WO2004015137A1 - Methode de comparaison du niveau d'expression de genes - Google Patents

Methode de comparaison du niveau d'expression de genes Download PDF

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WO2004015137A1
WO2004015137A1 PCT/CN2003/000648 CN0300648W WO2004015137A1 WO 2004015137 A1 WO2004015137 A1 WO 2004015137A1 CN 0300648 W CN0300648 W CN 0300648W WO 2004015137 A1 WO2004015137 A1 WO 2004015137A1
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gene expression
gene
genes
primers
source
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Guohua Zhou
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Huadong Rearch Institute for Medicine and Biotechnics
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Huadong Rearch Institute for Medicine and Biotechnics
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Priority to EP03783905A priority Critical patent/EP1536022A4/en
Priority to US10/523,953 priority patent/US20070105097A1/en
Priority to AU2003255104A priority patent/AU2003255104A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the invention relates to a method for quantitatively determining the content of each DNA fragment in a mixture of DNA fragments, in particular to a method for simultaneously measuring the expression level of genes from various sources by using a bioluminescence method, which can be used for comparative analysis of the expression levels of genes from each source.
  • the first step of the genome project is to complete the structural analysis of the genome
  • the second step is to analyze the function of the gene, that is, to understand the distribution status of the gene transcription product mRNA and the mRNA translation product protein in the cells and organ tissues. And effect.
  • the function of unknown genes can be estimated, and the interactions between genes and proteins can be clarified [3] . Therefore, the analysis of gene expression has gradually become the main subject of DNA analysis.
  • the RT-PCR method first reverse transcribes mRNA into DNA, and then uses specific primers of each gene to perform PCR amplification reaction with polyadenine, and then separates each DNA fragment by electrophoresis, according to the depth of the electrophoretic band of each sample. Determine the expression of relevant genes and the expression of each gene. Although the sensitivity of this method is high, the repeatability is poor. Although the internal standard can be quantified, the quantification is not good because the linear relationship between the amount of PCR products and the amount of DNA substrate is poor, and the measurement results cannot reflect the actual gene expression.
  • Sequencing is a method of calculating gene expression by large-scale determination of the base sequence of cDNA.
  • the results of this method are accurate and reliable.
  • the former method uses a sequencer to determine the frequency of occurrence of cDNA sequences representing each gene in the sample. The workload is large and many expensive instruments are required. The latter method is an improved method.
  • Sequencing method First, use restriction enzymes to cut the cDNA fixed on the surface of the microspheres (beads) into fragments with viscous nicks, and then divide the DNA connected to the microspheres into two halves, and add them separately with viscous properties.
  • the nicks are connected to DNA A and B (denoted as Liker- A and Liker- B) and ligated, and then cut into 9-12 bases with ⁇ S endonuclease (labeling enzyme).
  • Fragments, labeled fragments are short nucleotide sequences used to identify genes, because type II S endonucleases can cut into specific lengths of labeled base fragments starting from specific digestion sites.
  • the two parts containing Linker-A and B were mixed and ligated with ligase. Add primer A (the part in Linker A) and primer B (the part in Linker-B) for PCR amplification.
  • the PCR product contains two gene-specific markers.
  • the above-mentioned endonuclease was used to cut the PCR product into cDNA fragments containing 4 base sticky nicks, and then cloned after ligation. Each clone contained 10 to 50 gene marker fragments, and one between each two markers contained four Short base fragments (specific sequences for endonucleases) are separated. Finally, the sequence of each clone was determined, and the expression amount of each gene was calculated based on the frequency of occurrence of each marker in the sequence.
  • Microarray method is to connect oligonucleotide fragments (20 ⁇ 30 bases) or cDNA on the surface of solid phase, and then extract and transcribe mRNA from biological samples into cDNA and then hybridize with it.
  • Source samples are hybridized, each sample is labeled with a different fluorophore, and the relative content of mRNA in two different source samples is compared by the difference in color of the hybridization point on the chip.
  • the disadvantages of this method are low sensitivity, poor quantification, special software is required to process the data, and the instrument is expensive.
  • Pyrosequencing is a bioluminescence method for determining DNA sequences, which can accurately determine only 10 to 30 base sequences, and is mainly used in SNP analysis [121] . This method has good quantitative performance, high sensitivity, simple operation, no electrophoresis, no labeling reaction, no laser source, no special or expensive reagents, and simple instruments.
  • Pyrosequencing is a sequencing technology based on the determination of pyrophosphate (PPi) by luminescence analysis. The principle of the determination is as follows:
  • a complementary dNTP Under the action of nucleic acid polymerase, if a complementary dNTP is added, the single-stranded MA annealed with the primer will undergo an extension reaction, releasing an equimolar amount of PPi; PPi is converted under the catalysis of adenosine triphosphate ⁇ T ⁇ acidase (ATP sulfurylase) It is adenosine triphosphate (ATP); under the catalysis of luciferase, ATP reacts with luciferin to emit light, which can be detected by photomultiplier tube (PMT) or charge coupled device (CCD). Since the intensity of the optical signal is directly proportional to the amount of PPi, the sequence of the target DNA can be determined based on the type and signal intensity of the added dNTP.
  • PMT photomultiplier tube
  • CCD charge coupled device
  • the purpose of the present invention is to solve the above-mentioned problems, and propose a method for comparative analysis of gene expression with high sensitivity, good quantification, low price, and convenient operation.
  • a method for comparative analysis of gene expression characterized in that it has the following characteristics: (a) The messenger ribonucleic acid (mRNA) from different sources is labeled with an appropriate method, mixed in equal amounts, and used as a polymerase chain reaction (PCR) Substrate (b) PCR amplification using primers corresponding to each source of the gene and gene-specific primers;
  • base types represent different sources of genes, and signal intensity represents gene expression in each source.
  • mRNA from different sources means different individuals of the same gene in the same biological species or different organs of the same gene in the same individual, such as mRNAs in a certain organ of a healthy person and a patient; it may also refer to the same gene in the same individual Gene expression under different chemical or physical stimuli. '
  • the "specific primers corresponding to each source” refers to a set of primers having different sequences but composed of the same kind and number of bases, and each kind of primer represents a source of a gene.
  • the "labeling with an appropriate method” refers to a method for distinguishing genes from various sources by using DNA fragments of an appropriate length.
  • the first method is to use reverse-transcription PCR (RT-PCR) to make mRNA from different sources into double-stranded complementarity.
  • RT-PCR reverse-transcription PCR
  • cDNA DNA
  • cDNA DNA
  • adapter selective adapter
  • different sources of mRNA are connected with different adapters
  • the second method The microspheres with polythymidine primers were respectively hybridized with mRNAs from different sources and made into first-strand cDNA.
  • the complementary strands (second-strand cDNA) were made with anchor primers containing 5 sequences at the ends corresponding to each source.
