CN1294279C

CN1294279C - Method for comparing expression amount of idential gene in different origin using base sequence determination method

Info

Publication number: CN1294279C
Application number: CNB2004100627512A
Authority: CN
Inventors: 周国华
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-04-05
Filing date: 2004-07-09
Publication date: 2007-01-10
Anticipated expiration: 2024-07-09
Also published as: CN1580286A

Abstract

The present invention relates to an analytical method for quantitatively comparing expression amount of identical gene in different origins, wherein the gene expression amount difference of the identical gene in different source tissues or cells are quantitatively compared, by adopting the quantitatively specific characters of bioluminescence method and the principle that different deoxyribonucleoside triphosphates (dNTP) are added therein one by one. The concrete steps are as follows: the messenger RNAs (mRNA) from different sources are inverse transcribing into cDNA, and a section source specificity DNA sequence for cDNA from each source is signed; the signed cDNA from different sources are mixed in a tube, as a substrate of a polyase chain reaction (PCR); a common primer and a gene specificity primer are used to execute PCR enlargement; bioluminescence analytical method is adopted to determine the base sequences, wherein the base species represent different sources of genes, and the signal intensity represents the expression amount of genes form each source. Said method exhibits great significance for disease related gene selecting, clinical early diagnosis and creation of specific medicines for treating diseases.

Description

Adopt the method for the expression amount of the more same gene of base sequence assay method in different sources

Technical field

The present invention relates to the method for the same gene of a kind of quantitative comparison relative expression quantity in the tissue of different sources or cell, is the method that a kind of method with the base order-checking is measured each dna fragmentation relative content in the dna fragmentation mixture of Different Alkali basic sequence mark simultaneously specifically.

Background technology

Progress along with molecular biology and analytical instrument, the examining order of the Human Genome Project is finished, now turn to functional analysis [1] from tectonic analysis, promptly understand the distribution of gene transcription product mRNA and amount, distribution situation and the effect of translation product protein in cell and organs and tissues of mRNA.Gene expression amount by healthier and disease between individual can be sought and find disease related gene, and then be used for clinical early diagnosis [2].In drug screening, measure the related gene expression amount of administration group and not administration group, can find the action target spot of medicine, seek the specifics [3] of initiative treatment disease.Therefore, the variance analysis of gene expression amount becomes one of the main task in " back order-checking epoch " gradually.Developed country has successively dropped into very big financial resources and material resources, strives seizing the commanding elevation in this field, carries out technical monopoly.The analytical procedure that is used for icp gene expression amount difference at present mainly contains SAGE method [4], RT-PCR method [5] and microarray method (being gene chip) [6] etc., but these methods still have some shortcomings, mainly show the difference that once can only compare two gene expression amounts between individuality, instrument costs an arm and a leg, complicated operation and quantitative poor performance etc.As the SAGE method, to measure quite cumbersomely, step is many, is difficult to grasp, and cost is also high, is difficult to popularize; The mensuration of RT-PCR method need be used instrumentation, and needs to use interior mark, measure cumbersome, poor repeatability; Little display method is a kind of high-throughout measuring method, though measure big in batches, can on chip piece, measure several genes simultaneously, but need to use the fluorochrome label sample, sensitivity is low, and instrument costs an arm and a leg, and data processing is also complicated, main problem is its quantitative poor performance, is difficult to accurately more a certain gene expression amount difference between different sources.

Adopting bioluminescence technique to measure base sequence is a kind of brand-new technology [7-8] that newly-developed gets up, and this method is easy fast, the instrument low price, and cost of determination is low, is easy to automatization.But owing to only can measure 10-30 base sequence, so this method only limits to the sudden change of analyzing gene and the polymorphism of gene [9].

Summary of the invention

Purpose of the present invention is exactly the method for the relative expression quantity of the same gene of technical study in different sources that adopts base sequence mensuration, promptly how measure the relative expression quantity of gene by the sequence of measuring several bases, set up good, cheap, the easy to operate method of a kind of highly sensitive, quantitative property, and be used for clinical diagnosis.

Technical solution of the present invention is as follows, and its measuring principle as shown in Figure 1.

