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

Abstract

The cDNA subtractive hybridization differential display method based on the cDNA library is a molecular biology gene cloning analysis method and belongs to the field of molecular biology. It utilizes the restriction endonuclease cutting site of the cDNA library carrier, and the restriction endonuclease selection represents the preparation of the amplicons of the two libraries, and performs a bidirectional round of subtractive hybridization between the amplicons of the two libraries. Bidirectional two-round subtractive hybridization, after two rounds of differential product labeling with isotopes, mRNA differential display technology was introduced for differential analysis. This method provides a new application prospect for cDNA library, and provides an effective means for cloning low-abundance and weakly differential genes.

Description

cDNA Subtractive Hybridization Differential Display Method Based on cDNA Library

The cDNA subtractive hybridization difference display method (SHDD) based on cDNA library is a molecular biology gene clone analysis method, which belongs to the field of molecular biology.

There are many existing gene cloning methods, and according to literature reports, there are two most common methods, one is cDNA representative difference analysis method (cDNA RDA), and the other is mRNA differential display method (mRNA DD).

(1) cDNA Representative Difference Analysis (cDNA RDA)

The genomic subtractive hybridization method is the working basis of cDNA RDA. The so-called genomic subtractive hybridization is to separate the specific sequence of the detection DNA from two related DNA samples (detection DNA and driver DNA). A certain amount of detection DNA ( Tester DNA) hybridizes with excess driver DNA (Driver DNA), so that most of the sequences in the test DNA that are identical to the driver DNA undergo hybrid annealing, and the unique DNA fragments in the test DNA undergo self-annealing and are separated . The cDNA RDA method introduces a kinetic enrichment process based on PCR amplification on the basis of the classic genome subtractive hybridization method. Identification of genes differentially expressed in tissue cells before and after induction by different developmental stages, different cycle phases, drugs, cytokines, or pathogens has been widely used in research fields. However, this method has the following disadvantages: (1) The classic cDNA RDA method uses ethidium bromide to develop the bands, usually three rounds of subtractive hybridization are required to obtain obvious differential bands, the higher ratio of subtractive hybridization and each round of hybridization After multiple rounds of PCR amplification, the distribution trend of fragment groups of driver DNA, first-round differential product (DP1), and second-round differential product (DP2) decreased successively. The problem of disequilibrium subtractive hybridization between fragment populations of first- and second-round differential products. The overlapping region between the driver DNA and the first and second rounds of differential products, that is, the longer region of the differential product fragment group is extracted by a higher subtractive hybridization ratio and the amount of information is lost; the shorter region of the differential product fragment group is in the driver DNA There are only few corresponding sequences in , which suffer from weak extraction effect and appear as false positive information. This finding provides a reasonable explanation for the low information content and false positives in the differential products obtained by the classical cDNA RDA method. (2) The classic cDNA RDA method can only detect the difference between the DNA of two paired samples, and can only obtain the mRNA difference information of the increased expression level in the detected DNA, and cannot obtain the information of the down-regulated expression level at the same time. (2) mRNA differential display method (mRNA DD)

The mRNA DD method uses random primers and anchor primers to perform polymerase chain reaction amplification (PCR) on mRNA information groups from two different sources, and the amplified products are subjected to denaturing polyacrylamide gel electrophoresis to compare and select differentially expressed fragments. Using the mRNA DD method, the difference in gene expression between multiple samples can be compared at the same time, and the "up-regulated" and "down-regulated" genes can be detected at the same time; the amplification of the target sequence depends on different combinations of random primers and anchor primers, while It is not dependent on the abundance of the target sequence, and the application of isotope is also conducive to screening to obtain information of lower abundance differential expression. Since the establishment of the mRNA DD method in 1992, people have obtained a considerable number of tumor-related genes by using this technology and further improved methods on this basis. After several years of practice, people have also found some shortcomings of this technology, mainly including:

(1) High false positive rate: Many studies have shown that more than 70% of the differential bands presented by mRNA differential display technology cannot be confirmed by methods such as Northern blot hybridization and reverse Northern blot hybridization. The reasons for the analysis are mainly related to the following factors: low specificity of random primers; mRNA samples are prone to degradation or DNA contamination; due to the use of 3′ end anchor primers, most of the differential bands obtained by this method only contain 3′ ends A short piece of information in the untranslated region (3'-UTR), and the sequence of this region is relatively conserved, which may cause false positives in Northern blot hybridization using the 3'-terminal cDNA fragment as a probe.