  • the anchoring primer 5 is used to distinguish the different sources of the gene; the third method: directly use the anchoring primer with a sequence corresponding to each source at the 5, and hybridize with the mRNA, and make the first strand
  • the construction of cDNA, anchored primer 5, and end are the same as the second method.
  • the present invention solves the problems that the existing methods for comparing mRNA expression levels between individuals have different shortcomings and are not easy to popularize.
  • the method mainly uses the principle of coke sequencing method to quantitatively measure and compare samples from different sources by using bioluminescence
  • the expression level of mMA can reach the purpose of finding genes related to the disease and used for clinical diagnosis.
  • the invention has the advantages of high sensitivity, good quantification, low price and convenient operation.
  • Fig. 2 is a schematic diagram showing the expression of genes in two bodies by enzymatic hydrolysis.
  • Figure 3 is a schematic diagram of the structure of a DNA adapter.
  • Figure 4 is a schematic diagram of a reaction module.
  • Figure 5 is a map of gene expression of two different sources by bioluminescence method.
  • Fig. 6 is a schematic diagram of an apparatus for simultaneously measuring multi-gene expression levels using a 96-well reaction plate.
  • Figure 7 is a schematic diagram comparing the average gene expression levels in two groups of mixed samples.
  • Fig. 8 is a diagram showing the results of measuring a sample in Example 1 using a PP i assay solution containing no adenosine triphosphate diphosphatase.
  • the method of the present invention consists of three steps: (1) Transcription and identification of cDNAs from various sources; (2) Mixing cDNAs from various sources in equal amounts as a template for PCR amplification; and (3) determining the sequence by bioluminescence.
  • the measurement principle is shown in Figure 1.
  • the first key point of the present invention is how to identify the cDNA of each source by an appropriate method before PCR amplification without affecting the PCR proportional amplification.
  • the second key point of the present invention is how to change the sequence in the result measured by the focal measurement method to information of each source and the signal intensity to the expression level of mRNA of each source.
  • the present invention achieves this objective by introducing several bases with different sequences into the cDNA (PCR amplification template) before PCR amplification, but the total number and type of the introduced bases are the same, only The sequences are different. In this way, the PCR reaction after mixing can achieve equal proportion amplification. Finally, a primer mixture corresponding to each cDNA source is added for sequencing reaction. Different sequences represent different sources, and the peak height represents the expression amount. The following uses examples to illustrate.
  • This example describes a method for restriction amplification of a cDNA fragment and ligating it with a DNA adapter, and then mixing the source-A and source-B cDNA in equal amounts for PCR amplification.
  • the measurement process is shown in Figure 2.
  • the human P53 gene was used as an example.
  • the mRNA was extracted using the standard method of Gibico TRI20LLS- Reagent TM kit.
  • cDNA samples of each source are prepared separately. Take 1 source-A or source-B mRNA samples, add 0, 5 ⁇ g 01igo (dT) 12 -is, 5.5 ⁇ ⁇ 2 0, and mix well Using a cDNA first-strand synthesis kit (Gibico Super Script TM Preampl if icat ion System for First Strand cDNA Synthesis), incubate at 10 ° C for 10 min, and then add a previously prepared mixed solution (4 ⁇ 5 times buffer) (I), 2 ⁇ of 0.1 M dithreitol, 1 ⁇ of 10 mM dNTPs, 2 ⁇ ⁇ 20 and 1 ⁇ of 200 U / ⁇ reverse transcriptase), incubate at 42 ° C for 50 min, and then Incubate at 70 ° C for 15 min. Store at 10 ° C after the reaction.
  • a cDNA first-strand synthesis kit Gibco Super Script TM Preampl if icat ion System for First Strand
  • I buffer 200 mM Tris-HCl (pH 8.5), 100 mM MgCl 2 , 10 mM DDT and 1000 mM KC1) 20 ⁇ , sterilized distilled water 30 ⁇ , the total amount is 150 ⁇ 1.
  • DNA adapters are used to distinguish gene expression from different sources.
  • a double-stranded cDNA was cut into a wedge-shaped structure with a four-base "ctag" protruding at the 5 'end by using Mbol restriction enzymes as described above.
  • the "Y" DNA adapter consists of two partially complementary strands.
  • the 5 'end arm of strand "a” is used to distinguish the origin of each gene.
  • Each 5' end arm contains the same number and type of bases. Base, but the alignment order is different; the 3 'end of the strand "b” is lifted up, that is, it is not complementary to the strand "a”.
  • the purpose is that during the PCR amplification reaction, the strand "b” does not undergo an extension reaction, and its 5' There are four prominent bases at the end, which are the restriction sites, the sequence is "gate", and the 3 'end is phosphorylated.
  • the DNA adapters-A and-B were used to identify the genes A and B respectively. The difference between them is mainly the 5 'end of the strand "a", and the rest of the sequences are the same.
  • P-1 5 '-ccc cac t tct tgt t etc teat cagg cgc ate act eg— 3'
  • P-2 be the "a" chain of adapter -B, the sequence is:
  • a source of the enzyme cDNA solution was added 10 pmol of P-1 and P- 3, was added 2 P- 10 pmol of the enzyme and P- 3 B source of cDNA solution. And contains Tris- HC1 at pH 7.6, 6.5 mM MgCl 2 , 0.5 mM ATP, 0.5 mM DDT and 2.5% polyethylene glycerol- 800, react at 70 ° C for 10 minutes, and then slowly cool to 16 ° C. T 4 DNA ligase was added and reacted at 16 ° C for 2 h. The ligation product is the substrate for the next PCR reaction.
  • the cDNA restriction enzyme fragments from sources A and B were mixed in equal amounts with the ligation product of the adapter to serve as a substrate for the PCR reaction.
  • the 5 'end of the adapter is used as the PCR reaction primer, that is, a mixture of primers (calculated at the 5' end) consisting of the first 21 bases in P-1 and P-2, and recorded as MP-1 and MP-2, respectively.
  • a sequence in the P53 gene was selected as a specific primer, denoted as GSP, and biotin was used at the 5 'end.
  • biotin label An equal amount of MP-1, MP-2 and GSP were mixed and used as PCR primers.
  • the substrate is 1 ⁇
  • the primers are lpmol
  • the buffer is 20 mM Tris-HCl, 50 mM KC1, 0.2 mM dNTPs, and 1.25 U DNA polymerase.
  • the PCR reaction conditions were: 94 ° C 30s, 58 ° C lmin, 72 ° C 30s, and 30 cycles of reaction.
  • the final product is biotinylated double-stranded DNA. Streptavidin-coated microspheres
  • reaction medium is 5 mM Tris-HCl, pH7.5, 0.5 mM EDTA, 1.0 M NaCl, remove the supernatant and add 0.1 M NaOH solution, react for 5 min, rinse, Store in microsphere wash buffer (5 mM Tris-HCl (pH7.5), 0.5 mM EDTA and 1.0 M NaCl) for later use.
