CN102533752B

CN102533752B - Oligo dT primer and method for constructing cDNA library

Info

Publication number: CN102533752B
Application number: CN201210047909.3A
Authority: CN
Inventors: 盛司潼
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-02-28
Filing date: 2012-02-28
Publication date: 2015-01-21
Anticipated expiration: 2032-02-28
Also published as: CN102533752A

Abstract

The invention relates to the field of molecular biology and provides Oligo dT primer, wherein Oligo dT primer is a single-stranded DNA molecule containing continuous dT sequences and recognition sites for cutting. The invention further provides a method for constructing a cDNA library on the basis of the Oligo dT primer. The Oligo dT primer is excellent in applicability, can be applied to various different library schemes, and can further accurately locate the initiation site of mRNA molecule in poly A tail. The method for constructing the cDNA library can ensure that all mRNA molecules in a sample can show specificity in the constructed cDNA library.

Description

A kind of method in Oligo dT primer and construction cDNA library

Technical field

The present invention relates to genetically engineered field, more particularly, relate to a kind of Oligo dT primer, and carry out the method for cDNA library structure based on this Oligo dT primer.

Background technology

The analysis of gene expression dose, plays vital effect to research gene function.Expressed sequence analyzes (serial analysis of gene expression, SAGE), it is a kind of technology of real-time analysis gene expression information, it is by quick and detailed analysis thousands of expressed sequence tag (Expressed Sequenced Tags, EST) the SAGE sequence label that gene expression abundance is different is found out, thus close to intactly obtaining genomic expression information.SAGE technology is the modal gene expression profile research method of order first two together with gene chip.Along with the development of s-generation sequencing technologies, by construction cDNA library, then utilize the high-throughput advantage of s-generation sequencing technologies to check order to mRNA library, and then the method for carrying out gene expression spectrum analysis occupy more and more consequence in gene expression profile research.

There is cap sequence in 5 ' end of Eukaryotic mRNA molecule, and contain secondary structure toward contact, therefore, to successfully build 5 ' end cDNA library (label to be measured that in this library, each molecule carries derives from 5 ' end of 5 ' each mRNA molecule), must overcome the impact of the secondary structure of 5 ' end cap minor structure and 5 ' end existence, difficulty is larger.In addition, even if obtain 5 ' end cDNA library, because mRNA molecule 5 ' end GC content is higher, the corresponding relation of the label to be measured that in this library, each molecule carries and each mRNA molecule is poor, make the application difficulty of this library in multiple scientific research higher, the result obtained during expression pattern analysis for mRNA molecule is inaccurate.Further, 5 ' the end cDNA library 5 ' end performance not enough (undellrepresentation) building gained is the inherent limitations of current technology, and is caused by many factors.Wherein most important a kind of factor is caused by the random failure of the prolongation process of reversed transcriptive enzyme.Because reversed transcriptive enzyme moves to 5 ' end from 3 ' of mRNA, so the reversed transcriptive enzyme of certain percentage may dissociate from deleting RNA template, thus cDNA is caused to synthesize premature end.The factor that another kind works is that reversed transcriptive enzyme suspends in mRNA secondary structure region, slows down or stop.Also have a kind of factor to be is introduced by the RNA enzyme of contaminative, if becoming in the process of doublestranded cDNA molecule by mRNA molecule reverse transcription, have the contaminative RNA enzyme of denier, so, 5 ' end of part mRNA molecule can the RNA enzyme liberating removal of contaminated property.The impact of above-mentioned situation on long mRNA molecule is especially serious.Therefore, in the prior art in current construction cDNA library, building 3 ' end cDNA library is main flow.And the normal Oligo dT primer adopted is the single strand dna containing continuous print dT sequence in prior art, sequence can be abbreviated as: 5 '-(dT) _n-3 ', in the building process in whole library, only have separation and purification mRNA molecule, the reverse transcription of mRNA molecule is become the effect of double-strand cDNA, comparatively large to the subsequent operations restriction formed after double-strand cDNA, applicable banking process is single.

At present, the method building 3 ' end cDNA library is roughly as follows:

(1) utilize Oligo dT primer by mRNA reverse transcription and synthesize dscDNA;

(2) grappling enzyme (anchoring enzyme, AE) NlaIII enzyme cuts dscDNA, collects the fragment between the poly A/T part of dscDNA and nearest restriction enzyme site, obtains 3 '-cDNA;

(3) in 5 ' the termination top connection 1 of 3 '-cDNA, must contain the cDNA of joint 1, this joint 1 is double chain DNA molecule, comprises Mme I restriction endonuclease recognition sequence, and its 3 ' end is containing CATG tetra-base protruding terminus;

(4) Mme I enzyme suits the cDNA having joint 1, and reclaims the digestion products containing joint 1;

(5) in 3 ' termination top connection 2 of the digestion products containing joint 1, thus obtain the cDNA library that two ends are connected with different joint sequence, this joint 2 is double chain DNA molecule, and its 5 ' end is protruding terminus, containing two universal base.

Grappling enzyme in aforesaid method is sequence-specific restriction enzyme, and when restriction enzyme site without this grappling enzyme on some mRNA molecule, these mRNA molecules cannot embody in final cDNA library; Or the restriction enzyme site of grappling enzyme is improper on some mRNA molecule, make follow-up IIs type restriction enzyme Mme I cannot realize enzyme to cut, or realize position that Mme I enzyme the cuts poly A/T part at dscDNA, the specific sequence comprised in the cDNA library molecule that these mRNA molecules are formed is too short, and what cannot realize with these mRNA molecular specificities is corresponding.That is, aforesaid method cannot ensure that mRNA molecules all in sample all can be embodied in cDNA library, and then makes follow-up gene expression spectrum analysis result inaccurate.

Therefore need a kind of new Oligo dT primer, this primer suitability is wide, and the restriction of doublestranded cDNA molecule to subsequent operations formed based on this primer is little, can be applied in multiple banking process; Need a kind of new cDNA library construction process in addition, this cDNA library construction process can make mRNA molecules all in sample obtain specific embodiment in the cDNA library built.

Summary of the invention

The object of the present invention is to provide a kind of Oligo dT primer, solve the Oligo dT primer scope of application of the prior art little, the performance constraint formed after doublestranded cDNA molecule based on Oligo dT primer of the prior art is large, applies the problem that its banking process is single.

In order to realize goal of the invention, the invention provides a kind of Oligo dT primer of particular design, this Oligo dT primer, suitability is good, can be applicable to multiplely different build storehouse scheme; Further, this Oligo dT primer construction is simple, can be positioned at the initiation site of mRNA molecule poly A tail accurately; And utilize this Oligo dT primer to carry out the structure of cDNA library, can avoid part mRNA molecule in the cDNA library built, can not get the appearance of the phenomenon of specific embodiment.

A kind of Oligo dT primer, be the single strand dna containing continuous print dT sequence, it contains the recognition site for cutting.

Wherein, this recognition site being used for cutting is uridylate, Hypoxanthine deoxyriboside, phosphorothioate bond or IIs type restriction endonuclease recognition sequence, and described IIs type restriction endonuclease recognition sequence is at 5 ' end of Oligo dT primer.

Wherein, this Oligo dT primer sequence is:

5’-(dT) _xU(dT) _y-3’；

Or 5 '-(RERS)-(dT) _z-3 ';

Or 5 '-(dT) _α(P-S) (dT) _β-3 ';

Or 5 '-(dT) _γ(I) (dT) _δ-3 ';

Wherein, x, y, z, α, β, γ, δ are natural number, and RERS is the nucleotide sequence containing IIs type restriction endonuclease recognition sequence, and P-S is phosphorothioate bond, and I is Hypoxanthine deoxyriboside.

Wherein, 3 ' end of this Oligo dT primer is the Nucleotide M containing universal base, and M is A or G or C.

Further, 3 ' end of this Oligo dT primer is two Nucleotide containing universal base, and be followed successively by M and N from 5 ' end to 3 ' end, N is A or G or C or T or U or I; Described Oligo dT primer sequence is:

5’-(dT) _xU(dT) _yMN-3’；

Or 5 '-(RERS)-(dT) _zmN-3 ';

Or 5 '-(dT) _α(P-S) (dT) _βmN-3 ';

Or 5 '-(dT) _γ(I) (dT) _δmN-3 ';

Further, x+y >=14, z >=15, alpha+beta >=14, γ+δ >=14.

Wherein, described Oligo dT primer contains marker, and described marker adopts at least one in vitamin H, antigen, antibody, acceptor, part, polyhistidine.

Another object of the present invention is to provide a kind of new cDNA library construction process, is intended to solve prior art and can not ensures that in sample, all mRNA molecules all can obtain the problem of specificity embodiment in cDNA library.

Based on the Oligo dT primer of above-mentioned particular design, the invention provides a kind of cDNA library construction process, comprise the following steps:

A. utilize Oligo dT primed reverse transcription mRNA, form Article 1 cDNA chain, obtain RNA/DNA hybrid molecule; This Oligo dT primer is the single strand dna containing continuous print dT sequence, and it contains the recognition site for cutting;

B. the Article 1 cDNA chain in RNA/DNA hybrid molecule is increased, obtain double-strand cDNA;

C. utilize IIs type digestion with restriction enzyme double-strand cDNA, the fragment of corresponding sequence must be held containing mRNA molecule 3 ';

D. hold the fragment of corresponding sequence to be connected with the first linkers containing mRNA molecule 3 ', obtain cDNA library.

Wherein, this recognition site being used for cutting is uridylate, phosphorothioate bond or Hypoxanthine deoxyriboside; This step C comprises the following steps:

C01. cutting agent cutting double-strand cDNA, obtains double-strand cutting fragment;

C02. double-strand cutting fragment is connected with the second linkers, obtains the first connection product;

C03. utilize an IIs type digestion with restriction enzyme first to connect product, the fragment of corresponding sequence must be held containing mRNA molecule 3 ';

Described cutting agent is used for second phosphodiester bond that specificity cutting uridylic, phosphorothioate bond or Hypoxanthine deoxyriboside 3 ' is held.

Further, this Oligo dT primer sequence is:

5’-(dT) _xU(dT) _y-3’；

Or 5 '-(dT) _α(P-S) (dT) _β-3 ';

Or 5 '-(dT) _γ(I) (dT) _δ-3 ';

Wherein, x, y, α, β, γ, δ are natural number, 14≤x+y≤17,14≤alpha+beta≤17,14≤γ+δ≤17, and P-S is phosphorothioate bond, and I is Hypoxanthine deoxyriboside.