(1) the same gene of base sequence method mark different sources

Have two kinds of methods to realize: first method is a DNA adapter labelling method, promptly extract total RNA or mRNA in different sources tissue or the cell earlier, and its reverse transcription become double-stranded cDNA, with restriction enzyme the cDNA in each source is cut into the dna fragmentation of different lengths again, but the cDNA enzymolysis product with each source is connected with the DNA adapter of distinguishing different respectively, makes the different DNA adapter of sequence on each cDNA mark of originating.The DNA adapter is made up of two incomplete complementary single stranded DNAs, its structure as shown in Figure 2, promptly an end contains and above-mentioned restriction enzyme otch complementary sequence 1, can be connected with double-stranded cDNA enzymatic fragment under the ligase enzyme effect; The other end is " Y " shape structure, by one section not

complementary base sequence

2 and 3 form, also sequence 3 can be designed to and sequence 2 complementations, but 3 ' end of this time series 3 in addition suitable modification make it and can not under the effect of polysaccharase, extension take place.Contain a fragment gene specific sequence 4 of originating in the sequence 2, and between 5 ' end of this section sequence and this chain, contain one section base sequence 5 that does not change with gene source.Only different gene source specific DNA adapter design can be become base sequence difference, and it is identical with number to form the base kind of this section sequence at sequence 4 places.

Second method is a reverse transcription primer mark method, promptly extracts total RNA or mRNA in different sources tissue or the cell earlier, and the primers different with sequence become cDNA with its reverse transcription respectively, make the different dna fragmentation of sequence on each cDNA mark of originating.The structure of reverse transcription primer as shown in Figure 3, it 3 ' holds promptly among the figure sequence 1 to be made up of a plurality of thymine alkali bases, between 3 ' end and 5 ' end, contain a fragment gene specific sequence 2 of originating, and between 5 ' of sequence 2 and this chain held, contain one section base sequence 3 that does not change with gene source.Only different gene source specificity reverse transcription design of primers can be become base sequence difference, and it is identical with number to form the base kind of this section sequence at sequence 2 places.

(2) the same gene fragment of PCR equal proportion amplification different sources

Usually by the expression amount of the goal gene that extracts in the tissue seldom, must pass through pcr amplification and just can detect.How the same gene at the above-mentioned different sources through mark of single tube moderate proportions amplification is one of gordian technique of this patent.At first according to gene-specific primer GSP of target gene sequences design; Design another general primer CP simultaneously, the sequence 3 among the sequence 5 among its sequence and Fig. 2 (with DNA adapter mark time) or Fig. 3 (with the reverse transcription primer mark time) is identical.In the presence of primer CP and GSP, if having goal gene in certain source, then GSP anneals at first with it and extension takes place, and primer CP and the annealing of this extension products are also extended, if there is not the extension products of GSP, primer CP can not extend.Because adopt the same gene fragment of a pair of universal primer CP and GSP amplification different sources, and the Tm value of amplified production identical (length is identical with the base kind), so can guarantee the equal proportion amplification of PCR.

If arm 3 and arm 2 complementations among Fig. 2, then arm 3 will extend under the effect of archaeal dna polymerase, produces for primer CP annealed template, so just can not realize the equal proportion pcr amplification of different sources gene.

(3) sequencing of different sources gene amplification product

Common sequencing reaction is based on the principle of gel electrophoresis at present, but this method belongs to the qualitative test sequence, quantitatively poor performance; And can not measure the sequence of primer back base, promptly not be suitable for the sequence of measuring preceding 50 bases.Bioluminescence assay based on PPi mensuration, as burnt sequencing, be to add each dNTP by circulating one by one successively to carry out sequencing reaction, not only can directly measure the base sequence of primer back, and quantitatively performance is fine, can determine to repeat the number of base by measuring peak height.The present invention adopts the sequencing of measuring based on PPi to measure the sequence of different sources gene amplification product, distinguishes the respectively gene in source according to the kind of base in the sequence, just can judge the gene expression amount difference of different sources according to peak intensity.

The genetic expression component analysis is the important content of genomics research, and the present invention is intended to sequencing technologies is used for the comparative analysis of gene expression amount difference.Compared with prior art, its novelty shows: just can record the expression amount difference of homologous genes in a plurality of Different Individual simultaneously by once analyzing, and need not increase extra cost of determination.Be easy to instrumentation, do not need to use laser, gel, fluorescent mark and electrophoresis.Highly sensitive, the quantitative property of the inventive method is good, cheap, easy to operate, has great application prospect.The inventive method also can be used for researching human body, animal or cell under methods such as medicine are disposed, each Expression of Related Genes situation to the screening of disease related gene, the specifics of clinical early diagnosis and initiative treatment disease is significant.