(2) Low detection rate for low-abundance mRNA: in eukaryotic cells, a considerable amount of mRNA belongs to low-abundance information, and 86% of genes account for only 25% of the total amount of mRNA ^[10] . Due to the competitive amplification of PCR mRNA DD technology has a clear tendency to clone high-abundance mRNA ^[11] .

(3) Huge workload problem: Since the method does not subtract the equivalent information in the matched samples before the gel difference is displayed, the single band position displayed by the electrophoresis result contains 1-10 different cDNA sequences, with an average of 4 kinds. Further isolation and identification of these information has become the most time-consuming and labor-intensive step of mRNA DD. Moreover, because different random primers bind to specific regions of the same target sequence or different anchor primers are combined with the same random primer to amplify the same target sequence, the problem of repeated differential fragments further increases the workload.

The purpose of the present invention is to make full use of the abundant cDNA information contained in the cDNA library to analyze the gene expression difference between the libraries; to clone the expressed gene information with low abundance and weak difference, and to avoid the unbalanced subtractive hybridization caused by the classical cDNA RDA method. In order to solve the problems of low information content and false positives in the mRNA DD method, and the problems of false positives and huge workload in the mRNA DD method, a cDNA subtractive hybridization differential display method based on cDNA library was invented.

The purpose of the present invention is achieved through the following technical solutions. It utilizes the rich cDNA information contained in the cDNA library, and takes the effective extract of garlic (Allitridi) treated and untreated human gastric cancer cell line BGC823 cell cDNA library (Alli823cDNA library and BGC823 cDNA library) as samples to carry out allicin Clonal analysis of differentially expressed genes in human gastric cancer cell lines before and after treatment. Combined with the enzyme cutting sites of the library vector itself, the restriction endonucleases Xho I and BamH I were selected for double digestion to represent parallel PCR amplification to prepare the amplicons of the two libraries, and the amplicons of the two libraries were used as the detection DNA and driver DNA were subjected to bidirectional one-round subtractive hybridization and the hybridization ratio was reduced, and then the difference products of each round of subtractive hybridization were used as detection DNA and driver DNA for two-way two-way subtractive hybridization, and the difference products of the two rounds were respectively labeled with isotope α- ³⁵ S dATP Later, mRNA DD differential display technology was introduced, combined with reverse Northern blot hybridization for differential fragment screening. This method introduces differential display technology based on two rounds of two-way subtractive hybridization between two cDNA libraries, so it is named as Subtractive Hybridization Difference Display (SHDD) based on cDNA library.

Specific implementation steps:

1. Preparation of phage DNA from two cDNA libraries: take 1×10 ⁶ clones from allicin-treated BGC823 (Alli823 for short) and BGC823 parental cell cDNA libraries, respectively, and extract phage DNA from the two cDNA libraries, about 40 μg each;

