CN1130466C - cDNA reducing hybrid difference display method on cDMA library base - Google Patents

Abstract

A cDNA reducing hybridization difference display method on the base of a cDMA library base is a gene clone analysis method of molecular biology, which belongs to the technical field of molecular biology. Enzyme cutting sites of restriction enzymes of a cDNA library carrier is used, and enzyme cutting selection represents two prepared library amplicons for bidirectional reducing hybridization of a first round between the two library amplicons and bidirectional reducing hybridization of a second round between respective difference products of first round; the difference products of two rounds are marked with isotope, and then the mRNA difference display technique is guided for difference analysis. The method provides new application prospects for the cDNA library and provides an effective means for cloning genes with low abundance and weak difference.

Description

The cDNA reducing hybrid difference display method on cDMA library base

CDNA reducing hybrid difference display method on cDMA library base (SHDD) is a kind of molecular biology gene clone analytical procedure, belongs to biology field.

Existing gene clone method is a lot, and according to the literature method in common mainly contains two kinds, and a kind of is the representative difference analysis method (cDNA RDA) of cDNA, and a kind of is mRNA difference display method (mRNA DD).

(1) the representative difference analysis method (cDNA RDA) of cDNA

Genome subtractive hybridization method is the working foundation that cDNA RDA sets up, so-called genome subtractive hybridization is exactly to detect the specific sequence that DNA had in order to isolate from two kinds of relevant dna samples (detect DNA and drive DNA), a certain amount of detection DNA (TesterDNA) and excessive driving DNA (Driver DNA) are hybridized, make and detect the assorted and property renaturation of most of sequence generation identical among the DNA with driving DNA, self renaturation takes place in distinctive dna fragmentation among the DNA and detect, thereby is separated.CDNA RDA method has been introduced pcr amplification on classical genome subtractive hybridization method basis be main kinetics enrichment process, be equal to information, enrichment different information by subduing between two samples, with its higher specificity characteristics the clone, when identifying different developmental phases, different cycles before and after phase, medicine, cytokine or the pathogen-inducible research fields such as gene of histocyte differential expression be widely used.But there is following some deficiency in this method: (1) classical cDNA RDA method adopts ethidium bromide to show band, usually need just can obtain the evident difference band through the three-wheel subtractive hybridization, higher subtractive hybridization ratio and every many wheels pcr amplification of taking turns after the hybridization cause driving DNA, takes turns difference product (DP1), two and takes turns the segment group distribution trend of difference product (DP2) and successively decrease successively, two take turns and the three-wheel subtractive hybridization in just existed and drive DNA and and take turns and the two unbalanced subtractive hybridization problems of taking turns between the difference product segment group.Driving DNA and and take turns the overlap of taking turns difference product with two, also is that the long partially zone of difference product segment group suffers the extracting of higher subtractive hybridization ratio and quantity of information takes place loses; Only there is corresponding sequence seldom in the short partially zone of difference product segment group in driving DNA, occur as false-positive message thereby the extraction rate was acquired that suffers is more weak.This is found to be, and the quantity of information that exists in the difference product that utilizes classical cDNA RDA method to obtain is on the low side to provide a kind of proper explanations with the false positive problem.(2) adopt classical cDNA RDA method can only detect two differences between the paired sample DNA, and can only obtain to detect the mRNA different information that expression level increases among the DNA, can not obtain the information of expression level downward modulation simultaneously.(2) mRNA difference display method (mRNA DD)

The utilization of mRNA DD method is that random primer and anchor primer carry out polymerase chain reaction (PCR) amplification (PCR) to the mRNA ensemble of two kinds of different sourcess, the capable denaturing polyacrylamide gel electrophoresis of amplified production, and the differential expression fragment is selected in contrast.Adopt mRNA DD method can more a plurality of simultaneously samples between the difference of genetic expression, and can detect the gene that expression " rise " reaches " downward modulation " simultaneously; The amplification of aim sequence is depended on the various combination of random primer and anchor primer, rather than depend on the abundance of aim sequence, and isotopic application also helps the information that examination obtains low abundance difference expression.Since 1992 set up the foundation of mRNA DD method, people had utilized the method for this technology and further improvement on this basis to obtain the tumor-related gene of considerable number.Through the practice of several years, people have also found the some shortcomings part of this technology, mainly comprise:

(1) false positive rate height: many studies show that, the difference bar that the mRNA differential display technique presented have more than 70% and can not be confirmed by methods such as Northern blot hybridization and reverse northern blot hybridizations.Analyze reason, main relevant with following factor: the random primer specificity is low; The mRNA sample is easily degraded or is had the pollution of DNA; Owing to use 3 ' end anchor primer, the most of difference bands that obtain by this method only contain 3 ' end non-translational region (3 '-UTR) one section short-movie segment information, and this regional sequence is conservative relatively, makes that carrying out the Northern blot hybridization with 3 ' end cDNA segment as probe the false positive problem may occur.

(2) low to low abundance mRNA recall rate: in the eukaryotic cell, a considerable amount of mRNA belong to low abundance information, and 86% gene only accounts for 25% of mRNA total amount ^[10], because the amplification of the competitiveness of PCR, mRNA DD technology has tangible proneness to clone's abundant mRNAs ^[11]

(3) huge workload problem: since gel difference show before this method the information that is equal in the matched sample is not subdued, the different cDNA sequence of 1-10 kind is contained in the single band position that makes electrophoresis result show, average 4 kinds, further these information of isolation identification have become the step that mRNA DD is the most consuming time, consume power.And cause repetition differential fragment problem because different random primers is attached to the specific region of same aim sequence or different anchor primers and the combination of the same random primer same aim sequence that increases, further increased workload.

The objective of the invention is to carry out the gene expression difference analysis between the library for making full use of the abundant cDNA information that the cDNA library is comprised; For cloning the expressing gene information of low abundance and weakly heterogeneous, low and the false positive problem of the quantity of information of avoiding the unbalanced subtractive hybridization of classical cDNA RDA method to cause, and the false positive and the huge workload problem of the existence of mRNA DD method, invent a kind of cDNA reducing hybrid difference display method on cDMA library base.

The objective of the invention is to be achieved through the following technical solutions.The abundant cDNA information that it utilizes the cDNA library to be comprised, effective extract garlicin (Allitridi) processing and untreated SGC-7901 BGC823 cell cdna library (Alli823cDNA library and BGC823 cDNA library) with garlic are sample, carry out garlicin and handle front and back SGC-7901 Cloning of Differentially-expressed Genes analysis.The restriction enzyme site that self is had in conjunction with carrier library, select for use the parallel pcr amplification of restriction enzyme Xho I and BamH I double digestion choosing representative to prepare the amplicon in two libraries, carrying out two-way one as detection DNA with driving DNA respectively with two amplified library takes turns subtractive hybridization and reduces the hybridization ratio, then one taking turns the subtractive hybridization difference product and carry out two-way two and take turns subtractive hybridization as detecting DNA and driving DNA respectively separately, the two-wheeled difference product respectively label isotope α- ³⁵Introduce mRNA DD differential display technique behind the S dATP, and carry out the differential fragment screening in conjunction with the reverse northern blot hybridization.This method is to introduce differential display technique carrying out between two cDNA libraries on the double-wheel bidirectional subtractive hybridization basis, so called after cDNA reducing hybrid difference display method on cDMA library base (Subtractive Hybridization Difference Display, SHDD).

Concrete implementation step:

1. the preparation of two cDNA library phage DNAs: from garlicin treatments B GC823 (being called for short Alli823) and BGC823 parental cell cDNA library, get 1 * 10 respectively ⁶Clone shop dish extracts two cDNA library phage DNAs, each about 40 μ g;

