WO2004061129A1

WO2004061129A1 - A mothed for quantifying gene based polymerase chain reaction

Info

Publication number: WO2004061129A1
Application number: PCT/CN2004/000009
Authority: WO
Inventors: Dingbang Xu; Wenhui Xu; Defen Zhu; Wenkai Xie
Original assignee: Dingbang Xu; Wenhui Xu; Defen Zhu; Wenkai Xie
Priority date: 2003-01-06
Filing date: 2004-01-05
Publication date: 2004-07-22
Also published as: CN1316036C; CN1515683A

Abstract

The invention involves molecular biological technology ,that is,a mothed for quantifying gene based polymerase chain reaction. In the invention, the mothed of PCR successive transcription reaction consists of two steps , the first step is producing PCR less 5-10 cycles than conventional PCR cycles, during which the amplification product enrichment in linear, and stopping before nonexponential amplification . Because a RNA polymerase promotor sequence is inserted to one of PCR primers at 5' terminal , PCR amplification product can be used as transcription template for RNA synthesis in vitro. The second step is promoting RNA synthesis as soon as adding transcription reagent, the yield of RNA synthesis is in proportion to PCR amplification product therefore can quantify primal template in the sample. The yield of RNA synthesis is multiplication hundred and thousand than template which can quantified simply using ethidium bromide or other nucleotide dye. The said gene quantitation method don ' t use radioactive isotope , don ' t involve antigen-antibody reaction , and don' t carrying out nucleotide hybridization . The said mothed is simple and accurate, and can be used broadly.

Description

Technical method for gene quantification based on polymerase chain reaction

The invention relates to molecular biology technology, in particular to a method for quantifying genes based on a polymerase chain reaction. Background technique

Polymerase chain reaction (PCR) is a simple, specific, sensitive, and rapid method for amplifying specific DNA, but the function of standard PCR methods is limited to qualitative amplification, because the products of the previous stages of PCR are exponential Amplification, the product growth shows a straight line, and the cumulative amount of the target amplification product at this stage is still quite low. 'It is not yet possible to accurately quantify the band after electrophoresis with a fluorescent stain such as ethidium bromide. When the PCR reaction is continued and the total amount of amplified products is sufficient to be determined, the reaction has entered a non-exponentially increasing flat slope or saturation period. At this time, the total amount of amplified products is no longer proportional to the original template in the sample and is difficult to quantify. . Several methods have been developed over the years to overcome the drawbacks of standard PCR that cannot be quantified. One method is to use isotope-labeled primers or isotope-labeled deoxyribonucleotide substrates. The incorporation of isotopes into the amplification product greatly improves the detection sensitivity, so that the reaction can be stopped and measured before the PCR enters the flat slope. Although this method is relatively simple, the safety of the radioisotope and the series of operational problems brought about by it limit the application of this method. The second method is to label biotin or digoxin on the primers. The third method is to perform nucleic acid hybridization after PCR, and use the signal amplification of the labeled probe to perform the measurement. In terms of detection sensitivity, the latter two methods can stop the PCR reaction before entering the slope, but the subsequent detection of these two methods involves the reaction of biotin and avidin, the reaction of antigen and antibody, and the enzymatic color reaction The program is complicated, unstable and cumbersome to operate. Summary of the Invention

The purpose of the present invention is to provide a gene quantification method based on the polymerase chain reaction to overcome the defects that the existing quantitative analysis of genes requires labeling with radioisotopes or antigen antibodies or nucleic acid hybridization, and the methods are complicated and poorly accurate.

The inventive concept is as follows:

The gene quantification method of the present invention includes two steps of PCR reaction and transcription reaction, which can be completed by using only one test tube. The first step uses PCR to amplify the target gene, but the number of PCR cycles is 5-10 cycles less than usual. Although the total amount of amplification products in the reaction solution is small at this time, it cannot be directly determined by ordinary nucleic acid stains, but because the PCR reaction is still in the linear phase of exponential growth and has not entered the flat slope period, the total amount of amplification products is The amount is directly proportional to the number of original templates in the sample. The principle of the present invention is to use a transcription reaction to further synthesize RNA from the PCR amplification products in a linearly increasing manner. The basis for the realization of this idea is to discover the necessary components of the PCR reaction solution, such as deoxyribonucleotides, forward and backward citations. There is no measurable and significant effect on the transcription reaction in a wide range of concentrations such as chemicals and DNA polymerase. Therefore, it is possible to enter the transcription reaction without going through any separation step after completing the PCR reaction. Under the conditions that the RNA polymerase and ribonucleotide substrate are too halo, and the reaction temperature and time are fixed, the amount of RNA synthesis is proportional to the number of templates for the transcription reaction, that is, the number of PCR amplification products. Therefore, the final synthesized RNA The amount reflects the amount of original template DNA in the sample. The amount of RNA synthesized by the transcription reaction can reach hundreds to thousands of times the number of DNA templates, so that it can be conveniently determined quantitatively on an electrophoresis gel with ethidium bromide or other nucleic acid stains. If a fluorescent reagent that develops color with RNA is added during the transcription reaction, the generated RNA can be tracked intermittently or continuously for quantitative detection.

The technical solution of the present invention is-The present invention provides a method for quantifying genes based on the polymerase chain reaction, the reaction steps are as follows: (1) template denaturation; (2) primer annealing; (3) DNA polymerase catalysis Complement the complementary DNA strand by extension; (4) Perform the amplification reaction according to steps (1) to (3), for a total of 10-25 cycles; (5) Add reagents for transcription reaction to synthesize RNA by transcription reaction; In the polymerase chain Stop the cyclic reaction before entering the non-exponential growth, that is, the transcription reaction is performed after 10-25 cycles.

A preferred solution of the above-mentioned gene quantification method is that at least one primer of the polymerase chain reaction contains a 5 'end of the RNA polymerase promoter sequence, and the 3' end and the template sequence have at least 20-30 bases. The bases are complementary.

A preferred solution of the above-mentioned gene quantification method is that the final concentration of said polymerase chain reaction primer is 0.01 to 1 μM, the concentration of four kinds of deoxyribonucleotides is 0.01 to 0.2 mM, and the DNA polymerase is The dosage range is 0.5-5 units per 50 microliters.

Another preferred solution of the above-mentioned gene quantification method is that the volume of the PCR reaction solution is less than 10 microliters. In a preferred embodiment of the above-mentioned gene quantification method, the total volume of the transcription reaction solution is more than twice the volume of the PCR reaction solution.

Another preferred solution of the above-mentioned gene quantification method is to design two or more pairs of primers for two or more gene templates, the amplification products of each pair of primers have different lengths, and at least one primer in each pair of primers With RNA polymerase promoter sequence.

Another preferred method of the above-mentioned gene quantification method is to add a fluorescent reagent that develops RNA with the transcription reagent. Another preferred solution of the above-mentioned gene quantification method is that both the PCR primers and the reverse primers carry an RNA polymerase promoter sequence, and the obtained PCP amplification product can simultaneously synthesize complementary sense and antisense RNA during transcription. chain.

The invention also provides an application scheme of the above-mentioned gene quantification method for improving the specificity of the PCR reaction, which is to design two sets of primers for the outer sleeve and the inner sleeve for the same template, that is:

The outer primer can anneal at a temperature higher than 72 ° F, and the melting temperature of the amplified product is higher than that of the inner amplified product 2- A 10. C;

One primer in the inner primer carries the RNA polymerase promoter sequence. The maximum matching annealing temperature between the primer and the template matching sequence and the other inner primer is lower than 72t :.

The invention also provides an application scheme of the above-mentioned gene quantification method for improving the specificity of a PCR reaction, which is to design two pairs of primers for the same template:

The positive primer in the primer pair upstream of the template sequence carries the RNA polymerase promoter sequence; the reverse primer in the primer pair downstream of the template sequence carries the RNA polymerase promoter sequence;

And the sequence of the two amplified products has 100-500 bases overlapping each other.

In the following, some practical problems and the expanded forms of the above technical solutions are explained.