  • This product is a single-stranded mixture of DNA from sources A and B.
  • composition of the standard mixture for PPi determination is: 0.1 M Tris-HAc (pH 7.7), 2 mM EDTA, 10 mMMg (Ac) 2 , 0.1% albumin (BSA), ImM dithiothreitol (DTT), 3 ⁇ 5 , -Phosphoryl adenosine sulfate (APS), 0.4 rag / ml polyvinylpyrrolidone (PVP), 0.4 mM fluorescein, 200 mU / ml adenosine adenosine sulfatase, 2U / ml adenosine triphosphate bisphosphatase (apyrase) , 1U DNA polymerase Klenow without exonuclease activity, and an appropriate amount of luciferase.
  • a measuring device for one sample is designed according to the figure.
  • the key part of the instrument of the present invention is a reaction module, as shown in FIG. 4.
  • two dNTP reservoirs are connected to each other through capillaries.
  • dNTP or ddNTP flows to the reaction tank through the bottom pipe (capillary).
  • the resulting fluorescence enters the detector through the bottom material.
  • the detector can be a photomultiplier tube, a photodiode or a charge coupled camera (CCD).
  • dGTP and dCTP are added to two dNTP reservoirs respectively, and a sample and a PP i measurement standard mixed solution are added to the intermediate reaction tank.
  • a syringe is used above the dGTP and dCTP reservoirs
  • the signal generated by the reaction under pressure is shown in Figure 5. Based on the signal intensity, the relative expression of the genes from the two sources can be calculated.
  • Example 2 Simultaneous determination of the expression levels of 96 different genes using a 96-well reaction plate: This example mainly uses an ordinary 96-well reaction plate to simultaneously measure the expression levels of 96 different genes between the disease group and the healthy group.
  • the mRNA of source A and source B was extracted and made into double-stranded cDNA, and then cut with the restriction enzyme Mob I.
  • Two kinds of DNA adapters representing source A and B were added and ligated with ligase After completion, add 1 ⁇ 5 ⁇ 1 of the ligated solution as a substrate for PCR amplification in each well of a 96-well reaction plate, and finally add MP-1, MP-2 and corresponding GSP primers for each gene. Expand The 5 ′ end of the GSP primer was biotin repaired.
  • Single-stranded DNA was prepared according to the same method as in Example 1. Then, the same amount of MP-1 and MP-2 were added to each of the 96 wells. The mixture was used as a primer for a sequencing reaction, and the experimental method was the same as that in Example 1.
  • Measuring device The key point of this example is to purchase and build an instrument that can measure 96 samples simultaneously. As shown in Figure 6, using a differential pressure injection method, two liquid dispensers were made with capillaries according to the structure of a 96-well reaction plate. Each dispenser has 96 capillaries as the dNTP force of each reaction well. head.
  • One end of the capillary is connected to the dNTP or ddNTP reservoir.
  • the reservoir is above the 96-well reaction plate.
  • the dNTP filling head of the liquid dispenser is extended into the reaction solution through a lifting device.
  • dNTP or ddNTP flows into the reaction cell for the incorporation reaction.
  • the PP i released by the reaction is converted into adenosine triphosphate (ATP) under the catalysis of ATP sulf ury lase, ATP, luciferin and luciferase. The effect emits fluorescence.
  • ATP adenosine triphosphate
  • the light signals emitted from 96 reaction wells were detected by a charge-coupled device (CCD) or a photoelectric signal tube (PMT) or a photodiode.
  • CCD charge-coupled device
  • PMT photoelectric signal tube
  • this device is also suitable for simultaneous determination of multiple samples of the same gene.
  • Example 3 Comparison of gene expression levels from six different sources:
  • the display chip can only measure the expression levels of genes from two different sources. If one source is added, a different fluorescent marker needs to be added, which increases the measurement cost, and usually does not use more than four at the same time. A fluorescent label with different excitation wavelengths. Since the present invention uses a sequencing method to measure the gene expression levels of different sources, and the measured base sequences represent different gene sources, increasing the sample source will not increase any measurement cost. Since dATP is an analog of ATP, it has a high background and severely interferes with the measurement. Although dATPas, an analog of dATP, can be used instead, the cost of the measurement increases. The invention avoids the use of dATP.
  • the three dNTP addition sequences in addition to dATP are: dTTP, dGTP, dCTP, dTTP, and dGTP, which are added 7 times, according to the height of the 6 peaks in the measurement spectrum
  • the gene expression amount represented by each peak was calculated.
  • the calculation method is to solve simultaneous equations, which are automatically completed by computer software, and finally the relative gene expression of each gene source is given.
  • the measurement equipment is the same as in Example 1 except that an additional dNTP reservoir is required. If multiple samples are to be measured, the equipment in (Example 2) may be used. However, a 96-well sampling head must be added because there are three types of dNTPs. Need to be added to the reaction cell.
  • the purpose of this example is to determine the difference in average gene expression between two groups, such as the healthy group and the disease group.
  • two groups contain 100 samples each.
  • the conventional method such as the gene chip method, requires 100 measurements, and then gives the results of each sample.
  • the computer software is used to analyze the measurement data to obtain statistical results.
  • samples from the healthy group and the disease group containing 100 human parts are respectively mixed into two groups in equal amounts, namely, the healthy group and the disease group, and then the relative genes between the two groups are determined according to the method of measuring samples from two different sources. The amount of expression.
  • the result is the average amount of gene expression in a mixed sample.
  • the gene expression of the two groups was correlated with the occurrence and development of the disease, and the genes associated with the disease were found.
  • the efficiency of finding disease-related genes has been improved by two orders of magnitude.
  • the chip method measures 100 samples, but this method only needs to be performed once, and the measurement results are more accurate.
  • the measurement method is shown in Fig. 7.
  • the key to determine the average gene expression in mixed samples is: (1) the PCR method The trace cDNAs in the samples from the two sources were amplified in equal proportions; (2) 1 hundred losses ⁇ : the output ⁇ has good quantitative characteristics.
  • the present invention can just meet these two requirements, so it can be used to compare the average gene expression in mixed samples from different sources, and its measurement speed and result reliability will exceed the chip method.
  • the sample was prepared by the microsphere method, that is, polythymine immobilized on the microspheres was used to hybridize with mRNA, and then cDNA synthesis was performed. After the first strand of cDNA was synthesized, 5 was added, and the gene specificity of the anchor sequence was added to the end.
  • the primers are used to synthesize complementary strands.
  • the additional anchoring base sequence at the 5 'end of the primer is used to distinguish the source of each cDNA. In this example, the additional anchoring sequences used to distinguish the two sources of different genes are respectively P in Example 1. -The first 23 bases of 1 and P-2 (counted from the 5 'end).