2. two cDNA library phage DNA amplicon (Amplicon, abbreviation Amp) preparation: utilize Xho I (10U/ μ l, Promega) Alli823 and BGC823 library DNA being carried out enzyme cuts, enzyme is cut system 70 μ l: library DNA 50 μ l (40 μ g), 10 * damping fluid D (Buffer D), 7 μ l, Xho I 6 μ l, deionized water 7 μ l; 37 ℃ of 4 hours endonuclease reactions.Enzyme is cut product after phenol-chloroform-primary isoamyl alcohol extracting, connect Xho I 15,13 joints (10 μ M), the volume of ligation is 120 μ l (template and joint ratio=1: 10-30): template DNA 60 μ l (40 μ g), Xho I 15 joints (10 μ M) 15 μ l, Xho I 13 joints (10 μ M) 15 μ l, 10 * T4 ligase enzyme Buffer, 12 μ l mend deionized water to 120 μ l; The ligation system is positioned in 50 ℃ the warm water beaker, progressively reduces to 10 ℃ from 50 ℃ in 1 hour, adds T4 ligase enzyme (Biolab) 400U, puts in 16 ℃ of waters bath with thermostatic control 20 hours.(10U/ μ l Promega) digests, after phenol-chloroform-primary isoamyl alcohol extracting through BamHI to connect after product, again through QIAquick post (QIAGEN) purifying, the purifying after product connects 1RBam-12,24 joints, and ligation volume, template and joint ratio and ligation step are the same.To connect product is that template is carried out pcr amplification respectively, and reaction volume is that 100 μ l (connect after product 1.5 μ l, 2.5mM ribodesose nucleic acid (dNTP) 2 μ l, Taq archaeal dna polymerase (Taq DNA polymerase) 10 * buffer 10 μ l, 50mM MgCl ₂4 μ l, deionized water 71.5 μ l), the PCR response procedures be 72 ℃ 3 minutes → add Taq archaeal dna polymerase (Promega respectively, 5U/ μ l) → 72 ℃ of 1 μ l → 72 ℃ 5 minutes → add respectively 1RBam24 primer (10 μ M) 10 μ l → (95 ℃ 1 minute+72 ℃ 3 minutes) * 25 circulations are 10 minutes, amplified production promptly is (the Alli823 Amp of amplicon separately by the preparation of two library DNAs, BGC823 Amp), each needs preparation 20 μ g; (SHDD method amplicon preparation principle figure sees accompanying drawing 1A) 3. prepares detection DNA (Tester DNA) respectively and drive DNA (Driver DNA) with two amplified library: two amplified library (each about 20 μ g) cut except that joint and through the QIAquick column purification through BamH I enzyme respectively, and the purifying after product both had been the driving DNA separately by the preparation of two library DNAs; The purifying after product is got 1/10 (each about 2 μ g) respectively and is connected second bell and spigot joint (2JBam12,24), and the ligation step is the same, and connecting after product both had been two library DNAs detection DNA separately; (outline flowchart of step 3-6 is seen accompanying drawing 1B) 4. detects DNA and takes turns subtractive hybridization with the bidirectional equalization one that drives DNA: the detection DNA of 1 μ g is mixed (Tester: Driver=1: 20) with the driving DNA of 20 μ g, cold dehydrated alcohol-20 a ℃ precipitation is spent the night, be dissolved in 4 μ, 13 * EE Buffer after the centrifugal desalinization of soil by flooding or leaching, add 30 μ l autoclaving paraffin oils and cover liquid level, 95 ℃ of sex change 10 minutes, add 5M NaCl 1 μ l again, in thermostat container 67 ℃, hybridized 20 hours.Detect DNA and the amplification that drives after the DNA first round hybridizes, amplified reaction volume 100 μ l (hybridization after product 0.8 μ l, 2.5mM dNTP 2 μ l, 10 * Taq DNA Polymerase Buffer, 10 μ l, 50mM MgCl ₂4 μ l), the PCR response procedures be 72 ℃ 3 minutes → add Taq DNA Polymerase 1 μ l → 72 respectively ℃ 5 minutes → added → 72 ℃ of 2JBam24 primer (10 μ M) 10 μ l → (95 ℃ 1 minute+70 ℃ 3 minutes) * 15 circulations respectively 10 minutes; Be dissolved in after the PCR product ethanol sedimentation and the desalinization of soil by flooding or leaching among 30 μ l, 1 * TE.Use Semen Phaseoli radiati Germinatus nuclease (MBN) digestion single stranded DNA, add 2 μ l (Biolab, 10U/ μ l) MBN in the above-mentioned PCR product, 10 * NE Buffer, 4 μ l, 10 * ZnSO ₄Buffer 4 μ l; 30 ℃ digested 35 minutes.The digestion after product, is dissolved in the 10 μ l deionized waters after the ethanol sedimentation and the desalinization of soil by flooding or leaching through phenol-chloroform-primary isoamyl alcohol extracting.Pcr amplification reaction volume 80 μ l behind the digestion single stranded DNA: digestion single stranded DNA and deactivation MBN rear pattern plate 2.5 μ l, dNTP (2.5M) 2 μ l, 2JBam24 primer (10 μ M) 8 μ l, Taq DNAPolymerase 1 μ l, Taq DNA Polymerase Buffer (10 *) 8 μ l mend deionized water to 80 μ l; Reaction conditions: → 72 ℃ of 94 ℃ of 3 minutes → (94 ℃ 1 minute+70 ℃ 3 minutes) * 20 circulations 10 minutes; Amplified production is both for one taking turns subtractive hybridization difference product (DP1) separately as what detection DNA obtained respectively with two amplified library; 5. detect