Description of drawings

Fig. 1 is a gene expression amount difference detection schematic diagram of the present invention.

Fig. 2 is the organigram of DNA adapter.

Fig. 3 is a reverse transcription primer organigram.

Sequencing result figure when Fig. 4 is the P53 gene of using in DNA adapter method labelling human cancer of the brain tissue, healthy tissues and the liver cancer tissue.

Sequencing result figure when Fig. 5 is the P53 gene of using among reverse transcription primer method labelling human liver cancer cell and the human bladder cancer cell.

Embodiment

Set forth above method with concrete example below, main experimental procedure is as follows:

(1) the same gene of sign different sources.Extract total RNA or mRNA in related tissue in each individuality or the cell respectively, and measure its concentration.According to DNA adapter labelling method or reverse transcription primer mark method, make different DNA adapter or the different dna fragmentations of sequence of sequence on each cDNA mark of originating then.When with DNA adapter labelling method mark, at first the mRNA reverse transcription is become double-stranded cDNA, cDNA is cut into the fragment of certain-length with the restriction enzyme that can discern four base sequences (Mob I), be attached thereto with the DNA adapter that contains the gene source specific sequence respectively and connect, and then will mix through the cDNA fragment of respectively originating of DNA adapter mark, as the template of pcr amplification reaction.When with reverse transcription primer mark method mark, with each corresponding reverse transcription primer of originating, the mRNA reverse transcription is become cDNA respectively, mix behind the purifying, as the template of pcr amplification reaction.

(2) pcr amplification.The dna profiling in (1) is carried out pcr amplification reaction with an irrelevant general primer (CP) and the gene-specific primer (GSP) of gene source.Owing to adopt the increase same gene in a plurality of sources,,, and can not change along with the increase of the number of times that increases promptly with identical ratio amplification so the relative proportion of this gene in each source remains unchanged in amplification procedure with a pair of primer CP and GSP.As will measure the expression amount difference of a plurality of genes in above-mentioned different sources respectively the time, only need change gene-specific primer GSP and get final product.

(3) the gene fragment amplification product of the different sources of quantitative assay simultaneously.After with microballoon technology or enzyme incision technology amplification PCR products being prepared into strand, adding with template sequence complementary universal primer and anneal.Also can add with template sequence complementary universal primer and anneal directly with behind the PCR product purification.Measure the sequence of base then with the noclilucence sequencing, promptly in containing the solution of substrate, add and corresponding dNTP of gene source or ddNTP respectively, as dNTP or ddNTP and the template complementation that is added, then discharge pyrophosphate salt (PPi), PPi can change into ATP rapidly under the effect of enzyme, ATP and luciferin and luciferase reaction produce optical signal.Base sequence among the gained result is being represented the different sources of gene, the gene expression amount in each source of strength of signal representative.According to the difference of each individual gene expression amount, can judge the function of gene and the functional gene of discovery and disease-related rapidly.

The mensuration of P53 gene expression amount difference in [embodiment 1] people's healthy tissues, cancer of the brain tissue and the liver cancer tissue.

This example has been narrated the relative expression quantity with P53 gene in three different sources tissues of DNA adapter labelling method marker determination.At first design three kinds of different DNA adapters, be connected with the cDNA fragment of crossing through digestion with restriction enzyme respectively, be mixed into the performing PCR amplification then.

1.cDNA the preparation of sample

(1) extraction of total RNA: get people's healthy tissues, cancer of the brain tissue and each 0.1g of liver cancer tissue, add 1ml Trizol and in tissue grinder, grind, extract total RNA according to the operation of Trizol specification sheets.After electrophoresis is identified its 28s and the intact nothing degraded of 18s band, measure its concentration with ultraviolet absorption spectroscopy, again with sterilization DEPC-H ₂It is 1 μ g/ μ l that O regulates final concentration.

(2) cDNA first chain is synthetic: Oligo (dt) ₁₆(100pmol/L) 1 μ l and total RNA (1 μ g/ μ l) 3 μ l behind 70 ℃ of 10min, place on ice, the first chain damping fluid, the 4 μ l that add 5 times of concentration, 0.1mol/L DTT 2 μ l, RNase inhibitor (40U/ μ l) 1 μ l, dNTP mixture (each 2.5mmol/L) 4 μ l, DEPC-H ₂O 4 μ l, 37 ℃ of 2min add Superscript II (200U/ μ l) 1 μ l, in 42 ℃ of 1h, 70 ℃ of 10min, cooled on ice.