C11. cutting agent cutting double-strand cDNA, obtains double-strand cutting fragment;

C12. double-strand cutting fragment is connected with the second linkers, obtains the first connection product;

C13. utilize an IIs type digestion with restriction enzyme first to connect product, obtain not containing the endonuclease bamhi of the second linkers;

C14. the endonuclease bamhi not containing the second linkers is connected with the 3rd linkers, obtains the second connection product;

C15. utilize the 2nd IIs type digestion with restriction enzyme second to connect product, the fragment of corresponding sequence must be held containing mRNA molecule 3 ';

Further, this Oligo dT primer sequence is:

5’-(dT) _xU(dT) _y-3’；

Or 5 '-(dT) _α(P-S) (dT) _β-3 ';

Or 5 '-(dT) _γ(I) (dT) _δ-3 ';

Wherein, x, y, α, β, γ, δ are natural number, x+y >=14; 3≤x≤9, alpha+beta >=14,3≤α≤9, γ+δ >=14,3≤γ≤9.

Wherein, this recognition site being used for cutting is IIs type restriction endonuclease recognition sequence, and it is positioned at 5 ' end of Oligo dT primer; This step C is specially:

IIs type digestion with restriction enzyme double-strand cDNA, separation and purification goes out the digestion products of answering sequence containing Oligo dT primer pair, must hold the fragment of corresponding sequence containing mRNA molecule 3 '.

Further, this Oligo dT primer sequence is:

5 '-(RERS)-(dT) _z-3 '; Wherein, RERS is the nucleotide sequence containing IIs type restriction endonuclease recognition sequence, and z is natural number, 10≤z≤17.

C21.IIs type digestion with restriction enzyme double-strand cDNA, separation and purification goes out the endonuclease bamhi without IIs type restriction endonuclease recognition sequence;

C22. the endonuclease bamhi without IIs type restriction endonuclease recognition sequence is connected with the 4th linkers, obtains the 3rd and connect product;

C23. utilize the 3rd IIs type digestion with restriction enzyme the 3rd to connect product, the fragment of corresponding sequence must be held containing mRNA molecule 3 '.

Further, this Oligo dT primer sequence is:

5 '-(RERS)-(dT) _z-3 '; Wherein, RERS is the nucleotide sequence containing IIs type restriction endonuclease recognition sequence, and z is natural number, 15≤z≤30.

Wherein, before steps A, also steps A is comprised ': utilize Oligo dT primer separation and purification mRNA from total serum IgE.

Wherein, in above-mentioned any scheme, 3 ' end of described Oligo dT primer can be the Nucleotide M containing universal base, and M is A or G or C.

Further, 3 ' end of this Oligo dT primer can be two Nucleotide containing universal base, and be followed successively by M and N from 5 ' end to 3 ' end, N is A or G or C or T or U or I; Described Oligo dT primer sequence is:

5’-(dT) _xU(dT) _yMN-3’；

Or 5 '-(RERS)-(dT) _zmN-3 ';

Or 5 '-(dT) _α(P-S) (dT) _βmN-3 ';

Or 5 '-(dT) _γ(I) (dT) _δmN-3 ';

Further, x+y >=14, z >=15, alpha+beta >=14, γ+δ >=14.

Wherein, in above-mentioned any scheme, this Oligo dT primer is at 5 ' end containing marker, and this marker adopts at least one in vitamin H, antigen, antibody, acceptor, part, polyhistidine; Be preferably vitamin H or polyhistidine.

Wherein, this linkers contains marker, and this marker adopts at least one in vitamin H, antigen, antibody, acceptor, part, polyhistidine; Be preferably vitamin H or polyhistidine.

Wherein, after step D, also step D ' can be comprised: utilize the primer pair cDNA library identical or complementary with the known array at cDNA library two ends to carry out pcr amplification.

As from the foregoing, Oligo dT primer of the present invention, suitability is good, can be applicable to multiplely different build storehouse scheme, its structure is simple, carries out the structure of cDNA library based on it, and the phenomenon that part mRNA molecule in sample can be avoided in the cDNA library built to can not get specific embodiment occurs, further, the initiation site of mRNA molecule 3 ' end poly A can also be accurately positioned in.Based on the cDNA library construction process that Oligo dT primer of the present invention carries out, mRNA molecules all in sample can be made in the cDNA library built to obtain specific embodiment, and the demand of initial mRNA molecular weight can be reduced further, simplify experimental procedure, improve the success ratio of building storehouse, the determinacy of position is high in mRNA molecule for the label to be measured (namely deriving from the specific sequence of each mRNA molecule) that in constructed cDNA library, each molecule carries, be conducive to the interpretation of result of cDNA library after completing order-checking built, thus can further improve the accuracy of carrying out mrna expression spectrum analysis according to the cDNA library built, expression pattern analysis is can be used for by the cDNA library of the inventive method gained, mRNA molecule 3 ' terminal Sequence Analysis etc.

Accompanying drawing explanation

Fig. 1 is the structural representation of phosphorothioate ester key in one embodiment of the invention;

Fig. 2 is the method flow diagram that in one embodiment of the invention, cDNA library builds;

Fig. 3 is the structural representation of the joint with loop-stem structure in one embodiment of the invention;

Fig. 4 is the structural representation of single protruding terminus joint in one embodiment of the invention;

Fig. 5 is the structural representation of two protruding terminus in one embodiment of the invention;

Fig. 6 is the method flow diagram must holding the fragment of corresponding sequence in one embodiment of the invention containing mRNA molecule 3 ';

Fig. 7 is the method flow diagram must holding the fragment of corresponding sequence in another embodiment of the present invention containing mRNA molecule 3 ';

Fig. 8 is the method flow diagram must holding the fragment of corresponding sequence in another embodiment of the present invention containing mRNA molecule 3 '.

Embodiment

In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.

IIs type restriction enzyme of the present invention is the restriction enzyme of cleavage site outside recognition sequence, include but not limited to: Acu I, Alw I, Bbs I, BbV I, Bcc I, BceA I, BciV I, BfuA I, Bmr I, Bpm I, BpuE I, Bsa I, BseM II, BseR I, Bsg I, BsmA I, BsmB I, BsmF I, BspCN I, BspM I, BspQ I, BtgZ I, Ear I, Eei I, EcoP15 I, Fau I, Fok I, Hga I, Hph I, HpyAV, Mbo II, Mly I, Mme I, Mnl I, NmeAIII, Ple I, Sap I, SfaN I and TspDT I, be preferably Acu I, Bsg I, EcoP15 I or Mme I.

MRNA of the present invention derives from eukaryote, includes but not limited to animal, plant, fungi and protobiont.

A kind of Oligo dT primer, this Oligo dT primer is the single strand dna containing continuous print thymidylic acid (thymidine deoxynucleotide, dT) sequence, and it contains the recognition site for cutting.

It should be noted that:

The cutting carried out for the recognition site cut is utilized to occur in recognition site; Also can occur in outside recognition site, such as, at the specific position of 3 ' extreme direction of this recognition site.Utilize the cutting carried out for the recognition site cut can occur in this Oligo dT primer, also can occur in fragment that this Oligo dT primer amplification goes out.

Oligo dT primer of the present invention, because it contains the recognition site for cutting, carry out in the process of cDNA library structure utilizing it, various process can be carried out to the doublestranded cDNA molecule formed based on it as required, that is, can according to the difference of objective condition, adopt and multiplely different build storehouse scheme, thus reaching optimal design, Oligo dT primer suitability of the present invention is wide, can be applied to multiplely to build in the scheme of storehouse; And utilize this Oligo dT primer to carry out the structure of cDNA library, can avoid part mRNA molecule in sample in the cDNA library built, can not get the appearance of the phenomenon of specific embodiment.

Wherein, this recognition site being used for cutting includes but not limited to uridylate (uridine, U), Hypoxanthine deoxyriboside (deoxyinosine, I), phosphorothioate bond (P-S) or IIs type restriction endonuclease recognition sequence, described IIs type restriction endonuclease recognition sequence is at 5 ' end of Oligo dT primer.All can there is specific cutting under the effect of corresponding material in the above-mentioned recognition site for cutting.Corresponding relation for the recognition site the cut material corresponding to this includes but not limited to:

U---USER enzyme or UDG enzyme;

I---intestinal bacteria adjust restriction endonuclease V and homologue thereof or DNA glycosylase;

P-S---the cutting agent containing Ag, Hg, Cu, Mn, Zn or Cd atom;

IIs type restriction endonuclease recognition sequence---IIs type restriction enzyme.

It should be noted that:

One of phosphorothioate bond in such scheme bridge joint Sauerstoffatom referring to phosphodiester bond is replaced by sulphur atom.Phosphorothioate ester key can be that 5 ' shown in Figure 1A-S phosphorothioic acid ester connects (3 '-O-P-S-5 '), also can be that 3 ' shown in Figure 1B-S phosphorothioic acid ester connects (3 '-S-P-O-5 ').

Available various metallic material cutting phosphorothioate bond.Described metal can be Ag, Hg, Cu, Mn, Zn or Cd.Preferably, this material is to provide Ag ⁺, Hg ⁺⁺, Mn ⁺⁺, Zn ⁺or Cd ⁺water-soluble salt (also can adopt the salt of the ion that other state of oxidation is provided) of ion.Particularly preferably contain silver salt as Silver Nitrate (AgNO ₃) or other Ag is provided ⁺the salt of ion.The condition of cutting comprises such as 50mMAgNO ₃, about 22 ~ 37 DEG C, 10 minutes or longer time as 30 minutes.Preferably, pH is 4.0 ~ 10.0, more preferably 5.0 ~ 9.0, according to appointment 6.0 ~ 8.0, according to appointment 7.0.See Mag, M. etc., Nucleic Acids Res., 19 (7): 1437-1441,1991.

Further, this Oligo dT primer sequence is:

5’-(dT) _xU(dT) _y-3’；

Or 5 '-(RERS)-(dT) _z-3 ';

Or 5 '-(dT) _α(P-S) (dT) _β-3 ';

Or 5 '-(dT) _γ(I) (dT) _δ-3 ';

Wherein, 3 ' end of the Oligo dT primer in above-mentioned all schemes can be the Nucleotide M containing universal base, and M is A or G or C.

