentioned two embodiment, the step of the embodiment that joint is fixed on microballoon in advance reduces connection product and is connected with microballoon, conventional efficient is higher.

May be different from the density of the other compositions in step B reaction systems because being fixed with the density of the microballoon of joint, so, in connection procedure, whole reaction system is set periodically to shake, the joint efficiency of the joint and step A gained amplified productions being fixed on microballoon can be effectively improved, it is to avoid the appearance of the low phenomenon of joint efficiency that each composition is layered and causes in reaction system.

In addition, in the present invention, it is because the amplified production containing SNP site to be measured obtained by step A is directly connected with the joint in step B, i.e., not purified, reaction is directly attached, this causes that reaction system is larger, and DNA molecular concentration is relatively low, and joint efficiency is not high.In a preferred embodiment of the invention, PEG is added in the reaction system of the step B.

Because PEG can either improve the effective density of the molecule reacted in reaction system, the probability contacted between joint and corresponding amplified production is improved；The density of reaction system can be improved again, prevent the microballoon for being fixed with joint from settling；This programme effectively increases the joint efficiency of the joint and corresponding amplified production being fixed on microballoon in terms of two.In this programme, the specific concentration of PEG can be calculated according to the density of the density of microballoon in the step B and former reaction system, so as to the density of microballoon is essentially identical with the density for adding the reaction system after PEG be advisable.

Based on above-described embodiment, the present invention proposes an embodiment again, and the coupled reaction in the step B1 is carried out in cutting-coupled reaction system, and the cutting-coupled reaction system includes：Ligase, clastogen, the first joint being fixed on microballoon and connection buffer solution；Containing in the first general area can broken site；It is described can broken site can be cut agent specificity cutting so that based on the first primer pair obtain amplified production formed the first cohesive end；First joint is nucleic acid molecules, contains the second cohesive end matched with the first cohesive end complete complementary.

In this programme, based on the first general area in can broken site, amplified production is broken off after agent cuts out the first cohesive end, can be directly connected to the first joint containing the second cohesive end, improves joint efficiency.

In order to preferably realize the purpose of the present invention, the present invention proposes the 6th exemplary embodiments, and a kind of gene order surveying method is comprised the following steps：

A, using any of the above-described kind of primer sets, sample to be tested is entered performing PCR amplification, obtain the amplified production containing SNP site to be measured；

C, using any of the above-described kind of anchor primer, treat sequencing library molecule and be sequenced, obtain the sequence information of SNP site to be measured.

It should be noted that above-mentioned steps A, B are library construction step, it can carry out library construction using any one above-mentioned library constructing method.Hereinafter mainly it is further elaborated explanation by step C.Sequence measurement in step C can be alternatively synthesis sequencing for connection PCR sequencing PCR.

This programme is in sequencing procedure, anchor primer accurately can be anchored on target location, avoid because sequencing result is inaccurate caused by the particularity of sequence near SNP site to be measured or the appearance of failure phenomenon is sequenced, each anchor primer and target location is combined that situation is more consistent again, normalize, reduce because each anchor primer is combined the difference of situation with target location, so as to reduce the difference of each SNP site detection signal to be measured, the accuracy of sequencing is improved.

In one embodiment of the invention, sequence measurement in the step C is connection PCR sequencing PCR, also include step D between step B, C, using any one anchor primer in claim 7 to 11, the oligonucleotide probe marked using unstressed configuration replaces the oligonucleotide probe for having fluorescence labeling, treats sequencing library molecule and is once connected sequencing reaction.

It should be noted that the oligonucleotide probe of the unstressed configuration mark is compared with the oligonucleotide probe for having fluorescence labeling used in connection sequencing technologies, sequence is identical, and difference is only that whether there is fluorescence labeling；That is, the sequence oligonucleotide probe of unstressed configuration mark is（N-N-N……-N）N, the N are A, G, C or T, and n is positive integer.