  • the DNA strand is separated from the microspheres and used as a template for PCR amplification.
  • a biotin-modified gene-specific primer (GSP) and an anchor sequence portion that distinguishes each source were used as primers for PCR amplification, and the rest was the same as in Example 1. .
  • Example 5 Standard method for PP i determination of gene expression in the absence of apyrase bisphosphate (apyrase) in solution:
  • an enzyme "adenosine triphosphate diphosphate enzyme” that can simultaneously degrade dNTP and ATP must be added, which is also the key technology of the pyrosequencing method.
  • the added dNTPs are three different dNTPs, and they do not interfere with each other. That is, when the latter dNTP is added, the former dNTP does not add. Further extension reactions will occur; in addition, the ATP generated in the reaction solution does not need to be degraded, because the linear range of ATP measured by the luciferin method is very wide.
  • the ATP produced by the ATP The signal strength can still be controlled within a linear range.
  • Example 1 the sample in Example 1 is measured using a PP i measurement solution that does not contain adenosine triphosphate diphosphatase, and the result shown in FIG. 8 is obtained, which illustrates that it is feasible to add no adenosine triphosphate diphosphatase.
  • FIG. 8 the result shown in FIG. 8 is obtained, which illustrates that it is feasible to add no adenosine triphosphate diphosphatase.

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Description

基因表达量比较分析法 技术领域
本发明涉及一种定量测定 DNA片段混合物中各 DNA片段含量 的方法， 具体说是一种用生物发光法同时测定各来源基因表达量 的方法， 可用于各来源基因表达量的比较分析。
背景技术
近年来随着分子生物学的进步， 已测出几十种生物的全基因 组顺序，人类基因组计划也即将完成 [1' 2]。基因组计划的第一步是 完成基因组的构造解析， 第二步是进行基因功能解析， 即了解基 因转录产物 mRNA的分布状态及 mRNA的翻译产物蛋白质在细胞及 脏器组织中的存在量、 分布情况和作用。 通过比较基因转录产物 mRNA的表达量就可以推定未知基因的功能、解明基因间及蛋白质 间的相互作用 [3)。 因此基因表达量的分析已逐渐成为 DNA分析的 主要课题， 在临床医学中， 通过比较基因在不同个体 （健康和疾 病）（脑、 肝或心脏） 中的表达量， 可以寻找和发现疾病基因， 进而创造可治疗疾病的特效药， 在疾病预防中因为肿瘤、 糖尿病 和肥胖病等是多基因疾病， 用通常的方法很难做到及时诊断， 但 通过测定与疾病相关的基因在有关组织器官中的表达谱就早达 到及可预见患病的危险性， 以达预防的目的。 此外在分子生物学 研究中， 通过基因表达量的分析， 可以寻找功能基因， 为生物品 种的改良提供依据。 到目前为止可用于基因表达量的方法有： Nor thern 印迹法" ]、 RT-PCR 法[5]、 测序法 "' 7]、 微阵列法 ' 9' 1" ( mi croa r r ay ) 等。
Nor t hern 印迹法是经典的方法， 主要用于数个或数十个基 因的表达量分析。 但该法测定极其繁锁， 操作人员必须经过特殊 训练， 且需使用放射性物质， 对人和环境有危害， 测定效率不高。
1
替换页（细则第 26条) 此外该法的灵敏度也很低， 不能检出微小的基 表迟重。
RT-PCR法是先将 mRNA反转录成 DNA后， 用各基因的特异性 引物与多聚腺嘌呤进行 PCR扩增反应， 然后用电泳分离各 DNA片 段， 根据各样品的电泳条带的深浅判断有关基因表达情况和各基 因表达量。 该法灵敏度虽高， 但重复性差， 虽然内标可以定量， 但定量性不好，因为 PCR产物量与 DNA底物量之间的线性关系差， 测定结果不能很好地反映实际基因表达情况。
测序法是通过大规模测定 cDNA 的碱基序列而计算基因表达 量的方法。 主要有整体测序法 （Body Mapp ing法） 和基因表达系 歹1 J分析法 ( Ser i a l Ana l ys i s of Gene Expres s i on , 筒称 SAGE 法）。 该法结果准确可靠， 前一种方法是用测序仪分别测定代表 各基因的 cDNA序列在样品中的出现频率， 工作量很大， 且需要 许多昂贵的仪器； 后一种方法， 是经改进的测序法， 即先用限制 性内切酶把固定于微球 ( bead ) 表面的 cDNA 切成带有粘性切口 的片段， 然后， 将与微球相连的 DNA分成二等分， 分别加入带有 粘性切口的连接 DNA A和 B (记为 L iker- A 和 L iker- B) , 并使 之连接， 然后再用 Π S 内切酶 （标记酶） 切成带有 9 - 12个标记 碱基的片段， 标记片段是用于鉴别基因的短核苷酸序列， 因为 II S型内切酶能从特异性酶切位点开始切割成一定长度的标记碱基 片段。 将含有 Linker-A和 B的两部分混合， 并用连接酶连接。 加入引物 A ( L inker A中的部分）和引物 B ( Linker-B中的部分 ) 进行 PCR扩增， PCR产物中含有两个基因特异性标记。 再用上述 内切酶将 PCR产物切割成含有 4个碱基粘性切口的 cDNA片段， 连接后进行克隆， 每个克隆中含有 10 ~ 50 个基因标记片段， 每 两个标记之间有一个含有四个碱基的短片段 (内切酶的特异性序 列） 相隔。 最后测定各克隆的序列， 根据各标记在序列中的出现 的频率计算各基因的表达量。 该法虽然不需分别测定各 cDNA 的 序列， 但工作量仍然很大， 且操作较为繁锁， 需要使用价格昂贵 的测序仪， 普通实脸室无法做到， 非常规测定方法。