The present invention uses primers containing DNA-dependent RNA polymerase promoter sequences such as T7, T3, or SP6 for PCR 10-25 cycles, and stops the cycle reaction before the polymerase chain reaction enters non-exponential growth, and then does not undergo any purification or other Step: Start the transcription reaction directly after adding the pre-configured transcription reagent. In order to eliminate non-specific amplification products, the present invention has the following features in terms of primer design and reaction conditions:

First, the polymerase chain reaction of the present invention uses a pair of primers, one of which contains an RNA polymerase promoter sequence at the 5 'end, and the bow I is 40-50 bases long. The 3 'end of the primer exactly matches the sequence of the target gene and is 20-30 bases in length. The other primer is 31-40 bases long, and its sequence exactly matches the target gene. Its rigor, melting temperature, and specific binding strength to the template are close to those of the primer with RNA polymerase promoter sequence. As a result, the entire primer has high rigor, high melting temperature, and high specific binding strength to the template.

Second, the volume of the PCR reaction solution can be usually 50 or 100 microliters. After the reaction, take a certain volume to another test tube and add the transcription reaction reagent at a ratio of 1: 1 to 1: 4. In order to enable the PCR and transcription reactions to be completed in the same test tube and save a large number of tests, the volume of the PCR reaction solution can be reduced to 5 or 10 microliters. After the reaction is completed, 15 or 1C microliter of transcription reagent is added to make the transcription reaction solution total. The volume is usually 20 microliters.

Third, the final concentration range of PCR reaction solution primers is 0.01-1 μM, the concentration of four kinds of deoxyribonucleotides is 0.01-0.2mM, and the amount of DNA polymerase can be 0.5-0.5 microliters of reaction solution. 5 units. The lower limit of the concentration range of these three reagents is lower than the normal range of standard PCR. This is because the PCR of the present invention only needs to perform 15-2C cycle, and the consumption of primers and deoxyribonucleotides is a little less than that of normal PCR. Ten to several hundred times, no matter the initial concentration of the reagent is low or low, its consumption relative to the starting amount is negligible.

Fourth, magnesium ions are required for both PCR reactions and transcription reactions, but the optimal concentrations vary. Since magnesium ions are not consumed in the reaction cattle, the concentration before and after the reaction remains unchanged, and the amount of magnesium ions brought into the transcription reaction by the PCR reaction solution is not different between the tubes and does not affect the accuracy of the entire measurement. In order to make both the PCR reaction and the transcription reaction at the optimal magnesium ion concentration For example, you can increase the ratio of the volume of the transcription reaction to the volume of the PCR reaction, or take into account the amount of magnesium ions that the PCR reaction will carry when preparing the transcription reaction solution.

Fifth, the optimal pH of the PCR reaction is 0.5-1 unit higher than that of the transcription reaction. The addition of the PCR reaction solution will not affect the accuracy of the measurement, but it will bias the pH of the transcription reaction and affect the reaction speed. In order to make the transcription reaction work at the optimal pH, the concentration of the PCR buffer may be appropriately reduced or the concentration of the transcription reaction buffer may be appropriately increased to reduce the effect of the PCR reaction solution on the pH of the transcription reaction, or increase the volume of the transcription reaction solution to the PCR reaction. The ratio of the liquid volume or the pH of the transcription buffer is set to be slightly lower than the optimal reaction pH. The optimal pH of the transcription reaction is reached after adding the pre-formulated transcription reagent to the PCR reaction tube.

The gene quantification method for the final detection of RNA by the PCR-sequenced transcription reaction of the present invention includes various expansion and change forms, and has a wide range of applications.

One of the extensions and variants. Two or more pairs of primers are designed for two or more different gene templates. The amplification products of each pair of primers have different lengths. One primer in each pair is designed to carry RNA polymerase. Promoter sequence. Two or more target amplification products can synthesize two or more synonymous or different synonymous RNA fragments. RNA fragments of different lengths can be scanned and quantified separately by gel electrophoresis. This method can be used to compare the content of different genes (such as two viruses) and the expression of different genes in a sample.