Connection sequencing reaction is comprised the following steps each time：Anchor primer grappling, rinses（Remove unnecessary non-anchor primer）, probe connection, flushing（Remove excess probes, ligase etc.）, adopt figure（Obtain the sequence information of position corresponding to fluorescence labeling on probe）, denaturation wash-out connection product（To connect the grappling of anchor primer in sequencing reaction next time）.Connection sequencing reaction in step C, because using the oligonucleotide probe that unstressed configuration is marked, therefore can not carry out adopting figure step, to reduce experimental procedure, improve conventional efficient.Certainly, if carrying out adopting figure step, it may be verified that whether the fluorescence probe that this time is used is unstressed configuration mark, plays an effect reaffirmed.

Although, in theory, anchor primer can be eluted removal with the oligonucleotides case of unstressed configuration mark in step D, closing cannot be played a part of, but, present inventor contrasts discovery in specific experiment, before sequencing is connected, the step of probe that increasing an oligonucleotide probe replacement marked using unstressed configuration has fluorescence labeling is once connected sequencing reaction, it is effective to reduce the error signal occurred in follow-up connection sequencing, so as to reduce the interference signal during the analysis by sequencing initial results to final sequence information, improve the accuracy of SNP site detection.Concrete reason is probably that the target binding site of an active non-anchor primer and/or the target binding site of non-probe are closed by after step D, so that in follow-up connection sequencing experiment, anchor primer and/or probe more can be combined more accurately on target binding site, so as to reduce the generation of error signal, so as to improve the ratio of correct signal during genetic test, the accuracy detected to SNP site is improve.

Preferably, the n is between 6-10；More preferably between 7-9.

It is furthermore preferred that the oligonucleotide probe of the unstressed configuration mark is identical with the sequence oligonucleotide probe for having fluorescence labeling in step D, difference is only that whether there is fluorescence labeling.This programme can effectively reduce the design difficulty of the oligonucleotide probe of unstressed configuration mark.

In one embodiment of the invention, the step D is comprised the following steps：

D3, denaturation removal connection product.

It is single stranded nucleic acid molecule for being denatured the connection product for removing connection product it should be noted that connection product described in step D3 is the oligonucleotide probe that anchor primer is marked with unstressed configuration in step D3, can be matched with sequencing library complementary element is treated.The denaturation both can for example improve double chain acid molecule by the realization of the method for physics（Treat sequencing library molecule and connection product）Residing temperature, it is also possible to realized by the method for chemistry, for example, change double chain acid molecule（Treat sequencing library molecule and connection product）Residing pH value.Wherein, using chemical method, the reagent of acid or alkalescence need to be only added, without extra heater block, more simple and effective can realizes automation.

Preferably, the step D3 is：Rinsed with 0.05M-0.15M NaOH.

For above-mentioned each technical scheme, to further illustrate the technique effect and superiority of technical scheme described in the present invention, the present invention provides following specific embodiments.

In the first specific embodiment, the poba gene group DNA with 10 normal persons is template, while detecting CYP2C9 genes * 2（rs1799853）Site, ALDH2 gene rs671 sites, ADRB1 gene rs1801253 sites.

For these sites, a pair of inner primers and a pair of outer primers have been separately designed.It is described in detail below：

Rs1799853 inner primers to for：SEQ ID NO：1 and SEQ ID NO：2；Rs1799853 outer primers to for：SEQ ID NO：3 and SEQ ID NO：4；Rs671 inner primers to for：SEQ ID NO：5 and SEQ ID NO：6；Rs671 outer primers to for：SEQ ID NO：7 and SEQ ID NO：8；Rs1801253 inner primers to for：SEQ ID NO：9 and SEQ ID NO：10；Rs1801253 outer primers to for：SEQ ID NO：11 and SEQ ID NO：12.

The acquisition of the amplified production the, containing SNP site to be measured.

1st, first time PCR amplifications

With poba gene group DNA as template, using above-mentioned outer primer pair, sequence where each SNP site is expanded respectively, reaction system is：F primers（10μM）, 0.4 μ L；R primers（10μM）, 0.4 μ L；dNTP（Each 2.5mM）, 2 μ L；Poba gene group DNA, 50ng；Ex Taq（5U/μL）, 0.1 μ L；10 × Ex Taq Buffer, 2 μ L；ddH₂O adds to 20 μ L.

PCR reaction conditions are as follows：94℃ 5min；94 DEG C of 20s, 57 DEG C of 20s, 72 DEG C of 25s；Repeat 30 circulations；72℃ 3min.