DNA and take turns subtractive hybridization with the bidirectional equalization two that drives DNA: two libraries one are taken turns subtractive hybridization difference product (DP1) and are cut except that 2JBaml2 through BamH I enzyme, 24 joints and through the QIAquick column purification, purifying after product both be each the self-driven DNA by two library DP1 preparation; The purifying after product is got 1/10 (each about 2 μ g) respectively and is connected 3Nbaml2,24 joints, and the ligation step is the same, and connecting after product both had been the detection DNA separately that is prepared by two library DP1.The detection DNA of 0.1 μ g is mixed with the driving DNA of 20 μ g (Tester:Driver=1: 200), follow-up hybridization, MBN digestion single stranded DNA and pcr amplification prepare two library DNAs and two take turns that hybridization difference product (DP2) step is same takes turns hybridization separately; 6. one take turns and two take turns the isotopic labeling of difference product (DP1 and DP2) and the gel differences show: take turns and two take turns difference product, PCR reaction volume 20 μ l (template 10ng, 2J/3NBam24 (10 μ M) 1.4 μ l, 50mM MgCl with PCR method mark one ₂0.8 μ l, 25 μ MdC (GT) TP, 1.5 μ l, 25 μ M dATP, 1.2 μ l, Taq DNA Polymerase 0.4 μ l, TaqDNA Polymerase Buffer (10 *) 5 μ l, α- ³⁵S-dATP (10 μ M) 1.2 μ l mend deionized water to 20 μ l); PCR reaction conditions: 94 ℃ of 3 minutes → (94 ℃ 2 minutes, 70 ℃ 10 minutes) * 2 circulations.Sequencing gel difference shows: PCR product 3 μ l add 3 μ l termination reaction liquid after the isotopic labeling, the capable gel electrophoresis of sample is gone up in 80 ℃ of sex change after 2 minutes, it is unfixing that electrophoresis finishes back glue, directly adhere on the Whatman 3M filter paper, on cover preservative film, 80 ℃ of vacuum-dryings were drained in 3 hours, removed preservative film, the X-ray sheet is pressed in 3 punching location.Cut glue: three-point fix cuts the difference band, 0.1 * TE, 50 μ l soaked overnight.Getting an amount of supernatant does template and carries out PCR and increase reaction system 50 μ l again: cut glue soak solution 3 μ l, 2J/3NBam24 (10 μ M) 3 μ l, 50mM MgCl ₂1.5 μ l, 2.5mM dNTP 1 μ l, Taq DNA Polymerase0.4 μ l, Taq DNA Polymerase Buffer (10 *) 5 μ l mend deionized water to 50 μ l; Reaction conditions: → 72 ℃ of 94 ℃ of 3 minutes → (94 ℃ 1 minute, 70 ℃ 3 minutes) * 30 circulations 10 minutes, amplified production both one were taken turns and two were taken turns differential fragment for what use that the SHDD method obtains; (sequencing gel difference show and cut glue reclaim fragment again amplification see accompanying drawing 2A, 2B) 7. use the reverse northern blot hybridizations and one take turns and two take turns differential fragment and identify: get 100ng differential fragment PCR capable 1% agarose gel electrophoresis of amplified production more respectively, use the salt bridge transfer method fragment southern blotting technique is transferred to Hybond membrane what the SHDD method obtained; Do template (respectively getting 100ng) to make up the double-stranded cDNA that uses in Alli823 cDNA library and BGC823 cDNA library, with random priming go respectively α- ³²The P-dCTP mark; Prehybridization, hybridization step are with conventional Northern blot hybridization; (the reverse northern blot hybridization the results are shown in accompanying drawing 3) 8. the analysis of reverse northern blot hybridization confirms that application SHDD method clone obtains 10 cDNA fragments of inducing high expression level through garlicin, wherein 7 have been checked order and have confirmed to derive from respectively the supressor (XIP) of X protein of hepatitis B virus, folacin receptor α (FR α), calcium binding protein (Calcyclin), neuroglia-derived talin (GDN), cathepsin D (Cathepsin D), the sweet enzyme protection albumen of beta galactose (PPGB), the HADHA gene; Obtain 6 garlicins and induce the low cDNA fragment of expressing, wherein 3 confirm to derive from activated form G protein alpha subunit (Gpalpl) through order-checking, testis enhancing gene transcript/Bax supressor 1 (TEGT/BI-1), human interphotoreceptor stromatin polysaccharide (IMPG2) gene; And obtain on the garlicin abduction delivering to be in harmonious proportion two unknown cDNA fragments of downward modulation simultaneously.This result of study provides strong experimental basis for illustrating garlic mechanism of action antiviral, that resist multiple human tumor, neural system degeneration and diseases of cardiovascular and cerebrovascular systems.Having obtained two unknown cDNA fragments lays a good foundation for further disclosing prophylactic biological action of garlic active ingredient and molecular mechanism.(order-checking and database homology comparative result see Table 1)