The second and extended form is to add a fluorescent reagent, such as SYBR Green II, to the transcription reagent to determine the progress of the transcription reaction based on the fluorescence intensity as the endpoint method. Multiple or continuous measurements can be used instead of clotting. Gel electrophoresis followed by band scanning method to determine the RNA synthesized by transcription. Transcription reactions are performed at a constant temperature of 35-40 ° C. Continuous readings can be taken with a fluorescence colorimeter with a thermostat, or simple equipment with thermostat and U.V. devices can be used to record at regular intervals with a digital camera.

Third extension and variation. Two pairs of outer jacket and inner jacket primers can be designed for the same template, so that the outer jacket primer can be annealed at a higher temperature (for example, greater than 72 ° C), and the melting temperature of the amplified product is higher than that of the inner jacket. The product is several degrees high; one primer in the inner primer pair is designed to carry the RNA polymerase promoter sequence, and the maximum allowed annealing temperature of the primer and template matching sequence part and the other inner primer is lower (for example, less than 72 ° C) . According to the principle and method of selective low temperature denaturation, the denaturation temperature of the jacket primer amplification stage is higher than the melting temperature of the jacket amplification product, and the annealing temperature is higher than the maximum annealing temperature of the inner primer. The denaturation temperature in the inner primer amplification stage is controlled to be between the outer jacket amplification product and the inner jacket amplification product melting temperature ^, and the annealing temperature is lower than the maximum allowed annealing temperature of the inner primer. Nested PCR can be used to obtain specific target / amplification products, and specific target RNA can be obtained by transcription.

Expansion and variation of the fourth, PCR forward and reverse primer design Chengdu band RNA polymerase promoter sequence. The sequence of the polymerase promoters carried by the positive primers may be the same, if they are all T ₇ RNA polymerase promoter sequences, they may be different, such as If The primers carry the T ₇ RNA polymerase and the reverse primers carry the T ₃ RNA polymerase promoter sequence. The obtained PCP amplification product can simultaneously synthesize the sense and antisense RNA strands during transcription, and the two complementary strands can be annealed to form double-stranded RNA, which can be continuously detected with a fluorescent reagent that develops color with the double-stranded nucleic acid.

The fifth expansion and variation form, combined with the expansion forms three and four, obtain a specific amplification product containing two polymerase promoter sequences.

Sixth of the expansion and variation, two pairs of primers are designed for the same template. Most of the two amplification product sequences overlap each other, and the number of overlapping bases ranges from one to several hundred bases. The positive primer in the primer pair upstream of the template sequence was designed to carry the RNA polymerase promoter sequence; the reverse primer in the primer pair downstream of the template sequence was carrying the RNA polymerase promoter sequence. The length, base composition, melting temperature, and binding strength of the two pairs of primers are close to each other. First, divide two PCR reaction tubes into several cycles under the same conditions. Each reaction tube contains a pair of primers. After the PCR reaction is completed, the two reaction solutions are mixed and a transcription reagent and a double-stranded nucleic acid fluorescent reagent are added. The sense RNA strand and the complementary antisense RNA synthesized from the two target amplification products are annealed to form double-stranded RNA. The non-specific amplification product that each pair of primers may produce during the amplification stage, although it also carries the RNA polymerase promoter sequence and synthesizes single-stranded RNA during transcription, but the sequence of the RNA synthesized from the non-specific amplification product They are not complementary and cannot be annealed to form double-stranded RNA.

Beneficial effect

Compared with other existing PCR-based gene quantification methods, the present invention is simple, convenient and accurate. The entire detection process does not involve radioisotope manipulation, nor does it need to prepare probes and perform nucleic acid hybridization, nor does it require antigen-antibody action and enzymatic color reaction. Continuous measurement can also be performed by adding the fluorescence developed with RNA to the transcription reaction solution. The inflection point of the method of the present invention can be summarized as:..

1. The number of PCR cycles is 5-10 cycles less than usual. The dNTPs concentration range of the reaction system can be lower than that of standard PCR. The use of lower concentration reagents can enhance the rigor of PCR reaction parameters, which is beneficial to reduce non-specific products and reduce The concentrations of these reagents in the transcription reaction solution are shown.