微阵列法是在固相表面上连接寡聚核苷酸片段 （ 20~ 30 个 碱基）或 cDNA， 将生物样品中的 mRNA提取转录成 cDNA后与之杂 交， 同一块芯片可与两种不同来源的样品杂交， 每一样品标记有 不同荧光基团， 通过芯片杂交点颜色的差异比较两种不同来源样 品中 mRNA 的相对含量。 该法的缺点是灵敏度低， 定量性差， 需 要专门软件处理数据， 且仪器价格昂贵。
焦测序法[11]是一种采用生物发光的方法测定 DNA序列，仅能 准确测定 10 ~ 30个碱基序列， 主要用于 SNP分析中 [121。 该法定 量性能好， 灵敏度高， 操作简便， 不需电泳， 不需标记反应， 测 定时不需激光源，也无需使用特殊或昂贵的试剂，所需仪器简单。 焦测序法是一种基于用发光分析法测定焦磷酸盐 （PPi ) 的测序 技术， 测定原理如下：
在核酸聚合酶的作用下， 如果加入互补的 dNTP, 与引物退火 的单链 MA就发生延伸反应， 释放出等摩尔量的 PPi; PPi在三 磷酸腺苷^ Τυ酸化酶 （ATP sulfurylase ) 的催化作用下转化为三 磷酸腺苷 （ATP); 萤光素酶 （ luciferase) 催化作用下， ATP与 虫萤光素 （ luciferin) 反应发光， 可用光电倍增管 （ PMT ) 或电 荷耦合装置 （CCD) 检测。 由于光信号强度与 PPi 的量成正比， 所以根据加入的 dNTP种类和信号强度就可确定靶 DNA的序列。
但该法不能直接用来比较各来源的基因表达量。
发明内容
本发明的目的就是为了解决上述问题， 提出一种灵敏度高、 定量性好、 价格低廉、 操作方便的基因表达量比较分析法。
本发明的技术解决方案：
一种基因表达量比较分析法， 其特征在于它具有如下特征： (a) 将不同来源的信使核糖核酸 （mRNA) 用适当的方法标 记后， 等量混合， 作为聚合酶链反应 （PCR) 的底物； ( b ) 用与基因各来源相对应的引物和基因特异性引物进行 PCR扩增；
( c ) 用生物发光分析法测定序列， 以碱基种类代表基因的 不同来源， 信号强度代表各来源中基因表达量。
所述 "不同来源的 mRNA"是指同一基因在同一生物种的不同 个体或同一基因在同一个体的不同器官， 如健康人和病人某一器 官中的 mRNA;也可指同种个体中的同一基因在不同的化学或物理 刺激下产生的表达。 '
所述 "与各来源相对应的特异性引物" 是指一组序列不同， 但由相同种类和数目的碱基所组成的引物， 每一种引物代表一种 基因的来源。
所述 "用适当的方法标记" 是指用适当长度的 DNA 片段来 区别各来源基因的方法， 第一种方法： 将不同来源的 mRNA 用反 转录 PCR (RT-PCR) 法制成双链互补 DNA (cDNA) 后， 用限制性 内切酶将之切成适当长度的片段，用选择性适配器 （adap t e r) 与 之相连接， 不同来源的 mRNA连有不同的 adap t e r ; 第二种方法： 将带有多聚胸腺嘧啶引物的微球分别与不同来源的 mRNA 杂交， 并制成第一链 cDNA ,用 5，端含有与各来源相对应序列的锚定引物 进行互补链 (第二链 cDNA) 的合成， 锚定引物的 5，端用于区别基 因的不同来源； 第三种方法： 直接用 5，端含有与各来源相对应序 列的锚定引物与 mRNA杂交， 并制成第一链 cDNA , 锚定引物 5，端 的构造与第二种方法相同。
本发明解决了现有用于比较个体之间的 mRNA 表达量的方法 均存在不同的缺点及不易推广使用的问题， 主要借助于焦测序法 的原理， 用生物发光的方法定量测定和比较不同来源样品中的 mMA表达量，达到寻找与疾病相关基因的目的，并用于临床诊断。 本发明具有灵敏度高、 定量性好、 价格低廉、 操作方便的优点。 附图说明 图 1是本发明的测定原理图。
图 2是酶解法测定两个体中的基因表达量示意图。
图 3是 DNA适配器的构成示意图。
图 4是反应模块示意图。
图 5是生物发光法测定两个不同来源基因表达量的图谱。 图 6是使用 96 -孔反应板同时测定多基因表达量仪器装置示 意图。
图 7是两组混合样品中平均基因表达量的比较示意图。
图 8是用不含有三磷酸腺苷双磷酸酶的 PP i测定溶液测定实 施例 1中的样品结果示意图。
具体实施方式
下面结合附图对本发明作进一步说明。
本发明方法由三步组成： （ 1 ) 各来源 cDNA 的转录和标识； ( 2 ) 等量混合各来源的 cDNA, 并作为 PCR的扩增模板； （ 3 ) 用 生物发光法测定序列。 测定原理如图 1所示。
本发明第一个关键点是如何在 PCR扩增前用适当的方法标识 各来源的 cDNA , 而又不影响 PCR等比例扩增。 有几种技术可实现 这一方案： （ 1 )将 mRNA反转录成双链 cDNA后， 用限制性内切酶 消化成若干片段， 再用与可识别各 cDNA来源的 DNA适配器连接， 各 DNA适配器由相同碱基组成， 但序列不同， 连接后将各来源的 cDNA样品等量混合后， 进行 PCR扩增； （ 2 )用固定在微球上的多 聚胸腺嘧啶与 mRNA杂交， 然后再进行 cDNA合成， cDNA第一条链 合成完后， 加入 5， 端含有锚定序列的基因特异性引物进行互补 链的合成， 引物 5'端的附加锚定碱基序列用于区别各 cDNA 的来 源。 cDNA合成完后， 将该 DNA链与微球分离， 并作为 PCR扩增的 模板。 最后用基因特异性引物和区别各来源的锚定序列部分作为 引物进行 PCR扩增； （ 3 ) 用 5， 端带锚定碱基序列的基因特异性 引物与 mRNA杂交并进行 cDNA第一条链的合成， 其中引物中的锚 定碱基序列部分与基因各来源相对应， 各来源半链 cDNA 斧重 合后， 进行 PCR扩增， PCR扩增的引物为锚定碱基序列中用于区 别基因各来源的部分。
本发明第二个关键点是如何将焦测法所测结果中的序列变为 各来源的信息， 信号强度变为各来源 mRNA 的表达量。 本发明是 通过如下方法来实现这一目的的， 即在 PCR扩增前，在 cDNA(PCR 扩增模板） 中引入序列不同的数个碱基， 但所引入的碱基总数和 种类相同， 仅序列不同而已， 这样混合后进行 PCR反应可实现等 比例扩增， 最后加入与各 cDNA来源相对应的引物混合物进行测 序反应， 不同序列即代表不同的来源， 峰高代表表达量。 以下用 例子来说明。
实施例 1, 两个体中基因表达量的比较：
本例叙述了用限制性内切酶法切割 cDNA 片段后与 DNA适配 器连接， 然后等量混合来源- A和来源 -B的 cDNA进行 PCR扩增的 方法， 其测定过程如图 2所示。 以人 P53基因为例进行了说明， 其中 mRNA的提取采用 Gibico TRI20LLS- Reagent™试剂盒的标准 方法。