The differential expression cDNA fragment that table 1. utilizes the SHDD method to obtain

Garlicin abduction delivering fragment sequence number cDNA biological function

Raise the lysosomal significant proteolytic enzyme of downward modulation SH 2-3 Cathepsin D ++ +++

Proteolytic degradation, activation and antigen are only processed No. 14 karyomit(e)s of the SH 4-1 unknown gene sequence and the mankind +++

19 base homology SH 6-1 GDN thrombin inhibitorss+

The sweet enzyme protection albumen of neural axon elongation factor SH 7-1 PPGB beta galactose ++ SH 8-2 HADHA lipid acid β-Yang Hua +++SH 9-1 XIP suppresses the HBx transcriptional activation activity ++ ++

Suppress hbv replication and propagation SH 13-2 Gpalpl activation cAMP signal path +++

Promote the human interphotoreceptor of cell proliferation SH 14-1 IMPG2 ++

Stromatin polysaccharide SH 17-1 TEGT/BI-1 suppresses apoptosis ++ and SH 18-3 unknown gene sequence contains the S2H2 zinc fingers ++

Synthetic with multiple material in the fruit bat Scrt Gene Partial homology DP 3-1 FR α participation body ++ ++ DP 3-2 Calcyclin cell cycle associated protein +++

Participate in Calcium Signal conduction path SH 1 ND ++ ++ SH 3 ND ++ ++ SH 10 ND ++ SH 11 ND +++SH 16 ND ++ ++ SH 19 ND +++annotate: "+" represents the gene expression dose rise or reduces one times; Not order-checking of " ND " representative.

The present invention compared with prior art, its advantage is the following aspects: for ease of the narration, two libraries mRNA ensemble separately all is divided into three types: advantageous information; Be equal to information; Weak tendency information.For Alli823 cDNA library, it is advantageous information that garlicin is induced the information of rise, and the information of inducing downward modulation is weak tendency information; For the BGC823cDNA library, it is weak tendency information that garlicin is induced the information of rise, and the information of inducing downward modulation is advantageous information, and the information that garlicin does not influence is the information that is equal to of two library DNAs.

Reducing hybrid difference display method on cDMA library base (SHDD) is making full use of on the abundant cDNA information basis that the cDNA library provides, further combined with the advantage of cDNARDA and mRNA DD, enlarged advantage separately simultaneously again and avoided significant disadvantages separately as far as possible.