2. The number of cycles of PCR is 5-10 cycles less than usual, and the number of cycles to form primer dimers is correspondingly reduced. Because the primer concentration range of the reaction system is lower than that of standard PCR, the chance of primer dimer formation is also reduced. The fluorescence background is also low; because the primer dimer is double-stranded DNA, the single-stranded RNA product is detected with an RNA-specific fluorescent reagent, and the interference of the dimer is higher than the usual real-time quantitative PCR using SYBR Green I Method is low.

3. No fluorescent reagents or probes are added in the PCR reaction stage, which avoids the negative effects of the existing real-time fluorescent quantitative reactions in terms of amplification efficiency: stability and repeatability due to fluorescent reagents or probes.

4. There is a linear relationship between RNA concentration and template concentration in a wide range of quantification.

5. At the end of the PCR reaction, the concentration of the amplified product is 10-1000 times lower than the existing method, and the chance of lag pollution is significant ^ 04 000009 low. .

6. The transcription reaction is performed at a constant temperature, and the reaction is more stable than PCR amplification with periodic temperature changes. The equipment required to track fluorescence enhancement in a constant temperature culture strip is simpler than the current real-time fluorescence quantitative method.

7. The method of synthesizing two complementary RNA strands from two amplification products has both the specificity of a real-time fluorescent quantitative PCR method using a probe and the simplicity of a real-time fluorescent quantitative method using a fluorescent reagent. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the relationship between the amount of DNA template and the scanning value of RNA bands.

Figure 2 shows the relationship between the amount of cDNA added and the scanning value of RNA bands. detailed description

In the following, the quantitative determination of the glycerol-3-phosphate dehydrogenase gene (gene bank number XM_006959) is taken as an example, and the present invention will be further described in combination with specific embodiments, but the present invention is not limited thereto.

The primer design software Oligo 6.0 used in the present invention is produced by Molecular Biology lnsights, lnc. cDNA was synthesized via commercial human tissue total RNA (from BD CIontech) using a reverse transcription commercial kit (from BD CIontech). The gene numbers in the following examples are quoted from the Gene Bank of the National Center for Bioinformatics (NCBI).

Example 1. Compare the maximum allowable annealing temperature of two primer pairs under the same conditions.

The characteristics and order of primers for designing PCR reactions are listed in Table 1.

Table 1 Primer characteristics and sequence

Bottom line in the table is the T ₇ DNA polymerase promoter sequence

The first pair of primers (A5 'and A3') are excellent primers with high morbidity of about 20 bases selected by the primer design software Oligo 6.0. The second primer pair (B5 'is homologous to the first pair, where the B3' primer is a A 20-base RNA T ₇ polymerase promoter sequence is added to the end, while the B5 'primer extends 9 bases at the 5' end of the A5 'primer, and the melting temperature (Tm) of B5' is 86.78, Almost 17 ° C higher than A5 '.

The final concentration of the components of the PCR reaction solution is: buffer Tricine-KOH 40mM, pH 8.7, magnesium ion 35mM, primer concentration 0.5 μM, dATP, dCTP, dGTP and cH P concentration of four deoxyribonucleotides At 0.2 mM, DNA polymerase was 2.5 U per 50 microliter reaction solution, and cDNA was 5 microliter per 50 microliter reaction solution. The PCR reaction is denatured for the first time at 95 ° C for 1 minute, cyclic denaturation is 95 ° C for 30 seconds, and the extension is 72 ° C (at an annealing temperature of 63.4-72 ° C) or 74 ° C (at an annealing temperature of 73-74 ° C) 1 minute, 30 cycles of reaction. The PCR reaction solution was electrophoresed on an 8% polyacrylamide gel. The ethidium bromide was stained and scanned with an imager. The test results are listed in Table 2.

Detection results of PCR products for maximum allowable annealing temperature comparison test

The results showed that ordinary primer long primer pair A could anneal at a maximum of 71 ° C, while homologous RNA polymerase promoter sequence primer pair B could anneal above 73 ° C. Annealing at such high temperatures only forms the target amplification products that can be used as transcription templates, without any non-specific amplification products. Example 2. Compare the sensitivity of two primer pairs under the same conditions.

The composition and reaction conditions of the PCR reaction solution were the same as those in Example 1. The annealing temperature of primer pair A and primer pair B was separately 1. 70 and 72 ° C. The cDNA was added to the reaction solution in half dilution. The results are shown in Table 3.