(3) double-stranded cDNA's is synthetic: the second chain damping fluid, the 30 μ l that add 5 times of concentration in above-mentioned mixed liquid, dNTP mixture (2.5mmol/Leach) 12 μ l, Ecoli ligase enzyme (10U/ μ l) 1 μ l, dna polymerase i (4U/ μ l) 10 μ l, RNase H (2U/ μ l) 1 μ l adds DEPC-H ₂O is 150 μ l to cumulative volume, 16 ℃ of 2h, 70 ℃ of 10min.

2. identify the same gene of different sources

(1) endonuclease reaction: get double-stranded cDNA 10 μ l, add the damping fluid 2 μ l of 10 times of concentration, Mbo I TaKaRa restriction endonuclease (10U/ μ l) 1 μ l, sterile purified water 7 μ l, totally 20 μ l reaction systems place 37 ℃ of water-baths to react 2h, place 70 ℃ of 10min again, make Mbo I enzyme-deactivating.The characteristic of Mbo I restriction endonuclease be can discern among the DNA 5 ' → 3 ' the GATC order, and cut off and make it to form the sticky end that 5 ' distal process goes out GATC.

(2) ligation: the endonuclease reaction liquid of getting isopyknic each source, different with three kinds respectively DNA adapters links to each other, it is identical in three DNA adapters a chain adp-4 being arranged, contain four specific bases of gene source in another chain, these four bases only are the sequence differences, by c, t, g, c forms, and its sequence is respectively: adp-1:5 '-ccc cac ttc ttg ttctct cat GtcaCg cat cac tcg-3 '; Adp-2:5 '-ccc cac ttc ttgttc tct cat CtgaCg cat cac tcg-3 '; Adp-3:5 '-ccc cac ttc ttgttc tct cat AtcgCg cat cac tcg-3 '; Adp-4:5 '-gat ccg agt gatgcg cta ag-3 ', wherein the underscore italicized item is the specific base of gene source.Adp-1 and adp-4 constitute a DNA adapter 1, and adp-2 and adp-4 constitute DNA adapter 2, and adp-1 and adp-4 constitute another DNA adapter 3, and all have the structure that 5 ' distal process goes out four bases G ATC.

DNA adapter

1,2 and 3 is respectively applied for the P53 gene in labelling human healthy tissues, cancer of the brain tissue and the liver cancer tissue.Get enzyme and cut liquid 1 μ l, add each the 2 μ l of two strands (10pmol/L) that constitute the DNA adapter, 10 times of T4 dna ligase damping fluid 2 μ l, sterile purified water 11 μ l, place 70 ℃ of 10min, reduce to 16 ℃ with the speed of 0.2 ℃/s then, add T4DNA ligase enzyme (4U/ μ l) 2 μ l, reaction 2h.

3.PCR the preparation of amplification and strand

(1) pcr amplification: with the connection product of above-mentioned three different sourcess by 1: 1: 1 mixed in same reaction tubes, add general primer (CP respectively, 5 '-ccc cac ttc ttg ttctct cat-3 ') (10pmol/L) 2 μ l, the Auele Specific Primer of biotin labeled P53 gene (5 '-gga gca cta agc gag cac tg-3 ') is 2 μ l (10pmol/L), Mg ²⁺(25mmol/L) 3 μ l, dNTP Mixture (each 2.5mmol/L) 4 μ l, 10 * PCR Buffer5 μ l, TaKaRa Taq archaeal dna polymerase (5U/ μ l) 0.5 μ l adds sterile purified water to cumulative volume 50 μ l, carries out pcr amplification.The PCR reaction conditions is: 94 ℃ of 30s, and 60 ℃ of 30s, 72 ℃ of 30s react 35 circulations.The product that finally obtains is biotin labeled double-stranded DNA.

(2) preparation of strand: get 25 μ l M280 magnetic beads,, be dissolved in 2 * B﹠amp of 50 μ l according to the requirement washing of working instructions; Among the W Buffer (lavation buffer solution), add isopyknic PCR product, the reaction 30min, in the reaction process gently jolting make magnetic bead be in suspended state all the time.With the fixing magnetic bead of magnet, abandoning supernatant is with 1 * B﹠amp; Add 0.1mol/L NaOH solution 20 μ l after W Buffer washs 2～3 times, reaction 5min draws supernatant liquor to another test tube, and regulating the pH value with rare HCl is 6～7, stored refrigerated.Be dissolved in 1 * B﹠amp after the magnetic bead washing of solid phase; Preserve among the W Buffer, stand-by.