The Nucleotide M of universal base is contained by this, this Oliog dT primer is carrying out the initiation site that can be accurately positioned in mRNA molecule poly A tail in process of reverse-transcription, thus elimination Oligo dT primer when synthesizing Article 1 cDNA chain may be combined caused disadvantageous effect with the optional position of poly A tail, ensure that label to be measured (namely deriving from the specific sequence of each mRNA molecule) determinacy of position in mRNA molecule that in constructed cDNA library, each molecule carries, be conducive to the interpretation of result of cDNA library after completing order-checking built, further increase the accuracy of carrying out mrna expression spectrum analysis according to the cDNA library built.

3 ' end sequence of this Oligo dT primer can represent with following general formula:

5 '-M (N) _ε-3 '; Wherein, ε is natural number, and N is A or G or C or T or U or I.

Wherein, along with the increase of ε, the accuracy rate that this Oligo dT primer is carrying out being positioned in process of reverse-transcription mRNA molecule poly A tail improves.After the preparation cost considering accuracy rate and Oligo dT primer, ε is preferably 1 or 2.

Further, 3 ' end of described Oligo dT primer is two Nucleotide containing universal base, and be followed successively by M and N from 5 ' end to 3 ' end, M is A or G or C, N is A or G or C or T or U or I; Described Oligo dT primer sequence is:

5’-(dT) _xU(dT) _yMN-3’；

Or 5 '-(RERS)-(dT) _zmN-3 ';

Or 5 '-(dT) _α(P-S) (dT) _βmN-3 ';

Or 5 '-(dT) _γ(I) (dT) _δmN-3 ';

Further, x+y >=14, z >=15, alpha+beta >=14, γ+δ >=14.This programme ensure that the bonding strength of the poly A tail of Oligo dT primer and mRNA molecule, is conducive to carrying out smoothly of follow-up reverse transcription reaction.

Wherein, the Oligo dT primer in above-mentioned all schemes also can contain marker, and this marker adopts at least one in vitamin H, antigen, antibody, acceptor, part, polyhistidine.By the title complex with the marker adaptation on Oligo dT primer, can further improve Oligo dT primer ease for use in use, improve conventional efficient.

Fig. 2 shows the cDNA library construction process flow process in the first embodiment of the present invention, comprises the following steps:

S1. utilize Oligo dT primed reverse transcription mRNA, form Article 1 cDNA chain, obtain RNA/DNA hybrid molecule;

S2. the Article 1 cDNA chain in RNA/DNA hybrid molecule is increased, obtain double-strand cDNA;

S3. utilize IIs type digestion with restriction enzyme double-strand cDNA, the fragment of corresponding sequence must be held containing mRNA molecule 3 ';

S4. hold the fragment of corresponding sequence to be connected with the first linkers containing mRNA molecule 3 ', obtain cDNA library.

Wherein, the Oligo dT primer described in step S1 is any one Oligo dT primer of the present invention, and the single strand dna namely containing continuous print dT sequence, it contains the recognition site for cutting.

The present embodiment is by Oligo dT primer and relevant design, make mRNA molecules all in sample all obtain specific embodiment in the cDNA library built, and the label to be measured that in constructed cDNA library, each molecule carries (namely deriving from the specific sequence of each mRNA molecule) all derive from 3 ' end of mRNA molecule.

It should be noted that:

In the present embodiment, the Oligo dT primer in step S1 can include marker, and this marker includes but not limited to: vitamin H, antigen, antibody, acceptor, part, polyhistidine; By the title complex with the marker adaptation on Oligo dT primer, can further improve Oligo dT primer ease for use in use, improve conventional efficient.Such as, when this marker is vitamin H, can utilize and the Streptavidin of vitamin H specific binding or avidin, and realize the fast separating and purifying to RNA/DNA product in step S1, improve conventional efficient; Same, when this marker is antigen, antibody, acceptor or part, can utilize and the antibody of these marker adaptations, antigen, part or acceptor, separation and purification is fast completed to the product generated by Oligo dT primer.Certainly, these markers may cause certain influence to the extension of Oligo dT primer, but by the appropriate design to these markers position on Oligo dT primer, can be reduced to minimum by these impacts, even without impact; Generally, marker be arranged on Oligo dT primer 5 ' end its extension is all had no significant effect.Preferably, this marker is vitamin H or polyhistidine.Preferred, this marker is positioned at 5 ' end of Oligo dT primer.

Wherein, the recognition site for cutting includes but not limited to uridylate, Hypoxanthine deoxyriboside, phosphorothioate bond or IIs type restriction endonuclease recognition sequence, and described IIs type restriction endonuclease recognition sequence is at 5 ' end of Oligo dT primer.

All can there is specific cutting under the effect of corresponding material in the above-mentioned recognition site for cutting.Corresponding relation for the recognition site the cut material corresponding to this includes but not limited to:

U---USER enzyme or UDG enzyme;

P-S---the cutting agent containing Ag, Hg, Cu, Mn, Zn or Cd atom;

It should be noted that:

Available various metallic material cutting phosphorothioate bond.Described metal can be Ag, Hg, Cu, Mn, Zn or Cd.Preferably, this material is to provide Ag ⁺, Hg ⁺⁺, Cu ⁺⁺, Mn ⁺⁺, Zn ⁺or Cd ⁺water-soluble salt (also can adopt the salt of the ion that other state of oxidation is provided) of ion.Particularly preferably contain silver salt as Silver Nitrate (AgNO ₃) or other Ag is provided ⁺the salt of ion.The condition of cutting comprises such as 50mMAgNO ₃, about 22 ~ 37 DEG C, 10 minutes or longer time as 30 minutes.Preferably, pH is 4.0 ~ 10.0, more preferably 5.0 ~ 9.0, according to appointment 6.0 ~ 8.0, according to appointment 7.0.See Mag, M. etc., Nucleic Acids Res., 19 (7): 1437-1441,1991.

At above-mentioned four kinds of enumerating in the recognition site that cuts, the advantage of phosphorothioate ester key is, in use, cost is lower, and the speed completing cutting is faster, is simpler and more convenient to operate; But its pollution on the environment is comparatively large, and reacted treatment cost of waste liquor is high, and other three kinds of recognition sites for cutting, are all the cuttings completed by specific enzyme, there is not the problem of environmental pollution.

Further, this Oligo dT primer sequence is:

5’-(dT) _xU(dT) _y-3’；

Or 5 '-(RERS)-(dT) _z-3 ';

Or 5 '-(dT) _α(P-S) (dT) _β-3 ';

Or 5 '-(dT) _γ(I) (dT) _δ-3 ';

Wherein, x, y, z, α, β, γ, δ are natural number; RERS is the nucleotide sequence containing IIs type restriction endonuclease recognition sequence, and this nucleotide sequence can be DNA or RNA, is preferably DNA; P-S is phosphorothioate bond; I is Hypoxanthine deoxyriboside.

Wherein, to the size of x+y, z, alpha+beta, γ+δ all without particular restriction.Further, 9≤x+y≤44,9≤z≤44,9≤alpha+beta≤45,9≤γ+δ≤44.

In step sl, the source of mRNA is without particular determination.The separation and purification from total serum IgE of various prior art can be adopted to go out mRNA, conventional Oligo dT primer is such as adopted to catch mRNA from total serum IgE, this conventional Oligo dT primer can be fixed in fibre columns or with biotin labeling, so that the recovery purifying of the mRNA caught.

Preferably, before step S1, also step S1 ' is comprised: adopt Oligo dT primer separation and purification mRNA from total serum IgE.This Oligo dT primer is preferably Oligo dT primer of the present invention.

Preferred, this Oligo dT primer, with marker, then utilizes and carries out separation and purification with the title complex of this marker adaptation to the mRNA caught; Or this Oligo dT primer is fixed on medium, thus catches and purifying the mRNA molecule in total serum IgE at dielectric surface, and this medium is preferably fibre columns or gel column.

Preferred, this marker is at least one in vitamin H, antigen, antibody, acceptor, part, polyhistidine.

Such as, this Oligo dT primer has marker in 5 ' end band, and this marker is polyhistidine, and described step S1 ' is roughly as follows:

By the combination between polyhistidine and nickel ion (gel column with nickel ion), realize the separation and purification to the mRNA caught.

Or this Oligo dT primer is simultaneously with 2 kinds of markers, and a kind of is the polyhistidine held at this primer 5 ', and another kind is the vitamin H in the middle part of this primer sequence, and described step S1 ' is roughly as follows:

First, utilize the gel column with nickel ion to catch Oligo dT primer, recycling Oligo dT primer catches the mRNA molecule in total serum IgE;

Then, Oligo dT primer and the mRNA molecule of catching are eluted from gel column, and make their keep bonding state;

Finally, the purifying of magnetic bead realization to mRNA molecule with Streptavidin is recycled.

In the present embodiment, step S2 can adopt multiple existing method as required, includes but not limited to self-priming method, displacement synthesis method.Self-priming method is first with the mRNA chain in sodium hydroxide digestion RNA/DNA heteroduplex, 3 ' the end of the Article 1 cDNA chain obtained of dissociating is understood self and is formed a hairpin loop, and guide the polysaccharase depending on DNA to copy the second chain, link together between the double-strand now formed, and then utilize S1 nuclease junction (only this site is single-stranded structure) to be cut off the double-strand cDNA forming flat end construction.Displacement synthesis method realizes like this, mRNA chain in RNA/DNA heteroduplex first forms a lot of otch under the effect of RNA enzyme H, mRNA chain is just cut into drinks many small segments, and these small segments are the primer that the polysaccharase (as: e. coli dna polymerase I) depending on RNA provides synthesis second chain DNA.Depend on the cDNA fragment that the polysaccharase of RNA is a section complementation of templated synthesis with the first chain cDNA.These cDNA fragments and then connect into a chain under the effect of DNA ligase, thus obtain double-strand cDNA.But 3 ' end of the more difficult control of the reaction of self-priming method, especially Article 1 cDNA chain forms this reaction of hairpin loop and is difficult to control, and often needs repeatedly to grope experiment condition, and the mortality forming double-strand cDNA is higher.Preferably, step S2 adopts displacement synthesis method, and the easy control of reaction conditions of the method, success ratio is high.