2. Preparation of two cDNA library phage DNA amplicons (Amplicon, referred to as Amp): Use Xho I (10U/μl, Promega) to digest Alli823 and BGC823 library DNA, enzyme digestion system 70μl: library DNA 50μl (40μg ), 7 μl of 10× buffer D (Buffer D), 6 μl of Xho I, 7 μl of deionized water; enzyme digestion reaction at 37°C for 4 hours. After the digested product was extracted with phenol-chloroform-isoamyl alcohol, it was connected to the Xho I 15, 13 adapter (10 μM), and the volume of the ligation reaction was 120 μl (ratio of template to adapter = 1:10-30): template DNA 60 μl (40 μg ), Xho I 15 Adapter (10μM) 15μl, Xho I 13 Adapter (10μM) 15μl, 10×T4 Ligase Buffer 12μl, add deionized water to 120μl; ligation reaction system placed in a warm water beaker at 50°C, within 1 hour Decrease gradually from 50°C to 10°C, add 400 U of T4 ligase (Biolab), and place in a constant temperature water bath at 16°C for 20 hours. After ligation, the product was digested with BamHI (10U/μl, Promega), extracted with phenol-chloroform-isoamyl alcohol, and then purified by QIAquick column (QIAGEN). The ratio of linker and ligation reaction steps are the same as before. Carry out PCR amplification using the ligated product as a template, and the reaction volume is 100 μl (1.5 μl of the ligated product, 2 μl of 2.5 mM deoxyribonucleic acid (dNTP) , 10 μl of Taq DNA polymerase (Taq DNA polymerase) 10× buffer, 50 mM MgCl ₂ 4 μl, deionized water 71.5 μl), the PCR reaction program is 72 ° C for 3 minutes → add Taq DNA polymerase (Promega, 5U/μl) 1 μl → 72 ° C for 5 minutes → add 1RBam24 primer (10 μM) 10 μl → (95 ° C 1 minute + 3 minutes at 72°C) × 25 cycles → 10 minutes at 72°C, the amplification products are the respective amplicons (Alli823 Amp, BGC823 Amp) prepared from the DNA of the two libraries, and 20 μg each needs to be prepared; (SHDD method The schematic diagram of amplicon preparation is shown in Figure 1A) 3. Prepare the test DNA (Tester DNA) and driver DNA (Driver DNA) with the amplicons of the two libraries respectively: the amplicons of the two libraries (about 20 μg each) are separated by BamH I enzyme The adapters were cut off and purified by QIAquick column. The purified products were both the driver DNA prepared from the DNA of the two libraries; 1/10 (about 2 μg each) of the purified products were connected to the second set of adapters (2JBam12, 24), ligated The reaction steps are the same as before, and the product after ligation is the detection DNA of the two library DNAs; (see Figure 1B for a brief flowchart of steps 3-6) 4. Two-way balanced round subtractive hybridization of detection DNA and driver DNA: 1 μg of Mix the test DNA with 20 μg of driver DNA (Tester:Driver=1:20), precipitate in cold absolute ethanol at -20°C overnight, wash the salt by centrifugation, dissolve in 4 μl 3×EE Buffer, add 30 μl of high-pressure sterilized paraffin oil to cover the liquid surface, Denature at 95°C for 10 minutes, then add 1 μl of 5M NaCl, and hybridize for 20 hours in an incubator at 67°C. Amplification after the first round of hybridization between detection DNA and driver DNA, amplification reaction volume 100 μl (hybridized product 0.8 μl, 2.5 mM dNTP 2 μl, 10×Taq DNA Polymerase Buffer 10 μl, 50 mM MgCl ₂ 4 μl), PCR reaction program 72 3 minutes at ℃ → add 1 μl of Taq DNA Polymerase → 5 minutes at 72 ℃ → add 10 μl of 2JBam24 primer (10 μM) → (1 minute at 95 ℃ + 3 minutes at 70 ℃) × 15 cycles → 10 minutes at 72 ℃; ethanol precipitation of PCR products And wash the salt and dissolve in 30μl 1×TE. Mung bean sprout nuclease (MBN) was used to digest the single-stranded DNA, and 2 μl (Biolab, 10 U/μl) of MBN, 4 μl of 10×NE Buffer and 4 μl of 10×ZnSO ₄ Buffer were added to the above PCR product; digested at 30°C for 35 minutes. The digested product was extracted with phenol-chloroform-isoamyl alcohol, precipitated with ethanol and washed with salt, then dissolved in 10 μl deionized water. PCR amplification reaction volume after digesting single-stranded DNA: 2.5 μl template after digesting single-stranded DNA and inactivating MBN, 2 μl dNTP (2.5M), 8 μl 2JBam24 primer (10 μM), 1 μl Taq DNA Polymerase, Taq DNA Polymerase Buffer (10 ×) 8 μl, make up to 80 μl with deionized water; reaction conditions: 94°C for 3 minutes → (94°C for 1 minute + 70°C for 3 minutes) × 20 cycles → 72°C for 10 minutes; the amplification product is amplified with two libraries 5. Two-way balanced two-round subtractive hybridization of