Table 3 Detection results of PCR products for sensitivity tests

The results show that the primers containing the sequence of the RNA promoter are the same as their common common primer pairs. When the cDNA is diluted by 25 (fold), the target amplification product is still formed. Effect of Magnesium Ions on Transcriptional Response.

First, use primer pair B to obtain PCR products under the conditions shown in Examples 1 and 2. Use a PCR purification column to remove residues. Primers, deoxyribonucleotides, DNA polymerase, and buffer components were used to adjust the concentration of the purified PCR amplification product to 200 pg / l, which was used as a DNA template for the transcription reaction. The composition of the transcription reaction solution is shown in Table 4. Table 4 Composition and preparation of transcription reaction solution

After incubation at 37 ° C for 2 hours, electrophoresis was performed on 8M urea 5% polyacrylamide gel, and RN bands were scanned after ethidium bromide staining. The results showed that when the concentration of externally added magnesium ions was 0.5 and 2 mM, the concentrations of the four deoxyribonucleotides were 0.1, 0.2, and 0.4 mM, and the concentrations of the forward and reverse primers were 0.1, 0.2, and 0.4 μM, respectively. No effect. Example 4. The linear relationship between the amount of RNA synthesized and the amount of DNA template was tested.

The template concentration of the linearized plasmid used in the current in vitro transcription method to prepare the RNA probe is usually 500-1000 ng <, and the template amount of the PCR sequential transcription reaction method of the present invention is less than 1 ng. The DNA template used in this example is the same as in the example, the concentration is adjusted to 100 pg / μl, and 100-400 pg of DNA template is added to several test tubes, respectively. The composition, reaction conditions and measurement method of the transcription reaction ¾ are the same as those in Example 3. The intensity of the RNA band scan is shown in Figure 1.

Figure 1 shows a good linear relationship between the amount of RNA synthesized and the amount of DNA template added, R ² = 0.9771. Comparing the scanning value of the band with the standard RNA can measure the speed of RNA synthesis. The results show that under the test conditions, each DNA ¾ The plate can synthesize about 250 copies of RNA in 2 hours. Example 5. Explains the entire process of the method for quantifying genes for PCR-continuous transcription reactions of the present invention.

Primer pair B was used to amplify samples containing different amounts of original template. The primer concentration is 0.1 μM. The concentration of the four ribonucleotides is 0.1 mM, the buffer is Tris, 40 mM pH 8.3, and the magnesium ion concentration is 1.5 mM TaqDNA polymerase per 50 microliters of reaction solution. 2U PCR reaction volume per tube 5 μl. The PCR reaction conditions were the same as in Example 1. The annealing extension was 70 ° C for 2 minutes, and the reaction was performed for 20 cycles. After the PCR reaction is completed, 15 microliters of the previously prepared transcription reaction solution is added to each tube, so that the ribonucleotide concentration, magnesium ion concentration, RNA polymerase concentration, DDT concentration and nuclease inhibitor concentration in the mixed solution are as shown in the table. 4 shows the same. The concentration of the Tris buffer stock solution used to prepare the transcription reaction premix is the same as listed in Table 4, but the pH is 7.3. The pH of the PCR reaction solution is approximately 7.5 after mixing with the transcription solution. Method determination. The results are shown in Figure 2. The results show that when the amount of cDNA is 1 to 4 microliters per tube, the amount of cDNA added has a good linear relationship with the final RNA band scan value. R ² = 0.9748. Example 6. Applying two pairs of primers to two gene templates PCR, followed by transcription and synthesis of RNA

A pair of primers used the glycerol 3-phosphate dehydrogenase gene (gene bank number XM-006959) as a template. Primer sequence and characteristics are shown in primer pair B in Table 1. The other pair of primers uses the human beta actin gene (gene bank number BC016045: as template). The primer characteristics and sequence are shown in Table 5.