In the present embodiment, the enzyme of IIs type restriction enzyme to double-strand cDNA in step S3 has cut multiple implementation, includes but not limited to following two kinds of modes; The first, can be by the Oligo dT primer in step S1, IIs type restriction endonuclease recognition sequence is introduced in double-strand cDNA, thus make directly can to carry out enzyme to double-strand cDNA with the IIs type restriction enzyme of correspondence in step S3 and cut; Second, also can be carry out IIs type digestion with restriction enzyme step in step s3 before, utilize double-strand cDNA is cut for the recognition site cut on Oligo dT primer, then be connected with cleaved products by the linkers containing IIs type restriction endonuclease recognition sequence, thus introduce IIs type restriction endonuclease recognition sequence, and then with corresponding IIs type restriction enzyme, enzyme is realized to the product after connecting and cut.Specifically elaborate carrying out a step in follow-up embodiment.

In the present embodiment, the first linkers in step S4 is at least containing the nucleic acid molecule of a protruding terminus, the ends match that the IIs type digestion with restriction enzyme in this protruding terminus and step S3 is formed.

This nucleic acid molecule can be single stranded nucleic acid molecule, namely the joint (as shown in Figure 3) with loop-stem structure, this single stranded nucleic acid molecule comprises the first complementary pairing district 1, complementary pairing district 3 of Jing Huan district 2, second and protruding terminus 4, first complementary pairing district 1 can with the second complementary pairing district 3 complementary pairing, and the complementary pairing district that their are formed comprises at least one restriction enzyme enzyme recognition site, the ends match that the IIs type digestion with restriction enzyme in the position of this protruding terminus 4 and sequence and step S3 is formed.

Such as, when the IIs type restriction enzyme in step S3 is Acu I, this protruding terminus of this first linkers is positioned at 3 ' end of single stranded nucleic acid molecule, and this protruding terminus contains 2 Nucleotide containing universal base N, and N is A or G or C or T or U or I; When the IIs type restriction enzyme in step S3 is EcoP15 I, this protruding terminus of this first linkers is positioned at 5 ' end of single stranded nucleic acid molecule, and this protruding terminus contains 2 Nucleotide containing universal base N, and N is A or G or C or T or U or I; When the IIs type restriction enzyme in step S3 is Mme I, this protruding terminus of this first linkers is positioned at 3 ' end of single stranded nucleic acid molecule, and this protruding terminus contains 2 Nucleotide containing universal base N, and N is A or G or C or T or U or I.

When the first linkers is the joint of band loop-stem structure, first linkers holds the fragment of corresponding sequence to connect to generate and connect product with containing mRNA molecule 3 ', utilize restriction enzyme to cut the loop-stem structure (deriving from the joint of band loop-stem structure) of connection product, and then form cDNA library.This restriction enzyme can restriction enzyme enzyme recognition site on specific identification first linkers.

This nucleic acid molecule can also be double chain acid molecule, and this double chain acid molecule at least comprises a protruding terminus, the ends match that the IIs type digestion with restriction enzyme in this protruding terminus and step S3 is formed.According to the structure of this double chain acid molecule, can be divided into two classes, be single protruding terminus joint, two protruding terminus joint respectively.

This single protruding terminus joint (as shown in Figure 4) one end is that (a) or bifurcation structure (Fig. 4 b), the other end is the protruding terminus of the ends match formed with the IIs type digestion with restriction enzyme in step S3 to Fig. 4 to flat end.Single protruding terminus joint wherein with y splice can prevent joint from connecting.In order to prevent one end be single protruding terminus joint of flat end from connecting, 3 ' OH on flat end can be modified (including but not limited to close hydroxyl with amino), maybe 5 ' phosphate group on flat end is removed.

This pair of protruding terminus joint (as shown in Figure 5) is containing two protruding terminuses, these two protruding terminuses can on a nucleotide chain (Fig. 5 a) or on different nucleotide chains (Fig. 5 b), the ends match that the IIs type digestion with restriction enzyme in one of them protruding terminus and step S3 is formed.When these two protruding terminuses are in different nucleic acid chains, they are not complementary each other, in case occur that when connecting joint is from connecting.

In the present embodiment, also step S4 ' can be comprised after step S4:

Utilize the primer pair cDNA library identical or complementary with the known array at cDNA library two ends to carry out pcr amplification, obtain the cDNA library after amplification.To meet unit molecule amplification step in s-generation high-flux sequence method to the requirement of the amount of sequencing library.The amplification of this unit molecule refers to the multiple library molecule in sequencing library, spatially isolate (but these library molecule still belong to same reaction system on the whole) with the form of denier (even unit molecule), and in respective space, realize amplification, to promote the signal of various molecule in follow-up sequencing reaction.

For step S3, according to the difference of Oligo dT design of primers, different specific embodiments can be had.

Wherein, when the recognition site for cutting is uridylate, phosphorothioate bond or Hypoxanthine deoxyriboside, by corresponding cutting agent cutting double-strand cDNA, obtain double-strand cutting fragment, then double-strand cutting fragment is made to be connected with linkers, thus introduce IIs type restriction endonuclease recognition sequence, and then obtain the fragment of holding corresponding sequence containing mRNA molecule 3 '.This cutting agent is used for second phosphodiester bond that specificity cutting uridylic, phosphorothioate bond or Hypoxanthine deoxyriboside 3 ' is held.Along with the difference of Oligo dT primer length (i.e. x+y+1, alpha+beta, γ+δ+1), the present invention has multiple embodiments.

As shown in Figure 6, in the second embodiment of the present invention, step S3 comprises the following steps:

S301. cutting agent cutting double-strand cDNA, obtains double-strand cutting fragment;

S302. double-strand cutting fragment is connected with the second linkers, obtains the first connection product;

S303. utilize an IIs type digestion with restriction enzyme first to connect product, the fragment of corresponding sequence must be held containing mRNA molecule 3 '.

The present embodiment utilizes U or P-S on Oligo dT primer or I, sticky end is formed by cutting agent cutting, then this sticky end is connected with the second linkers adapted, and utilize the IIs type restriction endonuclease recognition sequence on the second linkers, formed the sheet holding corresponding sequence containing mRNA molecule 3 ' by an IIs type digestion with restriction enzyme, the first linkers that this fragment adapts with this fragment is again connected to form cDNA library.The present embodiment protocol step is simple, lower to the requirement of the amount of mRNA in step S1, and by the IIs type restriction endonuclease recognition sequence on adjustment second linkers, can ensure that the tag length to be measured that in the cDNA library constructed by the present embodiment scheme, each molecule carries is more than or equal to 9, make often kind of cDNA molecule and each mRNA built in the original material mRNA mixture in storehouse ensure theoretic one_to_one corresponding.The sequence of 9 bases can have 4 ⁹=262144 kinds of different permutation and combination, therefore the label of each 9 base just can the characteristic sequence of representatively a kind of mRNA molecule in theory.Certainly, along with the increase of tag length to be measured, the corresponding relation between itself and mRNA molecule is more special.

When x+y≤17, the present embodiment scheme is especially applicable.

It should be noted that:

The second linkers in step S302 is the nucleic acid molecule at least containing a protruding terminus, and this nucleic acid molecule is also containing double stranded region, and an IIs type restriction endonuclease recognition sequence is contained in this double stranded region; This protruding terminus is made up of dT and/or U, and the few nucleotide a that this protruding terminus comprises is relevant to the base number b of U or P-S in Oligo dT primer or I distance 3 ' end, a=b+1.Such as, when 3 ' end 3 bases of U or P-S or I distance Oligo dT primer, the dT number that the protruding terminus of the second linkers comprises is 4; When U or P-S or I is in 3 ' end of Oligo dT primer, the dT number that the protruding terminus of the second linkers comprises is 1; When 3 ' end 10 bases of U or P-S or I distance Oligo dT primer, the dT number that the protruding terminus of the second linkers comprises is 11.

As shown in Figure 7, in the third embodiment of the present invention, step S3 comprises the following steps:

S311. cutting agent cutting double-strand cDNA, obtains double-strand cutting fragment;

S312. double-strand cutting fragment is connected with the second linkers, obtains the first connection product;

S313. utilize an IIs type digestion with restriction enzyme first to connect product, obtain not containing the endonuclease bamhi of the second linkers;

S314. the endonuclease bamhi not containing the second linkers is connected with the 3rd linkers, obtains the second connection product;

S315. utilize the 2nd IIs type digestion with restriction enzyme second to connect product, the fragment of corresponding sequence must be held containing mRNA molecule 3 '.

The present embodiment, by twice IIs type digestion with restriction enzyme, obtains the fragment of holding corresponding sequence containing mRNA molecule 3 '.The scheme of the present embodiment is compared with the second embodiment, the design requirements to Oligo dT primer, the second linkers can be reduced further, the range of choice of the IIs type restriction endonuclease recognition sequence in the second linkers is wider, and the scope that Oligo dT primer sequence length optional is selected is also wider; And then relaxing to reaction conditions in step S1, step S3, the especially requirement of temperature of reaction, makes correlated response better can be balanced between specificity and speed of response.Certainly, the present invention also with reference to the design of this programme, by more II s type digestion with restriction enzyme, can obtain the fragment of holding corresponding sequence containing mRNA molecule 3 '.Thus the design requirements further reduced Oligo dT primer, the second linkers.

It should be noted that:

The 3rd linkers in step S313 is the nucleic acid molecule at least containing a protruding terminus, and this nucleic acid molecule is also containing double stranded region, and the 2nd IIs type restriction endonuclease recognition sequence is contained in this double stranded region; The terminal matching that this protruding terminus can be formed with an IIs type digestion with restriction enzyme.

One IIs type restriction endonuclease recognition sequence and the 2nd IIs type restriction endonuclease recognition sequence may be the same or different.

In order to ensure the bonding strength of the poly A tail of Oligo dT primer and mRNA molecule, be beneficial to carrying out smoothly of follow-up reverse transcription reaction, x+y >=14, namely the Oligo dT primer sequence length that combines with the poly A tail of mRNA molecule is more than or equal to 15; Preferably between 15 to 30.Further, 3≤x≤9, make the second linkers structure adapted with Oligo dT design of primers in second, third embodiment more reasonable, bonding force when can take into account connection, the factor such as cost and stability of the second linkers.

Wherein, when the recognition site for cutting is IIs type digestion with restriction enzyme recognition sequence, when it is positioned at 5 ' end of Oligo dT primer, directly can utilizes this IIs type restriction endonuclease recognition sequence, obtaining the fragment that mRNA molecule 3 ' holds corresponding sequence.Along with the difference of Oligo dT primer length, the present invention has multiple embodiments.