the detection DNA and the driver DNA: the difference product (DP1) of the two libraries is removed by BamH I digestion 2JBaml2, 24 adapters were purified by QIAquick column, and the purified products were the respective driver DNAs prepared from the two libraries DP1; 1/10 (about 2 μg each) of the purified products were connected to 3Nbaml2, 24 adapters, and the ligation reaction steps were the same as before, The products after ligation are the respective detection DNAs prepared from the two libraries DP1. Mix 0.1 μg of test DNA with 20 μg of driver DNA (Tester: Driver=1:200), the subsequent hybridization reaction, MBN digestion of single-stranded DNA, and PCR amplification to prepare the two library DNA’s two rounds of hybridization difference products (DP2) are the same 6. Isotope labeling and gel differential display of the first round and the second round of differential products (DP1 and DP2): label the first round and the second round of differential products by PCR method, PCR reaction volume 20 μl (template 10ng, 2J/3NBam24( 10 μM) 1.4 μl, 50 mM MgCl ₂ 0.8 μl, 25 μM dC(GT)TP 1.5 μl, 25 μM dATP 1.2 μl, Taq DNA Polymerase 0.4 μl, TaqDNA Polymerase Buffer (10×) 5 μl, α- ³⁵ S-dATP (10 μM) 1.2 μl , supplemented with deionized water to 20 μl); PCR reaction conditions: 94° C. for 3 minutes → (94° C. for 2 minutes, 70° C. for 10 minutes) × 2 cycles. Sequencing gel difference display: add 3 μl of stop reaction solution to 3 μl of isotope-labeled PCR product, denature at 80°C for 2 minutes, and then load the sample for gel electrophoresis. Vacuum dry at 80°C for 3 hours and drain, remove the plastic wrap, punch holes at three points for positioning, and press X-ray film. Gel cutting: three-point positioning to cut differential bands, soak overnight in 50 μl of 0.1×TE. Take an appropriate amount of supernatant as a template for PCR re-amplification. Reaction system 50μl: 3μl gel cutting soaking solution, 3μl 2J/3NBam24 (10μM), 1.5μl 50mM MgCl ₂ , 1μl 2.5mM dNTP, 0.4μl Taq DNA Polymerase, Taq DNA Polymerase Buffer (10×) 5 μl, supplemented with deionized water to 50 μl; reaction conditions: 94°C for 3 minutes → (94°C for 1 minute, 70°C for 3 minutes) × 30 cycles → 72°C for 10 minutes, the amplification product is applied SHDD The first and second rounds of differential fragments obtained by the method; (see Figure 2A and 2B for the results of sequencing gel differential display and gel-cut recovery fragment re-amplification) 7. The first and second rounds of reverse Northern blot hybridization pair SHDD method Identification of differential fragments: Take 100ng of differential fragment PCR reamplification products and perform 1% agarose gel electrophoresis, and apply the salt bridge transfer method to transfer the fragments to the hybridization membrane by Southern blotting; to construct Alli823 cDNA library and BGC823 cDNA library. Strand cDNA was used as a template (100ng for each), and was labeled with α- ^32P -dCTP by random primer method; the pre-hybridization and hybridization steps were the same as the conventional Northern blot hybridization; (reverse Northern blot hybridization results are shown in Figure 3) 8. Northern blot hybridization analysis confirmed that 10 allicin-induced high-expression cDNA fragments were cloned by SHDD method, 7 of which were sequenced and confirmed to be derived from hepatitis B virus X protein inhibitor (XIP), folate receptor α (FRα) , calcium-binding protein (Calcyclin), glial cell-derived talin (GDN), cathepsin D (Cathepsin D), β-galactosidase protective protein (PPGB), HADHA gene; six allicin-induced low Expressed cDNA fragments, 3 of which were sequenced and confirmed to be derived from the activated G protein alpha subunit (Gpalpl), testis enhancer gene transcript/Bax repressor 1 (TEGT/BI-1), human interphotoreceptor matrix proteoglycan (IMPG2) gene; and simultaneously obtained two unknown cDNA fragments whose expression was up-regulated and down-regulated by allicin. The results of this study provide a strong experimental basis for elucidating the mechanism of garlic's anti-virus, anti-a variety of human tumors, degenerative diseases of the nervous system and cardiovascular and cerebrovascular diseases. Obtained two unknown cDNA fragments laid the foundation for further revealing the biological effect and molecular mechanism of garlic active ingredients in disease prevention. (See Table 1 for the results of sequencing and database homology comparison)