Products therefrom is first time pcr amplification product.

2nd, second PCR amplification

With first time pcr amplification product as template, using above-mentioned inner primer pair, sequence where each SNP site is expanded respectively, reaction system is：F primers（10μM）, 0.4 μ L；R primers（10μM）, 0.4 μ L；dNTP（Each 2.5mM）, 2 μ L；First time pcr amplification product, 0.2 μ L；Ex Taq（5U/μL）, 0.1 μ L；10 × Ex Taq Buffer, 2 μ L；ddH₂O adds to 20 μ L.

PCR reaction conditions are as follows：95℃ 5min；94 DEG C of 20s, 40 DEG C of 20s, 57 DEG C of 25s；Repeat 5 circulations；94 DEG C of 20s, 57 DEG C of 20s, 72 DEG C of 25s；Repeat 30 circulations；72℃ 3min.

Products therefrom is second amplified production.

After the completion of second expands, detected through agarose gel electrophoresis, target molecule is contained in second amplified production of all samples, and gel electrophoresis figure shows that, without miscellaneous band, band is single；Illustrate that second PCR amplification have successfully been obtained required pcr amplification product.

2nd, the structure of sequencing library molecule is treated.

1st, the first joint is fixed on microballoon.

In order to distinguish different samples sources in detection process at the same time, this specific embodiment devises sequence label on the first joint, and the basic structure of the first joint is as shown in Figure 4（SEQ ID NO：13、SEQ ID NO：14）, the NNNN in the joint is sequence label, and sequence label is as shown in Figure 5 with the corresponding relation of samples sources and SNP site.

By above-mentioned each first joint respectively with the Myone magnetic beads modified with Streptavidin（Invitrogen）With reference to so that above-mentioned each first joint is separately fixed at magnetic bead surfaces, and reaction system and course of reaction are：By the joints of 200ng first and 4 μ L（About 4 × 10⁷Individual magnetic bead）The vibration of Myone magnetic beads spiral is mixed, and 30min is reacted, with appropriate TE buffer solutions（10mM Tris-HCl, pH8.0；1mM EDTA）Clean twice, centrifugation, the magnetic bead that will be obtained is with 4 μ L combination buffers（10mM Tris-HCl, pH7.5；1mM EDTA；1M NaCl；0.01% Triton X-100）Resuspended preservation, must be fixed with the magnetic bead of the first joint.

2nd, the connection of second pcr amplification product and the first joint being fixed on magnetic bead.

Following reaction systems are respectively configured by by the corresponding relation in table 2：The μ L of step one gained amplified production 20；USER enzymes（1U/ μ L, NEB, Cat#M5505S）, 10 μ L；Buffer solution, 8 μ L；T4 DNA ligases, 2 μ L；It is fixed with the magnetic bead of the first joint, 0.4 μ L；Plus ddH₂The μ of O to 40 L.

Wherein, the buffer solution is Tris containing 400mM, 100mM MgCl2,100mM DTT, 5mM ATP, 25% PEG 6000, the solution of pH value 7.8.

25 DEG C are reacted 20 minutes, obtain connection product, that is, treat sequencing library molecule.

After reaction terminates, 30 pipe connection products are merged into 1 pipe, 2500g is centrifuged 3min, and magnet adsorption magnetic bead removes supernatant, washs secondary with 50 μ L TE, and is finally suspended in 50 μ L TE, so as to will treat that sequencing library molecule is fixed on magnetic bead.

3rd, the addressable for treating sequencing library molecule being fixed on magnetic bead is fixed.

The load sample piece that step 2 products therefrom point sample to isothiocyano is modified（Slide）, in 37 DEG C of fixed 1h, that is, the addressable for treating sequencing library molecule for completing to be fixed on magnetic bead is fixed.

3rd, it is sequenced.

To the treating on solid phase carrier that be fixed on obtained by step 2, sequencing library molecule is sequenced.