1. the restriction enzyme digestion sites that utilizes abundant cDNA information that the cDNA library provides and carrier library to provide is based upon the subtractive hybridization difference display method on the basis, cDNA library, for existing a large amount of cDNA library provides new application prospect.

2. compare with cDNA RDA, there is following advantage in SHDD:

(1) cDNA RDA method is to discern the digestion with restriction enzyme choosing representative (average 1 point of contact/256 base pair (bp)) in 4 base sites, double-stranded cDNA (average 2,000 base pairs (kb)) on average can cut out the 7-8 bar segment, differential fragment is general short partially, specificity is lower, and exists several differential fragments to derive from the replication problem as a result of same aim sequence; And SHDD combines the restriction enzyme site that two cDNA carrier libraries self are had, utilize Xho I and BamH I double digestion choosing representative (average 1 point of contact/2kb), when guaranteeing the amplicon quantity of information, can obtain longer differential fragment again like this, help improving the specificity of follow-up Northern blot hybridization, sequence homology comparison and library screening;

(2) cDNA RDA method need be carried out three-wheel and even the four-wheel subtractive hybridization just can obtain the evident difference fragment, higher subtractive hybridization ratio and every many wheels pcr amplification of taking turns after the hybridization cause driving DNA, one takes turns difference product, the two segment group distribution trends of taking turns difference product successively decrease successively, two take turns and the three-wheel subtractive hybridization in just existed and drive DNA and and take turns and the two unbalanced subtractive hybridization problems of taking turns between the difference product segment group, drive DNA and and take turns the overlap of taking turns difference product with two, the long partially zone that also is the difference product segment group suffers the extracting of higher subtractive hybridization ratio and quantity of information takes place to lose, only there is corresponding sequence seldom in the short partially zone of difference product segment group in driving DNA, occur as false-positive message thereby the extraction rate was acquired that suffers is more weak; The SHDD method then be between two samples, carried out two-way one take turns subtractive hybridization and separately a bidirectional equalization two of taking turns between difference product take turns subtractive hybridization, thereby avoided the unbalanced quantity of information that is caused of subduing to lose and the false positive problem;

(3) two-way one take turns subtractive hybridization and given prominence to two samples information gap separately, and balanced two take turns subtractive hybridization and further amplify this information gap, the introducing of high resolving power isotopic differentiation technique of display has substituted cDNA RDA ethidium bromide again and has shown the method for being with, thereby can only adopt the two-wheeled subtractive hybridization and reduce the subtractive hybridization ratio, help poor subtracting and obtain low abundance and more weak different information;

(4) carrier library self sequence enzyme is cut four obvious banding patterns that about respectively 22kb, 11kb, 6kb and 850bp appear in the back, first three bar sequence is bigger, can not increase with the representative generation competitive PCR that aim sequence is selected but can be used as restriction endonuclease digests appropriate index, thereby has improved the comparability between the two sample amplicons;

(5) the 850bp fragment in carrier sequence source is represented the information that is equal to of the high copy number of two amplified library, can be used as the assessment foundation of subtractive hybridization extraction rate was acquired.

3. compare with mRNA DD, SHDD also has clear superiority:

(1) mRNA DD is owing to non-specific binding and other reasons of random primer and anchor primer and aim sequence make false positive results occupy higher ratio, and causes repetition differential fragment problem because different random primers is attached to the specific region of same aim sequence or different anchor primers and the combination of the same random primer same aim sequence that increases; And the difference of SHDD shows and to have adopted 24 high degree of specificity primers that base is long, has solved the problems referred to above effectively and has helped the amplification of low abundance information;

(2) mRNA DD causes the position of same band in the difference results may comprise several different information owing to self designing the problem that goes up existence, and further these information of isolation identification have become the step that mRNA DD is the most consuming time, consume power; And the SHDD method has been given prominence to separately advantageous information of two samples, has been equal to that the information equilibrium weakens, weak tendency information is significantly suppressed by bidirectional equalization two-wheeled subtractive hybridization, this improve the variance analysis result specific allow simultaneously the different information between the total mRNA of two samples concentrated on the plate sequencing gel carry out the difference display analysis, many kinds of random primers of mRNA DD make up with anchor primer, the screening of sequencing gel electrophoretic analysis and follow-up complexity repeatedly thereby greatly simplified;

(3) the 850bp fragment in carrier sequence source is owing to represented the information that is equal to of the high copy number of two amplified library, two take turns in the difference product through still being present in separately after the gentle subtractive hybridization extracting of two-wheeled, this just provides natural internal reference for follow-up difference display analysis, has improved the repeatability of experiment.