Table 1. Differentially expressed cDNA fragments obtained by using the SHDD method

Biological function

Up-regulation Down-regulation of SH 2-3 Cathepsin D Lysosomal marker protease +++++

                                                                                                                                                                                                                         , 

                                                                                                                                                                                           

Nervichalot extension factor SH 7-1 PPGB β-galactose glycase protection protein ++ SH 8-2 Hadha fatty acid β-oxidation +++ SH 9-1 XIP inhibitory activity +++++

Inhibition of HBV replication and proliferation SH 13-2 Gpalpl Activation of cAMP signaling pathway +++

                    Promoting cell proliferation SH 14-1     IMPG2       Human interphotoreceptor              

                                                                                                                                                                                 

Partially homologous to the Drosophila Scrt gene DP 3-1 FRα FRα Participated in the synthesis of various substances in the body ++++ DP 3-2   Calcyclin   Cell cycle-related proteins ++ +

Participate in calcium ion signal conduction channel SH 1 nd ++++ SH 3 nd ++++ SH 10 nd ++ SH 11 nd +++ sh 16 nd ++++ sh 19 nd +++ Note: "+" represents Gene expression levels were up-regulated or down-regulated by one-fold; "ND" means not sequenced.

Compared with the prior art, the present invention has the following advantages: for the convenience of description, the respective mRNA information groups of the two libraries are divided into three types: superior information; equivalent information; weak information. For Alli823 cDNA library, allicin-induced up-regulation information is dominant information, and allicin-induced down-regulation information is weak information; for BGC823 cDNA library, allicin-induced up-regulation information is weak information, and allicin-induced down-regulation information is dominant information, while allicin-induced down-regulation information is dominant information. The information not affected by allicin is the equivalent information of the DNA of the two libraries.

Library-based cDNA subtractive hybridization difference display method (SHDD) further combines the advantages of cDNA RDA and mRNA DD on the basis of making full use of the rich cDNA information provided by the cDNA library, and at the same time expands their respective advantages and avoids their respective advantages as much as possible. Obvious disadvantage.

1. Using the rich cDNA information provided by the cDNA library and the restriction endonuclease sites provided by the library vector, the subtractive hybridization differential display method is established on the basis of the cDNA library, which provides a new method for a large number of existing cDNA libraries. Application prospects.

2. Compared with cDNA RDA, SHDD has the following advantages:

(1) The cDNA RDA method is selected by restriction endonucleases that recognize 4 base sites (average 1 cut point/256 base pairs (bp)), double-stranded cDNA (average 2,000 bases) Base pair (kb)) can cut out an average of 7-8 fragments, the difference fragments are generally short, the specificity is low, and there is a problem of duplication of the results of several difference fragments originating from the same target sequence; and SHDD combines two cDNA The enzyme cutting sites of the library vector itself are selected by Xho I and BamH I double enzyme digestion (average 1 cutting point/2kb), so that longer differential fragments can be obtained while ensuring the amount of amplicon information. It is beneficial to improve the specificity of subsequent Northern blot hybridization, sequence homology comparison and library screening;

(2) The cDNA RDA method requires three or even four rounds of subtractive hybridization to obtain significant differential fragments. The distribution trend of the fragment groups of the differential products in the rounds decreases successively. In the second and third rounds of subtractive hybridization, there exists the problem of unbalanced subtractive hybridization between the driver DNA and the fragment groups of the first and second rounds of differential products. The overlapping region of the differential product, that is, the long region of the differential product fragment group is extracted by a high subtractive hybridization ratio and the information is lost, and the short region of the differential product fragment group has only a small correspondence in the driver DNA. Sequences suffer from weak extraction effects and appear as false positive information; while the SHDD method carries out a two-way subtractive hybridization between two samples and a two-way balanced two-round subtractive hybridization between two samples, thus avoiding Information loss and false positive problems caused by unbalanced reduction;

(3) One round of bidirectional subtractive hybridization highlights the difference in information between the two samples, while the second round of balanced subtractive hybridization further amplifies the difference in information, and the introduction of high-resolution isotope difference display technology replaces the cDNA RDA ethidium bromide band. Therefore, only two rounds of subtractive hybridization can be used and the ratio of subtractive hybridization can be reduced, which is beneficial to obtain lower abundance and weaker difference information by subtraction;