1. cDNA样品的制备 （各来源的 cDNA样品分别制备） 取 1 来源- A 或来源 - B 的 mRNA 样品， 加入 0, 5 μg 01igo(dT) 12-is , 5.5 μΐ Η20, 并混合均匀， 采用 cDNA第一链合 成试剂盒 ( Gibico Super Script™ Preampl if icat ion System for First Strand cDNA Synthesis ), 在 0°C下保温 lOmin后， 加入预先制备好的混合液 （ 4 μΐ 的 5 倍缓冲液 （ I ), 2 μΐ 的 0.1 Μ 二^苏糖醇， 1 μΐ的 10 mM dNTPs, 2 μΐ Η20和 1 μΐ 的 200 U/μΙ反转录酶）， 于 42°C下保温 50min， 之后在 70°C下保温 15min, 反应后保存于 10Ό。
在上述反应混合液中进一步加入 10 μΐ 10倍连接酶緩冲液， 70 μΐ Η20, 1 μΐ 1 mM dNTPs 混合液， 50U 聚合酶 I， 10U DNA 连接酶， 搅拌后加入 2U 核糖核酸酶 RNase H, 混勾。 在 10°( 下 保温 2hr, 然后于 70°C下保温 15min, 该混合物即为双链 cNDA 样品， 于- 20°C下冷冻保存。
取上述制备好的 cDNA溶液 150 μΐ, 加入 10倍限制性内切酶 6。 I緩冲液 （ 200 mM Tris-HCl (pH8.5) , 100 mM MgCl2, 10 mM DDT和 1000 mM KC1 ) 20 μΐ, 灭菌蒸馏水 30 μΐ, 总量为 150 μ1。 加入 60 U的限制性内切酶 Mbo I， 在 37°C的条件下保温反应一 夜， 反应完后， 于 75°C的条件下， 保温 15min, 使 1酶失活。
2. DNA适配器的制备
本例中， 使用 DNA适配器来区别不同来源的基因表达量。 首 先按上述方法用 Mbol限制性内切酶将双链 cDNA切割成 5'端突出 四个碱基 "ctag" 的楔型结构。 然后将 DNA适配器与之相连接。 在图 3中， "Y" 型 DNA适配器由 2条部分互补的链组成， 链 "a" 的 5'端臂用以区别各基因的来源， 每个 5'端臂含有相同数目和种 类的碱基， 但排列序引不同； 链 "b" 的 3'端翘起， 即与链 "a" 不互补， 目的是在 PCR扩增反应时， 链 "b" 不会发生延伸反应， 其 5'端有四个突出的碱基， 为酶切位点， 序列为 "gate", 3'端磷 酸化。
分别用 DNA适配器 - A和- B来识別基因来源 A和来源 B,它们 的区别主要是链 "a" 的 5'端部分， 其余序列相同。
设 P- 1为适配器- A的 "a" 链， 其序列为：
P-1： 5' - ccc cac t tct tgt t etc teat cagg cgc ate act eg— 3' 设 P-2为适配器 -B的 "a" 链， 其序列为：
P-2: 5'-cac etc teat ttct ccc tgtt gacg ogc ate act cg-3' 适配器 -A和- B链的 "b" 链相同， 用 P-3表示， 其序列为：
P— 3: 5'-gatccg agt gat gcg ct aag-3'
在来源 A的酶解 cDNA溶液中， 加入 10 pmol的 P-1和 P- 3, 在来源 B的酶解 cDNA溶液中加入 10 pmol的 P- 2和 P- 3。 并含有 pH7.6的 Tris- HC1, 6.5 mM MgCl2, 0.5 mM ATP, 0· 5 mM DDT和 2.5%聚乙烯甘油- 800, 在 70°C的条件下， 反应 10 min, 然后慢 慢冷却到 16°C。 加入 T4 DNA连接酶， 在 16°C下反应 2h。 连接产 物为下一步 PCR反应的底物。
3. PCR扩增 cDM限制性酶切片段及单链的制备
将来源 A和 B的 cDNA限制性内切酶片段与适配器的连接产 物等量混合， 作为 PCR反应的底物。 以适配器 5'端部分为 PCR反 应引物， 即由 P-1和 P- 2 中的前 21个碱基组成的引物 （以 5'端 计算） 混合物， 并分别记为 MP- 1和 MP- 2。 另外选取 P53基因中 的一段序列作为特异性引物， 记为 GSP, 并且在 5'端用生物素
( biotin) 标记。 取等量的 MP- 1、 MP-2 和 GSP 混合后作为 PCR 引物。在 10 μΐ PCR反应混合物中，底物为 1 μΐ,引物均为 lpmol, 緩冲液为 ρΗ8.0的 20 mM Tris-HCl, 50 mM KC1, 0.2 mM dNTPs 和 1.25 U DNA聚合酶。 PCR反应条件为： 94°C 30s, 58°C lmin, 72°C 30s, 反应 30 个循环。 最终得到的产物是生物素标记的双 链 DNA。 将其与抗生物素蛋白链菌素 （ streptavidin) 涂层微球
( Dynabeads M280 ) 于室温下反应 30 min, 反应介质为 5 mM Tris-HCl, pH7.5, 0.5 mM EDTA, 1.0 M NaCl, 除去上清后加入 0.1 M NaOH 溶液， 反应 5 min, 冲洗干净后， 在微球冲洗緩冲 液 （ 5 mM Tris-HCl ( pH7.5 ), 0.5 mM EDTA 和 1.0 M NaCl ) 中 储存备用。 该产物为来源 A和 B的 DNA单链混合物。
4. 生物发光法测定各来源基因表达量
取上述单链 DNA样品（步骤 3中的微球）制备成含有 25 mM Mg2+ 和 5 mM Tris ( H 7.7 ) 的溶液, 并分另 'J力口入 5 praol 的 MP— 1和 MP-2, 94°C加热 2min后自然冷却至室温。取 1 ~ 5 μΐ加入到 50 ~ 100 μΐ的 PPi测定标准混合液，依次加入 dGTP和 dCTP,或 ddGTP 和 ddCTP , 或它们的类似物， 进行测序反应。 加入 dGTP时所得信 号强度代表来源 A的基因表达量， 加入 dCTP 时所得信号强度代 表来源 B的基因表达量。
PPi测定标准混合液组成为： 0.1 M Tris- HAc ( pH 7.7 ), 2 mM EDTA, 10 mMMg(Ac)2, 0.1%白蛋白（ BSA ), ImM二硫苏糖醇（ DTT ), 3 μΜ 5， -磷酸化硫酸腺苷（APS) , 0.4 rag/ml 聚乙烯吡咯烷酮 (PVP), 0.4 mM荧光素， 200 mU/ml三碑酸腺苷硫酸化酶， 2U/ml 三磷酸腺苷双磷酸酶 ( apyrase ), 1U无外切酶活性的 DNA聚合酶 Klenow, 及适量荧光素酶。
5. 测定仪器装置
按图设计可供一个样品的测定装置， 本发明的仪器关键部分 为反应模块， 如图 4所示。 在图中正中间的反应池底部通过毛细 管分别连接两个 dNTP贮液槽， 当 dNTP贮液槽上方施以一下压力 时， dNTP或 ddNTP就通过底部的管道（毛细管）流向反应池。 反 应产生的荧光透过底部材料进入检测器。 检测器可以用光电倍增 管、 光电二极管或电荷耦合照相机 （ CCD )。
6. 测定结果