In the fourth embodiment of the present invention, step S3 is specific as follows: IIs type digestion with restriction enzyme double-strand cDNA, and separation and purification goes out the endonuclease bamhi of answering sequence containing Oligo dT primer pair, must hold the fragment of corresponding sequence containing mRNA molecule 3 '.

The present embodiment scheme is compared with the scheme of second, third embodiment, step is simpler, conventional efficient is high, and decrease the nucleic acid purification step that may exist between each step, and there is the problem of organic efficiency in nucleic acid in purge process, therefore, the mRNA amount that this programme can be required in lower step S1 further; In addition, this programme introduces IIs type restriction endonuclease recognition sequence by Oligo dT primer, all containing IIs type restriction endonuclease recognition sequence on all cDNA molecules, eliminate connection reagent, such as: ligase enzyme, linkers etc., and there is not the problem of joint efficiency, therefore, the efficiency in this programme construction cDNA library is higher, and cost is lower.

It should be noted that:

Needed for the method in the construction cDNA library in background technology of the present invention initial mRNA amount be at least 200ng, be generally advisable with 500ng to 1 μ g; Through contriver checking, in second, third embodiment of the present invention, the amount of required initial mRNA and the amount needed for background technology of the present invention basically identical; In the fourth embodiment of the present invention, the amount of required initial mRNA is minimum is down to 50ng.

Again because of the minimizing of step, and the reduction of required initial mRNA amount, the present invention can effectively improve the success ratio of building storehouse, reduces because step is numerous and diverse and required initial mRNA measures step and causes building the possibility of the phenomenon appearance of storehouse failure.

When z≤17, the present embodiment scheme is especially applicable.

As shown in Figure 8, in the fifth embodiment of the present invention, step S3 comprises the following steps:

S321.IIs type digestion with restriction enzyme double-strand cDNA, separation and purification goes out the endonuclease bamhi without IIs type restriction endonuclease recognition sequence;

S322. the endonuclease bamhi without IIs type restriction endonuclease recognition sequence is connected with the 4th linkers, obtains the 3rd and connect product;

S323. utilize the 3rd IIs type digestion with restriction enzyme the 3rd to connect product, the fragment of corresponding sequence must be held containing mRNA molecule 3 '.

It should be noted that:

4th linkers is the nucleic acid molecule at least containing a protruding terminus, and this nucleic acid molecule is also containing double stranded region, and the 3rd IIs type restriction endonuclease recognition sequence is contained in this double stranded region; The terminal matching that this protruding terminus can be formed with the IIs type digestion with restriction enzyme in step S321.

IIs type restriction endonuclease recognition sequence in 3rd IIs type restriction endonuclease recognition sequence and step S321 may be the same or different.

The present embodiment, by twice IIs type digestion with restriction enzyme, obtains the fragment of holding corresponding sequence containing mRNA molecule 3 '.The scheme of the present embodiment can reduce the design requirements to Oligo dT primer, the 4th linkers further, the range of choice of the IIs type restriction endonuclease recognition sequence in the 4th linkers is wider, and the scope that Oligo dT primer sequence length optional is selected is also wider; And then relaxing to reaction conditions in step S1, step S3, the especially requirement of temperature of reaction, makes correlated response better can be balanced between specificity and speed of response.Certainly, the present invention also with reference to the design of this programme, by more IIs type digestion with restriction enzyme, can obtain the fragment of holding corresponding sequence containing mRNA molecule 3 '.Thus the design requirements further reduced Oligo dT primer, the 4th linkers.

In order to ensure the bonding strength of the poly A tail of Oligo dT primer and mRNA molecule, be beneficial to carrying out smoothly of follow-up reverse transcription reaction, z >=15, namely the Oligo dT primer sequence length that combines with the poly A tail of mRNA molecule is more than or equal to 15; Preferably between 15 to 30, make the second linkers structure adapted with Oligo dT design of primers in the 4th, the 5th embodiment more reasonable, bonding force when can take into account connection, the factor such as cost and stability of the second linkers.

In above-mentioned all schemes, Oligo dT primer all can include marker with the linkers used by building in the process of storehouse, and this marker includes but not limited to: vitamin H, antigen, antibody, acceptor, part, polyhistidine; Then in the process of construction cDNA library, by the specific binding of marker and respective substance (as: vitamin H and streptavidin, vitamin H and avidin, antigen and antibody, acceptor and part or the specific binding between polyhistidine and nickel ion), letter assay step, improve and build storehouse efficiency, improve the organic efficiency of intermediate product, reduce and build Kucheng originally.

Preferably, linkers includes marker, and this marker is vitamin H or polyhistidine.

In order to improve the determinacy that Oligo dT primer is put with mRNA binding site molecule in process of reverse-transcription, the present invention can be realized by the annealing temperature improved in step S1 in transcriptive process,reversed, concrete preferred annealing region can according to the length of the sequence of the poly A tail complementation of holding with mRNA molecule 3 ' in used Oligo dT primer, and other actual experiment condition (as: Oligo dT primer with labeling pattern, the ThermoScript II etc. that uses) is determined.

In addition, the present invention can also realize this purpose by arranging at 3 ' end of Oligo dT primer containing the Nucleotide M containing universal base, and M is A or G or C.3 ' end sequence of this Oligo dT primer can represent with following general formula:

In an exemplary embodiments of the present invention, 3 ' end of Oligo dT primer is two Nucleotide containing universal base, and be followed successively by M and N from 5 ' end to 3 ' end, M is A or G or C, N is A or G or C or T or U or I, and its primer sequence is:

5’-(dT) _xU(dT) _yMN-3’；

Or 5 '-(RERS)-(dT) _zmN-3 ';

Or 5 '-(dT) _α(P-S) (dT) _βmN-3 ';

Or 5 '-(dT) _γ(I) (dT) _δmN-3 ';

The present embodiment is by two Nucleotide containing universal base of Oligo dT primer least significant end, make Oligo dT primer can be accurately positioned in the initiation site of mRNA molecule poly A tail in process of reverse-transcription, thus elimination Oligo dT primer when synthesizing Article 1 cDNA chain may be combined caused disadvantageous effect with the optional position of poly A tail, ensure that label to be measured (namely deriving from the specific sequence of each mRNA molecule) determinacy of position in mRNA molecule that in constructed cDNA library, each molecule carries, be conducive to the interpretation of result of cDNA library after completing order-checking built, further increase the accuracy of carrying out mrna expression spectrum analysis according to the cDNA library built.

To be further elaborated to cDNA library construction process of the present invention by specific embodiment below.Step is as follows:

One, with the preparation of the magnetic bead of Oligo dT primer

1. draw about 10 μ L magnetic beads (invitrogen, myOne TM Streptavidin Cl) in 0.6mL EP pipe, add 1%Triton X-1000.6 μ L (without RNA enzyme), mixing.Use magnet adsorption magnetic bead, carefully remove supernatant, note not being drawn onto magnetic bead.Clean 2 times with 10 μ L TE (10mM Tris-HCl, 1mM EDTA, pH7.5), magnet adsorption magnetic bead, removes supernatant with pipettor.

2. add the Binding buffer suspension magnetic bead of 10 μ L, and then add 1 μ L concentration be the 5 ' end of 100 μMs with the Oligo dT primer of twin thing element mark, spiral shaken well immediately.

3. be put in rotary turnplate by the EP pipe after concussion evenly and slowly run, hatch about 1 hour for 18 to 25 DEG C, every 10 to 15min flicks tube wall or of short duration vortex mixing magnetic bead, and vitamin H primer is fully attached on magnetic bead.Attention: magnetic bead is not bound on tube wall when flicking tube wall mixing.

4. utilize magnet adsorption to live magnetic bead, carefully remove supernatant, then clean magnetic bead 3 times with TE.Again use 10 μ L TE suspension magnetic beads, obtain the magnetic bead (save backup in 4 DEG C, at least can preserve 3 months) with Oligo dT primer.

Should be noted that:

Binding Buffer be containing 20mM Tris-HCl, 1.0M LiCl, 2mM EDTA, pH be the aqueous solution of 7.5.

5 ' end in step 2 is divided into two classes with the Oligo dT primer of twin thing element mark, and a class is the Oligo dT primer of control group, and its sequence is respectively: 5 '-(dT) ₁₅-3 ' (SEQ ID NO:1), 5 '-(dT) ₁₉-3 ' (SEQ ID NO:2), 5 '-(dT) ₂₅-3 ' (SEQ ID NO:3), 5 '-(dT) ₁₃-3 ' (SEQ ID NO:4); Another kind of is the Oligo dT primer of experimental group, and its sequence is respectively 5 '-(dT) ₈u (dT) ₆-3 ' (SEQ ID NO:5), 5 '-(dT) ₁₃u (dT) ₃mN-3 ' (SEQ ID NO:6), 5 '-AGAGCAGCAGC-(dT) ₂₅-3 ' (SEQ ID NO:7), 5 '-GAGCTGAAGATA-(dT) ₁₁mN-3 ' (SEQ ID NO:8); Corresponding above-mentioned 2 class 8 kinds of Oligo dT primers, obtain respectively with the magnetic bead of corresponding Oligo dT primer.

Two, separating mRNA from total serum IgE

Extract the total serum IgE in yeast saccharomyces cerevisiae Haploid.fa (S288C), Diploid.fa (BY4347) respectively, extracting method can refer to various routine techniques, such as Trizol method.

The concentration of adjustment total serum IgE, to 0.5-1.0 μ g/ μ L, is got 20 μ L and is carried out following experiment:

Heat 2 minutes to destroy secondary structure at 65 DEG C, be then placed on ice; Meanwhile, take out the magnetic bead of 10 μ L with Oligo dT primer respectively, and be placed in the pipe without RNA enzyme respectively; Use magnet adsorption magnetic bead, adsorb after 30 seconds, siphon away supernatant liquor; Wash magnetic bead once with 10 μ L Binding Buffer, then siphon away supernatant liquor; Magnetic bead is suspended in 10 μ L Binding Buffer, adds 10 μ l total serum IgE, fully mix, and annealing 10 minutes on the gyroscope being placed in room temperature; Use magnet adsorption magnetic bead again, remove supernatant liquor; Wash magnetic bead with 20 μ l Washing Buffer B, then use magnet adsorption, remove supernatant liquor, in triplicate, must containing the magnetic bead of mRNA molecule; The difference of the magnetic bead of the corresponding band Oligo dT primer used, has the magnetic bead of 2 class 8 kinds containing mRNA molecule.