(4) Four obvious band patterns of about 22kb, 11kb, 6kb and 850bp appeared after digestion of the sequence of the library vector itself. The first three sequences are relatively large and will not compete with the representative of the target sequence for PCR amplification, but they can be used as Moderate index of endonuclease digestion, thereby improving the comparability between the amplicons of the two samples;

(5) The 850bp fragment derived from the carrier sequence represents the equivalent information of the highest copy number of amplicons in the two libraries, which can be used as the basis for evaluating the extraction effect of subtractive hybridization.

3. Compared with mRNA DD, SHDD also has obvious advantages:

(1) mRNA DD has a high proportion of false positive results due to the non-specific binding of random primers and anchor primers to the target sequence and other reasons, and because different random primers bind to specific regions of the same target sequence or different The combination of anchor primers and the same random primers to amplify the same target sequence leads to the problem of repeating difference fragments; while the difference display of SHDD uses 24 bases long highly specific primers, which effectively solves the above problems and benefits low-abundance information the expansion of

(2) Due to the problems in the design of mRNA DD, the position of the same band in the difference results may contain several different information, and further separation and identification of these information has become the most time-consuming and labor-intensive step of mRNA DD; while the SHDD method Two-way balanced two rounds of subtractive hybridization highlighted the respective superior information of the two samples, weakened equivalent information balance, and significantly suppressed weak information, which allowed the differential information between the total mRNA of the two samples to be concentrated on one plate while improving the specificity of the differential analysis results Differential display analysis is performed on the sequencing gel, which greatly simplifies the combination of multiple random primers and anchor primers for mRNA DD, multiple sequencing gel electrophoresis analysis and subsequent complex screening work;

(3) Since the 850bp fragment derived from the vector sequence represents the equivalent information of the highest copy number of the amplicons of the two libraries, it may still exist in the two rounds of differential products after two rounds of mild subtractive hybridization extraction, which is the basis for subsequent differential display. The assay provides a natural endogenous control, improving experimental reproducibility.

The SHDD method is based on the cDNA library, and further combines the specific characteristics of cDNA RDA with the advantages of mRNA DD in two-way screening of differential fragments and high isotope detection sensitivity. In terms of specific technical route design, SHDD is characterized by the introduction of isotopic mRNA DD differential display technology on the basis of bidirectional one-round and two-round equalization and mild subtractive hybridization between two cDNA library samples, making this method more effective in cloning low-abundance and weakly differential Information, improve the specificity of differential analysis results, reduce false positives, reduce repeated differential results, increase the amount of information in differential analysis while reducing the workload, etc. It is obviously superior to the classic mRNA DD and cDNA RDA methods, and is suitable for cloning low-abundance and weak differences The gene provides an effective means; and at the same time provides a new application prospect for the cDNA library. (See Table 2)

Table 2. Comparison of advantages and disadvantages of SHDD and cDNA RDA, mRNA DD methods

Specific difference fragments quantitative workload CDNA RDA +++ ++ ++ mRNA DD+++++ ++++ shdd +++++ ++ ++

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

Accompanying drawing 1A is the preparation of the cDNA subtractive hybridization differential display method (SHDD) amplicon based on the cDNA library. Among them: λZAP II is the phage vector used for cDNA library construction; the black box represents the sequence of the library vector, and the white box represents the insertion sequence; Bam/B represents the restriction enzyme BamH I, and Xho/X represents the restriction enzyme Xho I; The Xho I linker contains the BamH I cutting point GGATCC.

Accompanying drawing 1B is the brief flowchart of cDNA subtractive hybridization differential display method (SHDD) based on cDNA library. Among them: Alli Amp is the amplicon prepared from the Alli823 library DNA, BGC Amp is the amplicon prepared from the BGC823 library DNA; T represents the test DNA (Tester DNA), D represents the test DNA (Driver DNA); Alli DP1 represents the Alli823 amplicon is used as a round of subtractive hybridization difference product obtained by detecting DNA, BGC DP1 represents a round of subtractive hybridization difference product obtained by using BGC823 amplicon as detection DNA; 1st SH represents a round of subtractive hybridization, 2nd SH represents two rounds of subtraction hybridize.