在图 4的反应的模块中，分别加入 dGTP和 dCTP于两个 dNTP 贮液槽中， 将样品和 PP i测定标准混合液加入中间反应池中， 分 别在 dGTP和 dCTP贮液池的上方用注射器加压， 反应产生的信号 如图 5。 根据信号强度， 可以计算出两个来源基因的相对表达量。
实施例 2，用 96-孔反应板同时测定 96个不同基因的表达量： 本实施例主要是采用普通 96-孔反应板， 同时测定疾病组与 健康组间的 96个不同基因的表达量。
1. 测定样品的制备
按实施例 1的方法， 提取来源 A和来源 B的 mRNA, 并制成双 链 cDNA, 然后用限制性酶 Mob I切割， 再加入两种分别代表来源 A和 B的 DNA适配器， 用连接酶连接好后， 分别于 96-孔反应板 中的每一个孔中， 加入 1 ~ 5μ1 连接好的上述溶液作为 PCR扩增 的底物， 最后加入 MP- 1、 MP-2和相应的各基因 GSP 引物进行扩 增反应， GSP 引物的 5 ' 端用生物素修 ， 按买施例 1相同的万 法制备单链 DNA , 然后在 96 孔中的每一个小孔中加入相同量的 MP - 1和 MP - 2混合物作为测序反应的引物， 实验方法同实施例 1。
2. 测定装置 本实施例的关键点在于购建可以供 96 个样品同时测定的仪 器装置。 如图 6所示， 采用压差进样法， 根据 96-孔反应板的结 构用毛细管制作两个液体分送器， 每个分送器有 96 根毛细管作 为每个反应孔的 dNTP力 p注头。
毛细管的一端与 dNTP或 ddNTP 贮液池相连， 贮液池在 96- 孔反应板的上方， 测定时， 通过升降装置将液体分送器的 dNTP 加注头伸入反应液中， 在压差的作用下， dNTP或 ddNTP流入反应 池中进行掺入反应，反应释放的 PP i在三磷酸腺苷硫酸化酶（ ATP s u l f ury l a s e )的催化作用下转化为三磷酸腺苷（ ATP ) , ATP与荧 光素及荧光素酶作用发出荧光。 用电荷耦合装置 （CCD ) 或光电 信增管 （PMT ) 或光电二极管检测 96个反应孔中发出的光信号。 当然本装置还适用于同一基因的多个样品的同时测定。
实施例 3 , 六个不同来源基因表达量的比较：
通常采用^ f敫陈列芯片仅能测定两种不同来源基因的表达量， 如果增加一种来源， 则需要增加一种不同的荧光标记物， 从而使 测定成本增加， 且通常同时不会使用超过四种激发波长不同的荧 光标记物。 由于本发明采用测序法测定不同来源的基因表达量， 测定的碱基序列代表不同的基因来源， 所以增加样品来源， 不会 增加任何测定成本。 由于 dATP是 ΑΤΡ的类似物， 背景高， 对测定产生严重干扰， 虽然可用 dATP的类似物 dATPas代替， 但测定成本增加。 本发明 避免使用 dATP。 当用本法比较两个不同来源基因表达量时， 分别 在 P-1和 P-2中采用 " cag" 和 " gac" 来控制。 因为最多可以加 入 "g"、 "c" 和 "t" 三种 dNTP来测序， 所以通过加入早个 dNTP 的办法最多只能测定三种不同来源的基因表达量。 由于本发明采 用的 PPi测定法具有很好的定量特性， 故在 DNA适配器的中间部 分 （PCR扩增引物后） 设计如下六种不同的序列用以控制基因的 不同来源， 每一个序列的第 4个碱基为 "T", 用于控制减基的进 一步延伸。
( 1 ) cgat; ( 2 ) gcat; ( 3 ) agct;
( 4 ) gact; ( 5 ) cagt; ( 6 ) acgt; 除 dATP外的三种 dNTP加入顺序为： dTTP、 dGTP、 dCTP, dTTP 和 dGTP， 共加入 7次， 根据测定图谱中 6个峰的高低计算出各峰 所代表的基因表达量。 计算方法为解联立方程， 由计算机软件自 动完成， 最终给出各基因来源的相对基因表达量。
测定仪器装置同 （实施例 1 ), 但需增加一个 dNTP贮液池， 如测定多个样品可采用 （实施例 2 ) 中的仪器装置， 但必须增加 一个 96孔加样头， 因为有三种 dNTP需加入到反应池中。
实施例 4, 混合样品中平均基因表达量的比较：
本例的目的是测定两组之间， 如健康组和疾病组， 基因平均 表达量的差异。设两组各含有 100个样本，常规法如基因芯片法， 需测定 100次， 然后给出每个样品的测定结果， 最后用计算机软 件对测定数据进行分析， 得出统计结果。 本发明将含有 100个人 份的健康组和疾病组的样品分别等量混合成两大组， 即健康组和 疾病组， 然后按照测定两个不同来源样品的方法， 测定两组之间 的相对基因表达量， 该结果为混合样品中的基因平均表达量。 然 后将两组的基因表达量与疾病的发生、 发展相关联， 找出与疾病 相关联的基因。 与基因芯片测定法相比， 寻找疾病相关基因的效 率提高了两个数量級， 芯片法要测定 100个样本， 而本法仅需一 次， 并且测定结果更为准确。 测定方法如图 7所示。
测定混合样品中平均基因表达量的关键是： （ 1 )用 PCR法将 两种来源样品中的微量 cDNA 进行等比例扩增； ( 2 ) 1百亏^：出^ 具有很好的定量特性。 本发明正好能满足这两点要求， 所以可用 于比较不同来源混合样品中的平均基因表达量， 其测定速度和结 果可靠性将超过芯片法。
样品制备采用微球法， 即用固定在微球上的多聚胸腺嘧啶与 mRNA杂交， 然后再进行 cDNA合成， cDNA第一条链合成完后， 加 入 5， 端含有锚定序列的基因特异性引物进行互补链的合成， 引 物 5 '端的附加锚定碱基序列用于区别各 cDNA的来源， 在本例中， 用于区别两种基因不同来源的附加锚定序列分别为实施例 1 中 P- 1和 P- 2的前 23个碱基（从 5'端计算）。 cDNA合成完后， 将该 DNA链与微球分离， 并作为 PCR扩增的模板。 最后用生物素修饰 的基因特异性引物（GSP)和区别各来源的锚定序列部分（同实施 例 1 中的 MP-1和 MP- 2)作为引物进行 PCR扩增，余下部分同实施 例 1。
实施例 5， PP i 标准测定溶中不加入三磚酸腺苷双磷酸酶 ( a pyr a s e ) 时的基因表达量测定法：
在焦测序法中， 因为需要得到连续的测定序列， 所以必须加 入一种可同时降解 dNTP和 ATP的酶 "三磷酸腺苷双磷酸酶"， 这 也是焦测序法的关键技术。 本发明中测定两个或三个不同来源的 基因表达量时， 加入的 dNTP为三种不同的 dNTP , 相互不会产生 干扰， 即当加后一种 dNTP时， 前一种已加入的 dNTP不会发生进 一步的延伸反应； 另外反应溶液中产生的 ATP也没有必要降解， 因为用虫萤光素法测定 ATP 的线性范围很宽， 当加入另外一种 dNTP或两种 dNTP时， 由 ATP产生的信号强度仍可控制在线性范 围内。
本实施例用不含有三磷酸腺苷双磷酸酶的 PP i测定溶液测定 实施例 1 中的样品， 得到图 8的结果， 说明不加三磷酸腺苷双磷 酸酶是可行的。 文中标引：
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3 Matsubara, K.， and K. Okubo. 1993. cDNA analyses in the human genome project. Gene 135: 265-74.