Washing Buffer B is 10mM Tris-HCl, 0.15M LiCl, and 1mM EDTA, pH are the aqueous solution of 7.5.

Three, the synthesis of cDNA

The synthesis of 1.cDNA first chain

Wash the magnetic bead containing mRNA molecule of step 2 gained with 10 μ l 1 × First Strand Buffer, then use magnet adsorption, remove supernatant liquor, repeat four times.

Before the supernatant liquor removing the 4th wash-out, configure cDNA first chain synthesis reaction liquid on ice, reaction solution system is as follows:

5×First Strand Buffer 3.0μl；

RNase inhibitor(Takala D2310A) 0.2μl；

DEPC Water 9.05μL；

0.1M DTT 1.5μL；

dNTP Mix(10mM each) 0.75μl；

Total Volume 14.5μl。

1 × First Strand Buffer washes four magnetic beads containing mRNA molecule, then suspend containing the magnetic bead of mRNA sample with the first chain synthesis reaction liquid of 14.5 μ L, flick tube wall or gently vortex vibration to mix solution (precipitate if any a small amount of magnetic bead, do not affect result).Then pipe is placed in 37 DEG C 2 minutes, to balance reagent.Add 0.5 μ L Superscript II reversed transcriptive enzyme (200U/ μ L, Invitrogen, Cat#18064-014), mix gently, 42 DEG C are reacted 1 hour, and every 10 to 15 minutes of period flicked pipe or the vibration of low speed vortex.Heat 15 minutes with stopped reaction at 70 DEG C, then lower the temperature on ice 2 minutes.Reaction product is stored in 4 DEG C.

The synthesis of 2.cDNA second chain

Following reaction solution is added in 15 μ L cDNA first chain synthesis reaction products:

DEPC Water 43.3μL；

10×NEBuffer2 6μL；

10mM ATP 7.5μL；

E.coli DNA Ligase(10U/μL，NEB，Cat#M0205S) 0.2μL；

E.coli DNA Polymerase(10U/μL，NEB，Cat#M0209S) 2.25μL；

E.coli RNase H(60U/μL，Takara，Cat#D2150) 0.75μL。

Wherein, E.coli DNA Ligase, E.coli DNA Polymerase, E.coli RNase H finally adds.Vortex vibration mixed reaction solution, each composition in reaction solution fully being mixed, then low-speed centrifugal, is that reaction solution concentrates at the bottom of pipe.16 DEG C are reacted 2 hours, every 10 to 15 minutes Eddy diffusions magnetic bead once.Between the reaction period, Wash Buffer C is placed on 75 DEG C of preheatings.After having reacted, reaction solution is placed on ice, adds 7.5 μ L 0.5M EDTA termination reactions.Then use magnet adsorption magnetic bead 1 to 2 minute, carefully remove supernatant liquor.The 100 μ L Wash Buffer C adding preheating make E.coli archaeal dna polymerase inactivation (this step is extremely important).Mixing, makes archaeal dna polymerase complete deactivation in 20 minutes by sample 75 DEG C of heating.Use magnet adsorption magnetic bead, remove supernatant.Three times are washed with 80 μ L Wash Buffer C.Add 100 μ L LoTE suspension magnetic beads.Bead suspension in pipe is all transferred in a new centrifuge tube, the 5 prime excision enzyme activity of e. coli dna polymerase can be avoided so as far as possible.Use magnet adsorption magnetic bead, remove supernatant, then wash magnetic bead once with 100 μ L LoTE, must containing the magnetic bead of double-strand cDNA product.In corresponding step 2, gained is containing the difference of the magnetic bead of mRNA, has the magnetic bead of 2 classes, 8 kinds of double-strand cDNA products.

Wash Buffer C: containing 10mM Tris-HCl, 50mM KCl, 1mM EDTA, 0.02%Triton X-100, pH are the aqueous solution of 7.5.

LoTE: be the aqueous solution of 7.5 containing 3.33mM Tris-HCl, 0.33mM EDTA, pH.

Four, construction cDNA library

1. the structure of first kind double-stranded cDNA library

With the first kind double-strand cDNA product of 4 of control group kinds of Oligo dT primer gained for starting material, react in the steps below respectively.

1) NlaIII enzyme is cut

By the magnetic bead containing double-strand cDNA product, with magnet adsorption, remove supernatant liquor; Then use 100 μ L1 × NEBuffer 4 to wash magnetic bead once, then in centrifuge tube, be sequentially added into following reagent:

LoTE 17.2μl；

100 × BSA 0.2 μ L (final concentration is 100 μ g/mL);

10×NEBuffer 4 2μL；

NlaIII(10U/μL，NEB，Cat#R0125S) 0.6μL；

37 DEG C of reactions 1 hour, within every 10 to 15 minutes, flick centrifuge tube; After having reacted, use magnet adsorption magnetic bead, remove supernatant liquor, then use 100 μ L 1 × T4 Ligation Buffer to wash five magnetic beads, obtain 3 '-cDNA.

2) jointing molecule 1

Following reagent is added in containing the centrifuge tube of magnetic bead:

Linkers 1 (20 μMs) 1 μ L;

LoTE 14μL；

10×T4ligation buffer 2μL；

10×ATP(10mM) 1μL；

Then 50 DEG C of heating two minutes, cool 15 minutes in room temperature, then add 2 μ L T4 DNA ligases (400U/ μ L, NEB, Cat#M0202L), room temperature reaction 2 hours; Then use magnet adsorption magnetic bead, remove supernatant liquor, then use 100 μ L 1 × NEBuffer4 to wash three magnetic beads, the cDNA magnetic bead of linkers 1 must be contained.

Linkers 1:

5’-TAGCGTGTCCGACATG-3’(SEQ ID NO：9)

3’-TATCGCACAGGCT-5’(SEQ ID NO：10)。

3) Mme I enzyme is cut

With the cDNA magnetic bead containing linkers 1 in magnet adsorption centrifuge tube, carefully remove supernatant liquor, and be placed on ice, in centrifuge tube, add following reagent:

LoTE 17.3μL；

10×NEBuffer 4 2μL；

SAM 0.3 μ L (final concentration 50 μMs);

By centrifuge tube 65 DEG C of preheatings 2 minutes, add 0.4 μ L Mme I (2U/ μ L, NEB, Cat#R0637S) and flick mixing.65 DEG C of reactions 1 hour, and frequently mix; After reaction, with magnet adsorption magnetic bead 1 ~ 2 minute.Do not abandon supernatant liquor! Carefully in centrifuge tube new for supernatant liquor dislocation one.With LoTE, liquid volume is mended to 150 μ L.Extract the digestion products containing linkers 1, and the digestion products containing linkers 1 is dissolved in 14 μ L LoTE.

4) jointing molecule 2

Following reagent is mixed in centrifuge tube:

Linkers 2 (20 μMs) 1 μ L;

Step 3) gained contains the digestion products 14 μ L of linkers 1;

10×T4 ligation buffer 2μL；

10×ATP(10mM) 1μL；

50 DEG C of heating 2 minutes, cold 15 minutes of room temperature; To add after 2 μ L T4 DNA ligases room temperature reaction 2 hours, obtain cDNA library.

Linkers 2:

5’-CGCCTCCCTGCAGTCCAG-3’(SEQ ID NO：11)

3’-NNGCGGAGGGACGTCAGGTC-5’(SEQ ID NO：12)。

2. the structure of Equations of The Second Kind double-stranded cDNA library

1)5’-(dT) ₈U(dT) ₆-3’(SEQ ID NO：5)

With Oligo dT primer: SEQ ID NO:5 prepares the double-strand cDNA of gained for starting material, reacts in the steps below.

A.USER enzyme enzyme is cut

By the magnetic bead containing double-strand cDNA product, with magnet adsorption, remove supernatant liquor; Then use 100 μ L LoTE suspension magnetic beads, then in centrifuge tube, be sequentially added into following reagent:

USER enzyme (1U/ μ L, NEB, Cat#M5505S) 10 μ L;

10×USER Buffer 20μL；

ddH ₂O up to 200μL。

Hatch 1h for 37 DEG C, reacted rear magnet adsorption magnetic bead, supernatant is transferred in 600 new μ L EP pipes, then purify with purification kit and reclaim double-strand USER enzyme digestion products.

B. jointing molecule 3

Ligation system is as follows:

Double-strand USER enzyme digestion products 2 μ g;

Linkers 3 (10 μMs) 1 μ L;

T4DNA ligase enzyme 2 μ L;

10 × T4 ligase enzyme damping fluid 2 μ L;

Add ddH ₂o to 100 μ L.

Linkers 3:

5’-CTTCAGCTATGCCGAT-3’(SEQ ID NO：13)

3 '-TTTTTTTGAAGTCGATACGGCTAAA-5 ' (SEQ ID NO:14), 5 ' end wherein on SEQ IN NO:14 chain contains biotin labeling.

Condition of contact, hatches 2h for 14 DEG C.After having reacted, add 10 μ L be with marked by streptavidin magnetic bead (invitrogen, myOne TM Streptavidin Cl) absorption connection product, hatch about 1 hour for 18 to 25 DEG C, every 10 to 15min flicks tube wall or of short duration vortex mixing magnetic bead, and the connection product of band vitamin H is fully attached on magnetic bead.After having reacted, use magnet adsorption magnetic bead, remove supernatant liquor; Rinse magnetic bead with 100 μ L 1 × NEBuffer 4, and then use magnet absorption magnetic bead, repeat 3 times, remove supernatant, obtain connection product.

C.Acu I enzyme is cut

The connection product of above-mentioned steps B gained is carried out enzyme and cuts being added in following reaction system:

10×NEBuffer 4 8μL；

Acu I(5U/μL，NEB，Cat#R0641S) 2μL；

SAM (3.2mM) 1.25 μ L (final concentration 50 μMs);

ddH ₂O up to 80μL。

Enzyme tangent condition: hatch 1h for 37 DEG C.After having reacted, use magnet adsorption magnetic bead, supernatant liquor is transferred in 200 new μ L EP pipes, reclaims to obtain double-strand Acu I digestion products.

D. jointing molecule 4

Ligation embodies as follows:

Double-strand Acu I digestion products 1 μ g;

Linkers 4 (10 μMs) 1 μ L;

T4DNA ligase enzyme 1 μ L;

10 × T4 ligase enzyme damping fluid 2 μ L;

Add ddH ₂o to 100 μ L.