Using the high flux gene sequencer Pstar II A sequenators of Shenzhen HYK Gene Technology Co., Ltd. in this specific embodiment, and it is sequenced using connection PCR sequencing PCR.In sequencing procedure,

For rs1799853 sites, the anchor primer for being used is SEQ ID NO：15；For rs671 sites, the anchor primer for being used is SEQ ID NO：16；For rs1801253 sites, the anchor primer for being used is SEQ ID NO：17.

In addition, in order to distinguish different samples sources, also needing to detect the sequence label on the first joint, the anchor primer for detecting sequence label is SEQ ID NO：18.

It should be noted that SEQ ID NO：5 ' the ends of 15-18 are phosphorylated modification.

Overall sequencing order is as shown in Figure 6.Wherein, SNP site anchor mixtures to be measured are SEQ ID NO：Mixture of this 3 kinds of anchor primers of 15-17 as obtained by identical molal quantity mixes.Wherein, Base1 is used to close anchor primer or probe and treats the nonspecific binding site on sequencing library molecule, and Base2 is used to detect SNP site to be measured that Base3-6 to be used to detect sequence label.

In addition, the present embodiment has been the first contrast experiment, same step 2 products therefrom is sequenced with the sequencing order in Fig. 7.That is, the Base1 in Fig. 6 is not carried out.

Fig. 8 shows the SNP site to be measured of sample 2 in above-mentioned specific embodiment --- the preliminary experimental results of rs1799853, rs671, rs1801253 in experimental group and the first contrast groups.Wherein, R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, W=A/T, H=A/C/T, B=C/G/T, V=A/C/G, D=A/G/T, N=A/C/G/T；N represents that signal is too weak, it is impossible to which which in A, C, G and T judgement be.

From experimental result, identical sample accounting highest SNP site sequence type all same in experimental group and the first contrast groups, but their accounting has significant difference；Accounting highest SNP site sequence type detects number more than 95% in experimental group, and accounting highest SNP site sequence type detects base sheet 85% or so in the first contrast groups.

In addition, also second amplified production is sequenced by Sanger for the present inventor being verified, as the second contrast experiment, as a result show, accounting highest SNP site sequence type is completely the same with Sanger sequencing results in above-mentioned experimental group and the first contrast groups.

In order to prove the technique effect of primer sets of the invention, anchor primer, this experiment inventor has also been the 3rd contrast experiment, and the equally poba gene group DNA with the normal person in above-mentioned specific embodiment is as template, while detecting CYP2C9 genes * 2（rs1799853）Site, ALDH2 gene rs671 sites, ADRB1 gene rs1801253 sites.

Rs1799853 inner primers to for：SEQ ID NO：19 and SEQ ID NO：20；Rs1799853 outer primers to for：SEQ ID NO：3 and SEQ ID NO：4；Rs671 inner primers to for：SEQ ID NO：21 and SEQ ID NO：22；Rs671 outer primers to for：SEQ ID NO：7 and SEQ ID NO：8；Rs1801253 inner primers to for：SEQ ID NO：23 and SEQ ID NO：24；Rs1801253 outer primers to for：SEQ ID NO：11 and SEQ ID NO：12.

1st, first time PCR amplifications.

Products therefrom is first time pcr amplification product.

2nd, second PCR amplification.

PCR reaction conditions are as follows：95℃ 3min；94 DEG C of 20s, 57 DEG C of 20s, 72 DEG C of 30s；Repeat 30 circulations；72℃ 3min.

Products therefrom is second amplified production.

2nd, the structure of sequencing library molecule is treated.

Sequencing library is built using upper specific embodiment identical method and will treat that sequencing library molecule addressable is fixed.

3rd, it is sequenced.

The same high flux gene sequencer Pstar II A sequenators using Shenzhen HYK Gene Technology Co., Ltd. in the 3rd contrast experiment, and be sequenced using connection PCR sequencing PCR.In sequencing procedure, for rs1799853 sites, the anchor primer for being used is SEQ ID NO：25；For rs671 sites, the anchor primer for being used is SEQ ID NO：26；For rs1801253 sites, the anchor primer for being used is SEQ ID NO：27.

It should be noted that SEQ ID NO：18th, the 5 ' ends of 25-27 are phosphorylated modification.