Fig. 2A is the result of sequencing gel differential display of the first-round and second-round differential products (DP1 and DP2) obtained by applying the SHDD method after being labeled with isotope α- ^35S -dATP. Among them: AlliDP1 represents the differential product of a round of subtractive hybridization obtained by using the Alli823 amplicon as the detection DNA, and BGC DP1 represents the differential product of a round of subtractive hybridization obtained by using the BGC823 amplicon as the detection DNA.

Figure 2B is the first round and second round of differential fragments obtained by applying the SHDD method.

Accompanying drawing 3 is the reverse Northern blot hybridization result of one round and two rounds of differential fragments obtained by applying the SHDD method. Wherein (A)A-K represent difference fragments SH 1, 2, 3, 21, 4, 5, 6, 22, 9, 8, 7 respectively. Except for fragment SH 21(D), other fragments are treated with allicin (Allitridi) The expression levels were all up-regulated. (B) A-L represent the differential fragments SH 13, 8, 14, 15, 23, 16, 24, 17, 18, 20, 19, 25. Fragments SH13, 14, 15, 16, The expression levels of 17, 18, 19 were down-regulated after allicin treatment. GAPDH was used as a reverse Northern blot hybridization control (Con). Probe (Probe) Alli823 was double-stranded synthesized by reverse transcription displacement of mRNA in BGC823 cells after allicin treatment cDNA (ds-cDNA); Probe (Probe) BGC823 is a double-stranded cDNA (ds-cDNA) synthesized by reverse transcription of parental BGC823 cell mRNA.

The best embodiment for realizing the invention:

1. Select two comparable cDNA libraries (such as drug-treated and untreated cell cDNA libraries or a certain organ tumor tissue and normal tissue cell cDNA library) as the object of gene expression difference analysis: such as the allicin treatment used in the study and untreated BGC823 cell cDNA library;

2. Choose a single restriction endonuclease or a combination of two endonucleases to digest the library DNA to select representatives. Endonuclease digestion should ensure the amount of information of the selected representatives, and at the same time, it should be noted that the endonuclease should not convert the carrier (Bacteriophage) DNA cuts out short fragments less than 800bp, and try to avoid the subsequent PCR competitive amplification of the vector DNA and the insert sequence; in this study, BamH I and Xho I double-enzyme digestion was used to select representatives, and the sequence of the library vector itself appeared differently after digestion. Four obvious band patterns of about 22kb, 11kb, 6kb and 850bp, the first three sequences are relatively large, and will not compete with the selected representative of the target sequence for PCR amplification, but can be used as an indicator of moderate endonuclease digestion;

3. Different cDNA library samples have different types, abundances and expression levels of differentially expressed genes. Therefore, it is necessary to select the ratio of the first round and the second round of subtractive hybridization according to the specific situation, so as to clone as much as possible low abundance, weak genes. Differentially expressed genes increase the amount of information in differential analysis results; this study used a subtractive hybridization ratio of detection DNA:driver DNA=1:20, and a second round of subtractive hybridization ratio detection DNA:driver DNA=1:200;

4. There should be good comparability between the difference products of the two samples in each round and between the difference products of the two rounds. The degree of PCR amplification should be based on the differential display of the sequencing gel to observe more clear difference bands. If the number of amplification cycles is too small, the differential information cannot be enriched to a sufficient degree, and if the number of amplification cycles is too high, the low-abundance differential information will be lost; Inconsistent, so the number of PCR amplification cycles needs to be adjusted to obtain the best differential analysis results; the number of PCR amplification cycles used in this study is 20 cycles;

5. On the basis of adjusting the trend comparability of product fragment groups, two rounds of differential products should be labeled with isotope α- ³⁵ S dATP by 1-2 cycles of PCR amplification; the template DNA is preferably 10ng, after 2 cycles 40ng of product can be obtained by PCR amplification of α- ³⁵ S dATP and the other four dNTPs (moles) according to which the ratio of α- ³⁵ SdATP: dNTP = 1: 30 is advisable; In order to increase the incorporation rate of α- ³⁵ S dATP on the basis of ensuring the integrity of the amplified fragment.

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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