Linkers 4:

5’-GGGCTTCTGAAGCG-3’(SEQ ID NO：15)

3 '-TAGCCCGAAGACTTCGCNN-5 ' (SEQ ID NO:16), wherein SEP ID NO:16 chain contains biotin labeling.

Condition of contact, hatches 2h for 14 DEG C.After having reacted, add 10 μ L be with marked by streptavidin magnetic bead (invitrogen, myOne TM Streptavidin C1) absorption connection product, hatch about 1 hour for 18 to 25 DEG C, every 10 to 15min flicks tube wall or of short duration vortex mixing magnetic bead, and the connection product of band vitamin H is fully attached on magnetic bead.After having reacted, use magnet adsorption magnetic bead, remove supernatant liquor; Rinse magnetic bead with 100 μ L 1 × NEBuffer 4, and then use magnet absorption magnetic bead, repeat 3 times, remove supernatant, obtain connection product.

E.Acu I enzyme is cut

The connection product of above-mentioned steps D gained is added in following reaction system and carries out enzyme and cut:

10×NEBuffer 4 8μL；

Acu I(5U/μL，NEB，Cat#R0641S) 2μL；

SAM (3.2mM) 1.25 μ L (final concentration 50 μMs);

ddH ₂O up to 80μL。

Enzyme tangent condition: hatch 1h for 37 DEG C.After having reacted, use magnet adsorption magnetic bead, remove supernatant liquor; Then use 100 μ L 1 × T4 ligase enzyme wash buffer magnetic beads, and then use magnet absorption magnetic bead, repeat 3 times, remove supernatant, obtain Acu I digestion products.

F. jointing molecule 5

Added in following reaction system by step e gained Acu I digestion products and carry out ligation, linked system is as follows:

Linkers 5 (10 μMs) 1 μ L;

10T4DNA ligase enzyme 2 μ L;

10 × T4 ligase enzyme damping fluid 2 μ L;

Add ddH ₂o to 100 μ L.

Condition of contact, hatches 2h for 14 DEG C.After having reacted, use magnet adsorption magnetic bead, remove supernatant liquor; With 100 μ L TE wash buffer magnetic beads, then use magnet adsorption magnetic bead, remove supernatant liquor, in triplicate, obtain cDNA library.

Linkers 5:

5’-TGACGCGCACGCGCTCG-3’(SEQ ID NO：17)

3’-NNACTGCGCATGCGCGAGC-5’(SEQ ID NO：18)。Wherein, 3 ' the end G of SEQ ID NO:17 is two deoxidation bases.

2)5’-(dT) ₁₃U(dT) ₃MN-3’(SEQ ID NO：6)

With Oligo dT primer: SEQ ID NO:6 prepares the double-strand cDNA of gained for starting material, with reference to above-mentioned 1) inner step reacts, and only need adjust accordingly according to SEQ ID NO:6 butt junction molecule 3, obtain cDNA library.

3)5’-AGAGCAGCAGC-(dT) ₂₅-3’(SEQ ID NO：7)

With Oligo dT primer: SEQ ID NO:7 prepares the double-strand cDNA of gained for starting material, reacts in the steps below.

A.EcoP15 I enzyme cuts double-strand cDNA

Utilize EcoP15 I (10U/ μ l, NEB Cat#R0646S) enzyme to cut the double-strand cDNA product of step 3 gained, reaction solution system is as follows: 100mMNaCl, 50mM Tris-HCl, 10mM MgCl2,1mMdithiothreitol, 100 μ g/mL BSA, 1mM ATP, pH7.9.Hatch 1h for 37 DEG C.After having reacted, use magnet adsorption magnetic bead, supernatant is transferred in 200 μ L PCR pipe, hatches 20 minutes for 65 DEG C, deactivation EcoP15 I, obtain EcoP15 I digestion products.

B. jointing molecule 6

Linked system is as follows:

EcoP15I digestion products 2 μ g;

Linkers 6 (10 μMs) 1 μ L;

T4DNA ligase enzyme 1 μ L;

10 × T4 ligase enzyme damping fluid 2 μ L;

Add ddH ₂o to 100 μ L.

Linkers 6:

5’-ATCGTAGTGCCTGAAG-3’(SEQ ID NO：19)

3 '-TAGCATCACGGACTTCNN-5 ' (SEQ ID NO:20), wherein, the 3 ' end of SEQ ID NO:20 is by hydroxylation, and the 5 ' end of SEQ ID NO:19 contains biotin labeling.

Condition of contact, hatches 2h for 14 DEG C.After having reacted, add 10 μ L be with marked by streptavidin magnetic bead (invitrogen, myOneTM Streptavidin C1) absorption connection product, hatch about 1 hour for 18 to 25 DEG C, every 10 to 15min flicks tube wall or of short duration vortex mixing magnetic bead, and the connection product of band vitamin H is fully attached on magnetic bead.After having reacted, use magnet adsorption magnetic bead, remove supernatant liquor; Rinse magnetic bead with 100 μ L 1 × NEBuffer 4, and then use magnet absorption magnetic bead, repeat 3 times, remove supernatant, obtain connection product.

C.Acu I enzyme is cut

10×NEBuffer 4 8μL；

Acu I(5U/μL，NEB，Cat#R0641S) 2μL；

SAM (3.2mM) 1.25 μ L (final concentration 50 μMs);

ddH ₂O up to 80μL。

Enzyme tangent condition: hatch 1h for 37 DEG C.After having reacted, use magnet adsorption magnetic bead, remove supernatant liquor; Then use 80 μ L 1 × T4 ligase enzyme wash buffer magnetic beads, then use magnet adsorption magnetic bead, remove supernatant liquor, in triplicate, obtain Acu I digestion products.

D. jointing molecule 7

Linkers 7 (10 μMs) 1 μ L;

10T4DNA ligase enzyme 2 μ L;

10 × T4 ligase enzyme damping fluid 2 μ L;

Add ddH ₂o to 100 μ L.

Linkers 7:

5’-CCTCCCTGCAGTCCTCTATGGGCT-3’(SEQ ID NO：21)

3’-NNGGAGGGACGTCAGGAGATACCCGA-5’(SEQ ID NO：22)。Wherein, the A of the 5 ' end of SEQ ID NO:22 is not with phosphate group.

The amplification in E.cDNA library

Utilize primers F (SEQ ID NO:67) and primer R (SEQ ID NO:68) to carry out pcr amplification to step 4 gained cDNA library, reaction system is as follows:

CDNA library 100 μ L;

Primers F (10 μMs) 100 μ L;

Primer R (10 μMs) 100 μ L;

10×Ex Taq Buffer 100μL；

Ex Taq(5U/μL) 8μL；

DNTP (each 2.5mM) 80 μ L;

ddH ₂O up to 1000μL。

PCR reaction conditions is as follows:

95℃3min；

94 DEG C of 20s, 58 DEG C of 20s, 72 DEG C of 20s; Repeat 15 circulations;

72℃7min。

Utilize PCR cleaning agents box, amplified production cleans, and removes the primer and dNTP that do not increase, reclaims the product after amplification, obtain the cDNA library after amplification.

4)5’-GAGCTGAAGATA-(dT) ₁₁MN-3’(SEQ ID NO：8)

With Oligo dT primer: SEQ ID NO:8 prepares the double-strand cDNA of gained for starting material, with reference to above-mentioned 3) in step react, and to adjust accordingly according to SEQ ID NO:8 butt junction molecule 6, obtain cDNA library.

Five, the order-checking of cDNA library

Utilize the cDNA library of s-generation high-throughput techniques to step 4 gained to check order, the technology of mensuration includes but not limited to: the synthesis sequencing based on polysaccharase, the connection sequencing based on ligase enzyme.

Synthesis sequencing based on polysaccharase is based on being with the Nucleotide can removing mark to carry out.In each building-up reactions, each template strand can only extend once at the most, and the roughly flow process based on the synthesis sequencing of polysaccharase is as follows:

A. sequencing primer is combined on the total known array of unit molecule amplified production (this unit molecule amplified production is fixed in primer-solid-phase carrier composite) by complementary pairing, under the effect of archaeal dna polymerase, the Nucleotide can removing mark with band carries out single-basic extension building-up reactions, collect the marking signal that this time adds Nucleotide, the base sequence information with the next bit of the unit molecule amplified production (being fixed in primer-solid-phase carrier composite) of sequencing primer 3 ' least significant end base complementrity can be obtained.

B. excision can remove mark, then under the effect of archaeal dna polymerase, the Nucleotide can removing mark with band proceeds single-basic extension building-up reactions, collect the marking signal adding Nucleotide, the base sequence information of lower two with the unit molecule amplified production of sequencing primer 3 ' terminal bases complementation can be obtained.

Repeat b step, till can not proceeding building-up reactions, thus obtain the full sequence information of unit molecule amplified production.

Connection sequencing based on ligase enzyme is carried out based on the fluorescently-labeled oligonucleotide probe of band.Wherein a kind of connection sequencing of ligase enzyme is based on that specific position carries out with fluorescently-labeled oligonucleotide probe, this oligonucleotide probe is with n base, a bit strip from its 5 ' terminal number has fluorescent mark, wherein different base pairs should specific fluorescent mark, because 3 ' end of this oligonucleotide probe has carried out specific modification, can not directly be interconnected between oligonucleotide probe, each ligation, each unit molecule amplified production can only connect an oligonucleotide probe.The roughly flow process of this connection sequencing is as follows:

A. sequencing primer to be combined on the total known array of unit molecule amplified production on (this unit molecule amplified production is fixed in primer-solid-phase carrier composite) by complementary pairing, utilize above-mentioned oligonucleotide probe, under the effect of ligase enzyme, nucleic acid probe is connected with above-mentioned oligonucleotide chain, then gather fluorescent signal, a bit base sequence information after 3 ' end of the known array had with unit molecule amplified production can be obtained.

B. the fluorescent mark on oligonucleotide is excised, under the effect of ligase enzyme, with above-mentioned oligonucleotide probe for raw material, proceed ligation, then fluorescent signal is gathered, thus the base sequence information of 2a position after obtaining 3 ' end of the known array that unit molecule amplified production has.

Repeat step B, till can not proceeding ligation, thus the base sequence information of a, 2a, 3a, 4a...... position after obtaining 3 ' end of the known array that unit molecule amplified production has.