4. the relative expression quantity of the more same gene of base sequence assay method in the different sources tissue

Getting above-mentioned single stranded DNA sample (microballoon in the step 3) is prepared into and contains 25mM Mg ²⁺With the solution of 5mM Tris (pH7.7), and add the general primer CP of 5pmol respectively, naturally cool to room temperature behind 70 ℃ of heating 10min.Get in the sequencing standard mixed solution that 1～5 μ l joins 100 μ l, add dNTP then successively, carry out sequencing reaction.

Sequencing standard mixed solution consists of: 0.1M Tris-HAc (pH7.7), 2mM EDTA, 10mM Mg (Ac) ₂0.1% albumin (BSA), 1mM dithiothreitol (DTT) (DTT), 3 μ M, 5 '-phosphorylation sulfuric acid adenosines (APS), 0.4mg/ml polyvinylpyrrolidone (PVP), 0.4mM fluorescein, the 200mU/ml adenosine triphosphate sulfurylase, 2U/ml apyrase (apyrase), 1U do not have the archaeal dna polymerase Klenow of 5 prime excision enzyme activity, and an amount of luciferase.

5. measurement result

Because

DNA adapter

1,2 and 3 are respectively applied for the P53 gene in labelling human healthy tissues, cancer of the brain tissue and the liver cancer tissue, the representative of gained strength of signal derives from the gene expression amount of people's healthy tissues when adding dGTP, the representative of gained strength of signal derives from the gene expression amount of human brain cancerous tissue when adding dCTP, and the representative of gained strength of signal derives from the gene expression amount of people's liver cancer tissue when adding dATP α S (analogue of dATP).Sequencing result as shown in Figure 4, first base " C " of sequence shown in the figure comes from DNA adapter 2, represents P53 expression of gene amount A1 in the cancer of the brain tissue; Second base " G " comes from DNA adapter 1, represents P53 expression of gene amount A2 in the healthy tissues; The 3rd base " A " comes from DNA adapter 3, represent P53 expression of gene amount A3 in the liver cancer tissue, the ratio of peak of these three base sequences is being represented the expression amount difference of P53 gene in these three kinds of sources, twice measurement result (A1: A2: A3) be respectively: 28.20: 24.9: 46.9 and 28.1: 22.4: 49.5, mean ratio (A1: A2: A3) be: 28.15: 23.65: 48.2.

The mensuration of P53 gene expression amount difference in [embodiment 2] human liver cancer cell and the transitional cell bladder carcinoma cell line.

Present embodiment mainly is to adopt reverse transcription primer mark method to measure the expression amount difference of P53 gene in human liver cancer cell and transitional cell bladder carcinoma cell line, promptly, make the different dna fragmentation of sequence on each cDNA mark of originating with the mRNA of the different primer difference reverse transcription different sources of sequence.And contrast with the measurement result of RT-PCR.

1. the preparation of working sample

By the method for [embodiment 1], from human liver cancer cell and transitional cell bladder carcinoma cell line, extract total RNA respectively, after electrophoresis calibrating quality is intact, its concentration of ultraviolet determination, regulating final concentration with DEPC-H20 then is 1 μ g/ μ l.With reverse transcription primer P-1 and P-2 human liver cancer cell is become cDNA with mRNA reverse transcription in the transitional cell bladder carcinoma cell line respectively.The sequence of reverse transcription primer P-1 and P-2 is P-1:5 '-ccc cac ttc ttg ttc tct cat CagTtt ttt ttt ttt ttt-3 '; P-2:5 '-ccc cac ttc ttg ttc tct cat GacTtt ttt ttt ttt ttt-3 '.

Reactions steps is: get primer P-1 or P-2 (10pmol/L) 3 μ l and total RNA (1 μ g/ μ l) 3 μ l, behind 70 ℃ of 10min, place on ice, the first chain damping fluid, the 4 μ l that add 5 times of concentration, 0.1mol/L DTT 2 μ l, Rnase inhibitor (40U/ μ l) 1 μ l, dNTP mixture (2.5mmol/L each) 4 μ l, DEPC-H2O2 μ l, 37 ℃ of 2min add SuperScript ^TMII RNase H ^-ThermoScript II 1 μ l, in 42 ℃ of 1h, 70 ℃ of 10min, cooled on ice.After being purified, getting equal-volume and mix, as the template of PCR reaction.