Then referring to the method in a upper specific embodiment, after changing anchor primer, it is sequenced according to the order of Fig. 6；In addition, it is same with this specific embodiment step 2 products therefrom, after changing anchor primer, it is sequenced according to the order of Fig. 7, as control.

Wherein, the sequencing and image collecting result such as Fig. 9 in rs671 and rs1801253 sites（Experimental group and control group）Shown, as shown in Figure 9, the two sites equal unstressed configuration in sequencing procedure twice occurs, sequencing failure；This sequencing and image collecting result with rs671 and rs1801253 in a upper specific embodiment（Figure 10 experimental groups and control group）There is significant difference.And rs1799853 sites experimental group sequencing and image collecting result as shown in figure 11, with a upper specific embodiment in experimental group rs1799853 sites sequencing and image collecting result（Figure 12）Compare, equally exist significant difference, fluorescence signal is substantially weaker.The sequencing and image collecting result of the control group sequencing and image collecting result in rs1799853 sites and control group in a upper specific embodiment, similar with experimental group, fluorescence signal is substantially weaker.

Analyzed through the present inventor, rs671 and rs1801253 sites in this specific embodiment, the reason for experimental group and control group are sequenced unsuccessfully are probably：There is labyrinth in the sequence of rs671 and rs1801253 the two location proximates, cause anchor primer cannot grappling, and then the connection of anchor primer and probe cannot be realized, so no signal when adopting figure.

And the sequencing and image collecting result of rs1799853 sites experimental group and control group in this specific embodiment is respectively compared with experimental group and the sequencing and image collecting result of control group in a upper specific embodiment, fluorescence signal is all substantially weaker.During reason is probably this specific embodiment, anchor primer and the combination effect of sequencing library molecule is treated, relatively go up a specific embodiment poor.

In the second specific embodiment of the invention, directly with the rs1799853 inner primers in above-described embodiment to for：SEQ ID NO：1 and SEQ ID NO：2；Rs671 inner primers to for：SEQ ID NO：5 and SEQ ID NO：6；Rs1801253 inner primers to for：SEQ ID NO：9 and SEQ ID NO：10.Enter performing PCR amplification to above-mentioned sample 1-10, then follow-up step two, three are carried out, experimental results and the first specific embodiment result are basically identical, accounting highest SNP site sequence type detects number more than 93% in experimental group, and accounting highest SNP site sequence type detects base sheet 80% or so in control group.Generally, the detection number of experimental group and control group and detection sum are low compared with a upper embodiment, and either in experimental group still in control group, accounting highest SNP site sequence type detects number proportion and decreases.

It should be noted that in above-mentioned specific embodiment, in the primer for using, can broken site be U, and design on sense primer, Cleavage sequences（It is corresponding with the first cohesive end）It is CGGU.Certainly, also can by can broken site design on anti-sense primer.The Cleavage sequences can be designed as needed, as long as the requirement of follow-up connection experiment can be met, for example：GCCU, AAAU, CGCU, GCCCU, CGGCU, TTTTU etc..With the change of the Cleavage sequences, the correlated series of first joint need to carry out related adjustment.

In addition, in above-mentioned specific embodiment, after the completion of sequencing library molecule construction, being provided without unimolecule amplification technique and being expanded, but follow-up sequencing steps are entered directly into, simplify experimental procedure.Certainly, present inventor has found that being treated after sequencing library molecule is expanded by unimolecule amplification technique and being sequenced again, experimental results are essentially identical with above-mentioned experimental result by experiment.Comparatively, sequencing and image collecting gained signal can be higher.

More than, the method for the present invention can efficiently while multiple SNP sites of multiple samples are detected simultaneously, and obtain accurate believable result, and the error signal in connection sequencing acquired results can be effectively reduced, improve the accuracy of SNP site detection.

It should be noted that the foregoing is only presently preferred embodiments of the present invention, it is not intended to limit the invention, all any modification, equivalent and improvement made within the spirit and principles in the present invention etc. should be included within the scope of the present invention.