Then sequencing primer and the oligonucleotide probe that connects thereof are eluted from sex change unit molecule amplified production, the primer using the few base of compared with sequencing primer before 3 ' end instead repeats above-mentioned reaction, thus the base sequence information of a-1,2a-1,3a-1,4a-1...... position after obtaining 3 ' end of the known array that unit molecule amplified production has.Repeat this step, finally obtain a-(a-1), 2a-(a-1), 3a-(a-1), 4a-(a-1) after 3 ' end of the known array that unit molecule amplified production has ... the base sequence information of position, thus the full sequence information obtaining single-stranded amplification product.

The connection sequencing of another kind of ligase enzyme is equally also carried out based on fluorescently-labeled oligonucleotide probe, this oligonucleotide probe is with n base, be divided into h (h≤n) group, the Different Alkali basic sequence of the corresponding same specific position of different fluorescent marks of same group of oligonucleotide probe, difference between different group is: the specific position that different fluorescent mark is corresponding is different, because 3 ' end of this oligonucleotide probe has carried out specific modification, can not directly be interconnected between oligonucleotide probe, each ligation, each unit molecule amplified production can only connect an oligonucleotide probe.The roughly flow process of this connection sequencing is as follows:

A. sequencing primer to be combined on the total known array of unit molecule amplified production on (this unit molecule amplified production is fixed in primer-solid-phase carrier composite) by complementary pairing, (base positions that fluorescent mark is corresponding is x to utilize in above-mentioned oligonucleotide probe one group, x≤h), under the effect of ligase enzyme, nucleic acid probe is connected with above-mentioned oligonucleotide chain, then fluorescent signal is gathered, xth bit base sequence information after 3 ' end of the known array had with single-stranded amplification product can be obtained, sequencing primer and the oligonucleotide probe that connects thereof are eluted from sex change unit molecule amplified production.

B. then again sequencing primer is combined on unit molecule amplified production, (base positions that fluorescent mark is corresponding is y to use the oligonucleotide probe group different from a step instead, y≤h), under the effect of ligase enzyme, nucleic acid probe is connected with above-mentioned oligonucleotide chain, then fluorescent signal is gathered, y bit base sequence information after 3 ' end of the known array had with single-stranded amplification product can be obtained, sequencing primer and the oligonucleotide probe that connects thereof are eluted from sex change unit molecule amplified production.

C. repeating step b, until h group oligonucleotide probe carried out a ligation respectively, thus after obtaining 3 ' end of the known array that unit molecule amplified production has the 1st, the base sequence information of 2......, h position.

The primer using 3 ' end compared with sequencing primer before many one or each universal base instead reacts by above-mentioned principle, and reading of 3 ' terminal base sequence of the known array that the unit molecule amplified production that can extend acquisition has is long.

This order-checking ratio juris of the connection based on ligase enzyme and specific embodiments can with reference to CN200710170507.1.The PStar II high-flux sequence instrument of preferred employing Shenzhen HYK Gene Technology Co., Ltd. exploitation carries out high-flux sequence, and analyzes sequence, and then obtains sequencing result.

Six, experimental result

1. control group experimental result

Search through pertinent literature, the genes involved of discovery yeast saccharomyces cerevisiae Haploid.fa (S288C), Diploid.fa (BY4347) has 5720.

Table 1 control group detected result

Table 2 experimental group detected result

Data in table 1, table 2 are all by obtaining with the comparison with reference to gene.

The gene detected refers to and detects through high-flux sequence the gene obtained.

Enliven the cDNA library that gene refers to certain sample corresponding, the gene of the number of times be detected in a high-flux sequence process more than 300 times.

Differential gene refers to when Oligo dT primer, cDN6A library constructing method are identical, the more another kind of obviously high gene of the expression of certain gene in above-mentioned a certain yeast saccharomyces cerevisiae (its screening principle see under).

Screening principle: adopt remarkable test of hypothesis, null hypothesis: gene A expresses in S288C and BY4347 does not have significant difference, alternative hypothesis: geneA expresses in S288C and BY4347 exists significant difference; The expression of relatively a whole set of reference gene, the expression of gene A can be similar to thinks obedience Poisson's distribution, carries out the derivation of equation on this basis and draws following Pvalue formula:

p (y | x) = {(\frac{N_{2}}{N_{1}})}^{y} \frac{(x + y)!}{x! y! {(1 + \frac{N_{2}}{N_{1}})}^{(x + y + 1)}} .

This formula is for calculating the similarity degree of gene A expression amount in S288C and BY4347; Parameter declaration: N1: unique comparison is to the reads number on S288C sample; N2: unique comparison is to the reads number on BY4347 sample; X: in S288C, unique comparison is to the reads number on gene A; Y: in BY4347, unique comparison is to the reads number on gene A.

The Pvalue calculated corrects and differential expression multiple (log through FDR (False discovery ratio) again ₂(Ratio) screening), just obtains significant difference expressing gene, corrects and screens formula and distinguish as follows:

FDR = \frac{N}{n} Pvalue; Ratio = \frac{RPKM (2)}{RPKM (1)};

Screening conditions are as follows: FDR≤0.001 AND|log ₂ratio|>=1;

Only have the gene meeting above-mentioned screening conditions just can be considered to differential gene.

From table 1, table 2, along with the increase of the poly A tail complementary region length of holding with mRNA molecule 3 ', the gene number detected also increases; When identical with the polyA tail complementary region length that mRNA molecule 3 ' is held, all comparatively control group is high for the gene number that experimental group detects, illustrate that the cDNA library prepared based on Ologo dT primer of the present invention can the more comprehensive kind of mRNA in response sample, will the part mRNA molecule in sample be caused because of the grappling enzyme built in the process of storehouse cannot to be embodied in the cDNA library of gained.

Should be noted that:

In above-described embodiment, as long as relate to the step of RNA, the reagent that this step uses and material all require without RNA enzyme, namely through the process of RNA enzyme-deactivating.

The present embodiment is only a specific embodiment of the present invention, acts on without any restriction the present invention.Oligo dT primer in the present embodiment can be selected as required, includes but not limited to: any one in SEQ ID NO:23 to SEQ ID NO:66.When the recognition site for cutting is I or P-S, the construction process carrying out cDNA library based on this Oligo dT primer can carry out with reference to above-described embodiment, the structure of the cDNA library that the Oligo dT primer being especially U based on the above-mentioned recognition site for cutting carries out.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

Claims

1. an Oligo dT primer, is characterized in that, described Oligo dT primer is the single strand dna containing continuous print dT sequence, and it contains the recognition site for cutting; The described recognition site for cutting is II s type restriction endonuclease recognition sequence, and described II s type restriction endonuclease recognition sequence is at 5 ' end of Oligo dT primer; Described Oligo dT primer sequence is: 5 '-(RERS)-(dT) _z-3 '; Wherein, z is natural number, 10≤z≤30, and RERS is the nucleotide sequence containing II s type restriction endonuclease recognition sequence.

2. Oligo dT primer according to claim 1, is characterized in that, 3 ' end of described Oligo dT primer is the Nucleotide M containing universal base, and M is A or G or C.

3. Oligo dT primer according to claim 2, is characterized in that, 3 ' end of described Oligo dT primer is two Nucleotide containing universal base, and be followed successively by M and N from 5 ' end to 3 ' end, N is A or G or C or T or U or I; Described Oligo dT primer sequence is: 5 '-(RERS)-(dT) _zmN-3 '.

4. Oligo dT primer according to any one of claim 1 to 3, is characterized in that, described Oligo dT primer contains marker, and described marker adopts at least one in vitamin H, antigen, antibody, acceptor, part, polyhistidine.

5. a cDNA library construction process, is characterized in that, comprises the following steps:

A. utilize Oligo dT primed reverse transcription mRNA, form Article 1 cDNA chain, obtain RNA/DNA hybrid molecule; This Oligo dT primer is the single strand dna containing continuous print dT sequence, and it contains the recognition site for cutting; The described recognition site for cutting is II s type restriction endonuclease recognition sequence, and described II s type restriction endonuclease recognition sequence is at 5 ' end of Oligo dT primer; Described Oligo dT primer sequence is: 5 '-(RERS)-(dT) _z-3 '; Wherein, z is natural number, 10≤z≤30, and RERS is the nucleotide sequence containing II s type restriction endonuclease recognition sequence;

C. utilize II s type digestion with restriction enzyme double-strand cDNA, the fragment of corresponding sequence must be held containing mRNA molecule 3 ';

6. cDNA library construction process according to claim 5, it is characterized in that, described step C is specially: II s type digestion with restriction enzyme double-strand cDNA, and separation and purification goes out the digestion products of answering sequence containing Oligo dT primer pair, must hold the fragment of corresponding sequence containing mRNA molecule 3 '.

7. cDNA library construction process according to claim 6, is characterized in that, 10≤z≤17.

8. cDNA library construction process according to claim 5, is characterized in that, described step C comprises the following steps:

C21. II s type digestion with restriction enzyme double-strand cDNA, separation and purification goes out the endonuclease bamhi without II s type restriction endonuclease recognition sequence;

C22. the endonuclease bamhi without II s type restriction endonuclease recognition sequence is connected with the 4th linkers, obtains the 3rd and connect product;

C23. utilize the 3 II s type digestion with restriction enzyme the 3rd to connect product, the fragment of corresponding sequence must be held containing mRNA molecule 3 '.

9. cDNA library construction process according to claim 8, is characterized in that, 15≤z≤30.

10. cDNA library construction process according to claim 6, is characterized in that, before steps A, also comprise steps A ': utilize Oligo dT primer separation and purification mRNA from total serum IgE.

11. cDNA library construction processs according to claim 5,6,8 or 10, is characterized in that, 3 ' end of described Oligo dT primer is the Nucleotide M containing universal base, and M is A or G or C.

12. cDNA library construction processs according to claim 11, is characterized in that, 3 ' end of described Oligo dT primer is two Nucleotide containing universal base, and be followed successively by M and N from 5 ' end to 3 ' end, N is A or G or C or T or U or I; Described Oligo dT primer sequence is: 5 '-(RERS)-(dT) _zmN-3 '.

13. cDNA library construction processs according to any one of claim 5 to 10,12, it is characterized in that, described Oligo dT primer is at 5 ' end containing marker, and this marker adopts at least one in vitamin H, antigen, antibody, acceptor, part, polyhistidine; And/or described linkers contains marker, this marker adopts at least one in vitamin H, antigen, antibody, acceptor, part, polyhistidine.