2.PCR the preparation of amplification and strand

Carry out pcr amplification and preparation strand according to the method for [embodiment 1], wherein general primer CP is all identical with [embodiment 1] with gene-specific primer.

3. the relative expression quantity of the more same gene of base sequence assay method in the different sources tissue

Get above-mentioned single stranded DNA specimen preparation and become to contain 25mM Mg ²⁺With the solution of 5mM Tris (pH7.7), and add the general primer CP of 5pmol respectively, naturally cool to room temperature behind 70 ℃ of heating 10min.Get in the sequencing standard mixed solution that 1～5 μ l joins 100 μ l, add dNTP then successively, carry out sequencing reaction.

Because reverse transcription primer P-1 and P-2 are respectively applied for P53 gene in labelling human liver cancer cell and the transitional cell bladder carcinoma cell line, therefore the gained strength of signal is represented the gene expression amount that derives from human liver cancer cell when adding dCTP; The representative of gained strength of signal derives from human bladder cancer cell's gene expression amount when adding dGTP.

4. measurement result

Sequencing result as shown in Figure 5, first base " C " of sequence shown in the figure comes from reverse transcription primer P-1, the gene expression amount A1 of representative's liver cancer cell; Second base " G " comes from reverse transcription primer P-2, the gene expression amount A2 of representative's transitional cell bladder carcinoma cell line.The ratio of peak of these two base sequences is being represented the expression amount difference of P53 gene in these three kinds of sources, twice measurement result (A1: A2) be respectively: 82.9: 17.1,87.4: 12.6,84.2: 15.8,89.5: 10.5, mean value is: 86: 14=6.14: 1, and measurement standard deviation is 3.0: 3.0.

Measure the P53 gene with the RT-PCR method and be respectively 126359 copy/μ l and 22093/ μ l at the expression amount of liver cancer cell and transitional cell bladder carcinoma cell line, ratio is 5.72: 1.

Compare the measurement result of two kinds of methods, relative average debiation illustrates that less than 2% method measurement result of the present invention is more accurate.

Index in the literary composition:

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Claims

1, a kind of method that adopts the expression amount of the more same gene of base sequence assay method in different sources, it has following feature:

(a) make in the reverse transcription product complementary DNA (cDNA) of messenger RNA(mRNA) (mRNA) of different sources with the different dna fragmentation of sequence and contain one section source specific sequence;

(b) will be mixed in through the cDNA that respectively originates of mark in the pipe,, carry out pcr amplification with a general primer and a gene-specific primer as the substrate of polymerase chain reaction (PCR);

(c) measure in the above-mentioned pcr amplification product and the corresponding one section base sequence of gene source with biloluminescence method, represent the different sources of gene, gene expression amount in its each source of strength of signal representative with the base kind.

2, by the method for the expression amount of the more same gene of the described employing base sequence of claim 1 assay method in different sources, it is characterized in that described different sources is meant different tissues or cell.

3, press the method for the expression amount of the more same gene of the described employing base sequence of claim 1 assay method in different sources, it is characterized in that described dna sequence dna labelling method is meant with the mRNA of the different primer difference reverse transcription different sources of sequence, makes the different dna fragmentation of sequence on each cDNA mark of originating; The primer that described sequence is different be meant its 3 ' end form by a plurality of thymine alkali bases, 3 ' end and 5 ' contain between a holding fragment gene specific sequence of originating, and between 5 of this section sequence and this chain ' end, contain one section base sequence that does not change with gene source.

4,, it is characterized in that described general primer is meant that its sequence is identical with full sequence in the base sequence that does not change with gene source described in the claim 3 by the method for the expression amount of the more same gene of the described employing base sequence of claim 1 assay method in different sources.

5,, it is characterized in that described biloluminescence method is meant the method for the pyrophosphate salt (ppi) that the quantitative assay extension produces by the method for the expression amount of the more same gene of the described employing base sequence of claim 1 assay method in different sources.

6, press the method for the expression amount of the more same gene of the described employing base sequence of claim 5 assay method in different sources, it is characterized in that described extension is meant that with the pcr amplification product in the claim 1 or its single stranded product be template, adding sequencing primer anneals, add triphosphate deoxy-nucleotide (dNTP) one by one by a graded then, or triphosphoric acid dideoxy nucleotide (ddNTP), under the effect of archaeal dna polymerase, when adding dNTP or ddNTP and template complementation, the polyreaction that is taken place.