SEQUENCE LISTING

<110>Sheng Sitong

<120>Primer sets, anchor primer, kit, library construction and gene order surveying method

<130>

<160> 27

<170> PatentIn version 3.3

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Claims

1. a kind of primer sets, including the first primer pair, it is characterised in that the sense primer in first primer pair is made up of the first general area and the first complementary region；First complementary region is connected with the 3 ' ends in the described first general area, first complementary region and First ray complete complementary；The First ray be SNP site to be measured one section of sequence in the sequence, the 3 ' ends in SNP site to be measured, 5 ' ends of the First ray are away from SNP site to be measured 1 to 7bp；The first general area is not complementary with the second sequence；Second sequence is one section of sequence being connected with 3 ' ends of First ray in sequence where SNP site to be measured.

2. primer sets according to claim 1, it is characterised in that the first complementary region sequence length is between 6 to 12bp.

3. primer sets according to claim 1, it is characterised in that the first general region sequence length is between 6 to 16bp.

4. primer sets according to claim 1, it is characterised in that the primer sets also include the second primer pair, first primer pair and the second primer pair are respectively the inner primer pair and outer primer pair expanded to sequence where SNP site to be measured.

5. primer sets according to claim 1, it is characterised in that the anti-sense primer in first primer pair is made up of the second general area and the second complementary region；Second complementary region is connected with the 3 ' ends in the described second general area；Second complementary region be SNP site to be measured one section of sequence in the sequence, the 5 ' ends in SNP site to be measured；The second general area is different from the 3rd sequence；3rd sequence is one section of sequence being connected with 5 ' ends of the second complementary region in sequence where SNP site to be measured.

6. primer sets according to claim 1, it is characterised in that contain U in the first general area.

7. a kind of anchor primer, it is characterised in that the anchor primer and the 4th sequence complete complementary are matched；4th sequence is single stranded nucleic acid molecule, be the first primer pair in claim 1 to 6 in any one primer sets amplified production in one section of sequence；

4th sequence includes First ray and the first normalization area, and the first normalization area is connected with 3 ' ends of First ray, and 5 ' ends of the 4th sequence are away from SNP site to be measured 1 to 7bp；Or the 4th sequence includes the first complementary region and the second normalization area, the second normalization area is connected with 5 ' ends of the first complementary region, and 3 ' ends of the 4th sequence are away from SNP site to be measured 1 to 7bp.

8. anchor primer according to claim 7, it is characterised in that the first complementary region sequence length is between 6 to 12bp.

9. anchor primer according to claim 7, it is characterised in that the length in the first normalization area is between 8 to 14bp.

10. a kind of kit, it is characterised in that including any one primer sets in claim 1 to 6.

11. a kind of kits, it is characterised in that including any one anchor primer in claim 7 to 9.

12. a kind of library constructing methods, it is characterised in that comprise the following steps：

A, using any one primer sets in claim 1 to 6, sample to be tested is entered performing PCR amplification, obtain the amplified production containing SNP site to be measured；

13. library constructing methods according to claim 12, it is characterised in that the step B is：

14. library constructing methods according to claim 13, it is characterised in that the step B is comprised the following steps：

15. library constructing methods according to claim 14, it is characterised in that the coupled reaction in the step B1 is carried out in cutting-coupled reaction system, the cutting-coupled reaction system includes：Ligase, clastogen, the first joint being fixed on microballoon and connection buffer solution；Contain U in the first general area；The clastogen is used for specificity cutting U, and the amplified production containing SNP site to be measured is formed the first cohesive end；First joint is nucleic acid molecules, contains the second cohesive end matched with the first cohesive end complete complementary.

16. library constructing methods according to claim 15, it is characterised in that contain PEG in connection buffer solution described in step B1.

17. a kind of gene order surveying methods, it is characterised in that comprise the following steps：

C, using any one anchor primer in claim 7 to 9, treat sequencing library molecule and be sequenced, obtain the sequence information of SNP site to be measured.

18. gene order surveying methods according to claim 17, it is characterised in that the sequence measurement in the step C is connection PCR sequencing PCR；Also include step D between step B, C, using any one anchor primer in claim 7 to 9, the oligonucleotide probe marked using unstressed configuration is replaced the oligonucleotide probe for having fluorescence labeling, treats sequencing library molecule and once connected sequencing reaction.

19. gene order surveying methods according to claim 18, it is characterised in that the step D is comprised the following steps：

D3, denaturation removal connection product.