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5 Karet, RE., D. S. Charnock- Jones, M. L. Harrison- Woolrych, G. O'Reilly, A. P. Davenport, and S. K. Smith. 1994. Quantification of mRNA in human tissue using fluorescent nested reverse-transcriptase polymerase chain reaction. Anal. Biochem. 220: 384-90.
6 Velculescu, V. Ε·, L. Zhang, B. Vogelstein, and K. W. Kinzler. 1995.
Serial analysis of gene expression. Science 270: 484-7.
7 Powell, J.2000. SAGE. The serial analysis of gene expression. Methods in Molecular Biology 99: 297-319.
8 Schena, M.， D. Shalon, R. W. Davis, and P. O. Brown. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA
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Hughes, E. Snesrud, N. Lee, and J. Quackenbush.2000. A concise guide to cDNA microarray analysis. Biotechniques 29 : 548-50, 552-4， 556 passim.
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Claims

权 利 要 求
1、 一种墓因表达量比较分析法， 其特征在于它具有如下特征：
( a ) 将不同来源的信使核糖核酸 （mRNA ) 用适当的方法标 记后， 等量混合， 作为聚合酶链反应 （PCR ) 的底物；
( b ) 用与基因各来源相对应的引物和基因特异性引物进行 PCR扩增；
( c ) 用生物发光分析法测定序列， 以碱基种类代表基因的 不同来源， 信号强度代表各来源中基因表达量。
2、 根据权利要求项 1 所述的基因表达量比较分析法， 其特 征在于所述 "不同来源的 mRNA"是指同一基因在同一生物种的不 同个体或同一基因在同一个体的不同器官， 如健康人和病人某一 器官中的 mRNA;也可指同种个体中的同一基因在不同的化学或物 理刺激下产生的表达。
3、 根据权利要求项 1 所述的基因表达量比较分析法， 其特 征在于所述"与各来源相对应的特异性引物 "是指一组序列不同， 但由相同种类和数目的碱基所组成的引物， 每一种引物代表一种 基因的来源。
4、 根据权利要求项 1 所述的基因表达量比较分析法， 其特 征在于所述 "用适当的方法标记" 是指用适当长度的 DNA 片段 来区别各来源基因的方法， 第一种方法： 将不同来源的 mRNA 用 反转录 PCR (RT-PCR) 法制成双链互补 DNA (cDNA) 后， 用限制 性内切酶将之切成适当长度的片段， 用选择性适配器 （adap t er) 与之相连接，不同来源的 mRNA连有不同的 adap t er ;第二种方法： 将带有多聚胸腺嘧啶引物的微球分别与不同来源的 mRNA 杂交， 并制成第一链 cDNA ,用 5，端含有与各来源相对应序列的锚定引物 进行互补链 （第二链 cDNA) 的合成， 锚定引物的 5，端用于区别基 因的不同来源； 第三种方法： 直接用 5，端含有与各来源相对应序 列的锚定引物与 mRNA杂交， 并制成第一链 cDNA , 锚定引物 5，端 的构造与第二种方法相同。
5、 根据权利要求项 4 所述的基因表达量比较分析法， 其特 征在于所述 "选择性适配器" 是指含有与权项 4中限制性内切酶 切口互补的序列， 并在连接酶作用下能与切口完全连接的楔形双 链 DNA , 其中一条链的 5'端含有权项 3中与各来源相对应的特异 性序列， 另一条链的 3'端舍有与上述链不互补的碱基， 或 3'端经 过适当修饰不能在 DNA聚合酶的作用下发生延伸反应。 两条链组 成 " Y" 型的结构， 一端不互补， 成叉型分开， 另一端成权项 4 第一种方法中限制性酶切口的形状。
6、 根据权利要求项 4 所述的基因表达量比较分析法， 其特 征在于所述 "选择性适配器" 和 "锚定引物" 中用于区别基因不 同来源的部分是指一段碱基种类和数目相同而排列顺序不同的 序列， 它们具有相同的熔解温度 （Tra值）。
7、 根据权利要求项 1 所述的基因表达量比较分析法， 其特 征在于所述 "生物发光分析法" 是指定量测定延伸反应产生的焦 磷酸盐 （ PP i ) 的方法。
8、 根据权利要求项 7 所述的基因表达量比较分析法， 其特 征在于所述 "延伸反应" 是指在权项 1中的 PCR扩增产物的单链 产物中， 加入特定的引物或引物混合物进行退火， 然后按一定次 序分别逐个加入三磷酸脱氧核苷酸 （ dNTP ) , 或三磷酸双脱氧核 苷酸 （ddNTP ), 或它们的类似物， 在 DNA聚合酶的作用下， 当所 加 dNTP或 ddNTP或它们的类似物与模板互补时， 所发生的聚合 反应。
9、 根据权利要求项 8 所述的基因表达量比较分析法， 其特 征在于所述 "单链产物" 是指用物理或化学的方法将权项 1中的 PCR 产物制成的单链产物， 其中 "物理学方法" 指用生物素 (biotin)标记 PCR中的一种引物，然后用固相法制备单链， "化 学方法" 是指用酶切法制备单链。
10、 根据权利要求项 7所述的基因表达量比较分析法， 其特征在 于所述 "延伸反应" 指权项 1中的 PCR扩增产物， 经酶处理去除 PCR反应中的 PPi, 过剩的 dNTPs 和过剩的引物后， 加入单链结 合蛋白 （如 SSB), 其余按项 8进行反应。
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