The SHDD method is based upon on the basis, cDNA library, and further combines the specificity characteristics of cDNA RDA and the mRNA DD advantage aspect two-way examination differential fragment and higher isotope detection sensitivity.In concrete technological line design, the characteristics of SHDD are exactly two-way one to take turns and two to take turns and introduced isotropic substance mRNA DD differential display technique on equilibrium, the gentle subtractive hybridization basis between two cDNA library samples, make this method in low abundance of clone and weakly heterogeneous information, the specificity that improves the variance analysis result reduces false positive, reduce and repeat difference results, the quantity of information that improves variance analysis reduces aspect such as workload simultaneously again and obviously is better than classical mRNA DD and cDNA RDA method, for the gene of cloning low abundance weakly heterogeneous provides effective means; And provide new application prospect for the cDNA library simultaneously.(seeing Table 2)

Table 2.SHDD and cDNA RDA, the contrast of mRNA DD method relative merits

Poor specificity heteroleptic quantity workload cDNA RDA +++++ ++ mRNA DD+++ ++ ++ +++SHDD +++++ ++ ++

Below in conjunction with drawings and Examples the present invention is done further and carefully to state.

Accompanying drawing 1A is the preparation of cDNA reducing hybrid difference display method on cDMA library base (SHDD) amplicon.Wherein: the phage vector that λ ZAP II uses for the cDNA library construction; Black surround is represented the carrier library sequence, and white edge is represented insertion sequence; Bam/B represents restriction enzyme BamH I, and Xho/X represents restriction enzyme Xho I; Comprise BamH I point of contact GGATCC in the Xho I joint.

Accompanying drawing 1B is the outline flowchart of cDNA reducing hybrid difference display method on cDMA library base (SHDD).Wherein: Alli Amp is the amplicon by the preparation of Alli823 library DNA, and BGC Amp is the amplicon by the preparation of BGC823 library DNA; The T representative detects DNA (Tester DNA), and the D representative detects DNA (Driver DNA); Alli DP1 represents with the Alli823 amplicon and takes turns the subtractive hybridization difference product as one of detection DNA acquisition, and BGC DP1 representative is taken turns the subtractive hybridization difference product with the BGC823 amplicon as one of detection DNA acquisition; 1st SH represents one to take turns subtractive hybridization, and 2nd SH represents two to take turns subtractive hybridization.

Accompanying drawing 2A for use that the SHDD method obtains one take turns and two take turns difference product (DP1 and DP2) through isotropic substance α- ³⁵Sequencing gel difference result displayed behind the S-dATP mark.Wherein: AlliDP1 represents with the Alli823 amplicon and takes turns the subtractive hybridization difference product as one of detection DNA acquisition, and BGC DP1 representative is taken turns the subtractive hybridization difference product with the BGC823 amplicon as one of detection DNA acquisition.

Accompanying drawing 2B one takes turns and two takes turns differential fragment for what use that the SHDD method obtains.

Accompanying drawing 3 is taking turns and the two reverse northern blot hybridization results that take turns differential fragment of using that the SHDD method obtains.Wherein (A) A-K represents differential fragment SH 1,2,3 respectively, 21,4,5,6,22,9,8,7. except that fragment SH 21 (D), all the other fragments expression level after garlicin (Allitridi) is handled is all seen rise. (B) A-L represents differential fragment SH 13 respectively, 8,14,15,23,16,24,17,18,20,19,25. fragment SH13 wherein, 14,15,16,17,18,19 are used as the reverse northern blot hybridization through expression level downward modulation .GAPDH after the garlicin processing contrasts (Con). and probe (Probe) Alli823 is BGC823 cell mRNA reverse transcription displacement synthetic two strands cDNA (ds-cDNA) after being handled by garlicin; Probe (Probe) BGC823 is by the double-stranded cDNA (ds-cDNA) of parent BGC823 cell mRNA reverse transcription displacement synthetic.

Realize the most preferred embodiment of invention:

1. select the strong cDNA library (as drug treating and untreated cell cDNA library or a certain internal organs tumor tissues and normal tissue cell cDNA library) of two comparabilities as the gene expression difference analytic target: to handle and be untreated the BGC823 cell cdna library as the garlicin that adopts in the research;

2. select single restriction enzyme or two kinds of restriction endonuclease combined utilization that library DNA is carried out enzyme and cut the choosing representative, endonuclease digestion should guarantee to select the quantity of information of representative, and notice that simultaneously restriction endonuclease should not cut out carrier (phage) DNA the short-movie section less than 800bp, avoid the competitive amplification of follow-up PCR of carrier DNA and insertion sequence as far as possible; BamH I and Xho I double digestion choosing representative are adopted in this research, carrier library self sequence enzyme is cut four obvious banding patterns that about respectively 22kb, 11kb, 6kb and 850bp appear in the back, first three bar sequence is bigger, and can not increase with the representative generation competitive PCR that aim sequence is selected but can be used as restriction endonuclease digests appropriate index;

3. different cDNA library samples, the degree difference of the kind of difference expression gene, abundance and expression level difference, thereby need select one to take turns the subtractive hybridization ratio of taking turns according to specific circumstances with two, to clone the gene that obtains low abundance, weakly heterogeneous expression as far as possible, increase variance analysis result's quantity of information; This research adopts one to take turns subtractive hybridization ratio detection DNA: drive DNA=1: 20, two take turns the subtractive hybridization ratio detects DNA: drive DNA=1: 200;

4. two samples one are taken turns between the difference product and two are taken turns between the difference product and should have comparability preferably separately separately, the degree of pcr amplification shows that with sequencing gel difference observing the more band of difference clearly is advisable, the very few different information of amplification cycles number can not obtain the enrichment of enough degree, and the amplification cycles number is crossed can cause losing of low abundance difference information at most; And the representative amplification trend of selecting different digestion with restriction enzyme to select is inconsistent, thereby need to adjust the pcr amplification cycle number to obtain best variance analysis result; The pcr amplification cycle number that this research is adopted is 20 circulations;

5. the two-wheeled difference product should be on the basis of adjusting product sheet stage group trend comparability, use 1-2 round-robin pcr amplification respectively label isotope α- ³⁵S dATP; Template DNA is advisable with 10ng, can obtain the product of 40ng through 2 round-robin pcr amplifications, calculate in view of the above α- ³⁵The consumption of S dATP and other four kinds of dNTP (mole number) ratio, with α- ³⁵SdATP: dNTP=1: 30 are advisable; Suitably reduce the consumption of the dATP of the plain mark of No Parity, with on the basis of guaranteeing the amplified fragments integrity, increase α- ³⁵The incorporation efficiency of S dATP.

Claims (1)

1. A kind of cDNA reducing hybrid difference display method on cDMA library base (SHDD), be based upon on the basis, cDNA library, and further combine representative difference analysis method of existing method cDNA (cDNA RDA) and mRNA difference display method (mRNA DD) advantage separately, it is characterized in that the restriction enzyme digestion sites that it utilizes abundant cDNA information in cDNA library and carrier to have, discharge the insertion sequence fragment as representative with suitable endonuclease digestion, the variance analysis of carrying out on this basis; And carry out two-way one as detection DNA with driving DNA respectively with two paired DNA (or cDNA) sample and take turns subtractive hybridization, obtain one to take turns the hybridization difference product separately; One take turns the hybridization difference product and prepare respectively and detect DNA and drive DNA separately with two samples respectively, further carry out two-way two on this basis and take turns subtractive hybridization and obtain two to take turns the subtractive hybridization difference product separately; Carrying out sequencing gel difference behind the two-wheeled difference product label isotope shows.

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