Table 5 Example 6 primer characteristics and sequence

Bottom line in the table is the T ₇ DNA polymerase promoter sequence

The lengths of the amplified products of primer pairs B and C were 472 and 314 base pairs, respectively. The amplified products were used as templates to be transcribed into synonymous RNA with lengths of 450 and 292 bases, respectively. The cDNA preparation and PCR conditions and transcription method were the same as those in Example 5. One microliter of cDNA was added to each reaction tube. Transcription products were subjected to denaturing gel electrophoresis and ethidium bromide staining to obtain two distinct RNA bands. Example 7. Detection method of adding a fluorescent reagent to a transcription reaction system.

The primers and PCR reaction conditions used in Example 7 are the same as those in Example 5, but the fluorescent reagent SYBR Green II was added to the transcription reaction solution so that the final concentration of the fluorescent reagent in the reaction system was 1/2000. The transcription reaction system was incubated at 37 ° C for 2 hours, and the fluorescence was observed under the excitation of ultraviolet light every 30 minutes. As a result, the fluorescence continued to increase as the reaction time increased. Example 8. Nested PCR of two pairs of primers, in which the amplification product of the inner set of primers is followed by a transcription reaction.

The two pairs of primers use the human beta actin gene (gene bank number BC016045) as the template. The characteristics and sequence of the primers are shown in Table 6.

Table 6 Example 8 primer characteristics and sequence

Bottom line _ in the table is the T ₇ DNA polymerase promoter sequence

The lengths of the outer and inner amplification products were 756 and 302 base pairs, respectively, and the melting temperatures of the amplification products were 87.4 and 82.0 ° C, respectively. The melting temperature of the 21-base 3 'end of the inner primer that matches the template is 63.6 ° C. The concentration and the like of the primer and the inner primer are the same as those in Example 6. The denaturation temperature for the first 10 cycles of PCR was 9CTC for 15 seconds, and the withdrawal and extension temperature was 72 ° C for 1 minute; the denaturation temperature for the 10 cycles after PCR was 85 to 15 seconds, and the annealing and extension temperature was 62 to 1 minute. After the PCR was completed, transcription was performed according to the method of Example 5, and a specific target RNA band was obtained from the results of electrophoresis and staining. The other reaction was the same as the above experiment, but the two-stage PCR was increased from 10 cycles to 15 cycles each. After 30 cycles of PCR, direct electrophoresis and staining revealed a specific 302 base long DNA amplification band. Example 9. Both the forward and reverse primers carry an RNA polymerase promoter sequence, and the amplified product simultaneously transcribes sense and antisense RNA.

The primers used the human beta actin gene (gene bank number BC016045) as a template. The characteristics and sequence of the primers are shown in Table 7. Example 9 Primer Characteristics and Sequence

Bottom line in the table is the T ₇ DNA polymerase promoter sequence

The primers and PCR reaction conditions used in Example 9 are the same as those in Example 7, but the fluorescent reagent SYBR Green I was added to the transcription reaction solution so that the final concentration of the fluorescent reagent in the reaction system was 1/2000. The transcription reaction system was incubated at 37 ° C for 2 hours and observed under UV light excitation at intervals of 30 minutes. As a result, the fluorescence continued to increase as the reaction time increased. Example 10. Nested PCR, the inner and reverse primers carry the RNA polymerase promoter sequence, and the amplified products simultaneously transcribe the sense and antisense RNA.

The two pairs of primers use the human beta actin gene (gene bank number BC016045) as a template. The outer primer pair is shown in Table 6 and the primer pair is "D outer", and the inner reverse primer is shown in Table 6 "D inner 3,". The forward primer is 44 bases in length from 1174 to 1217, and the sequence is TTG TAA TAC GAC TCA CTA TAG GGC TTC TAG GCG GAC TAT GAC TT (the nucleotide with the bottom line is the T ₇ DNA polymerase promoter sequence).

The lengths of the outer and inner amplification products were 756 and 325 base pairs, respectively, and the melting temperatures of the amplification products were 87.4 and 82.7 ° C, respectively. The melting temperatures of the 21-base 3 'end of the inner and outer primers that match the template are 63.8 ° C and 63.6 ° C, respectively. The concentrations of the outer and inner primers were the same as in Example 8. The denaturation temperature for the first 10 cycles of PCR was 9CTC for 15 seconds, and the annealing and extension temperature was 72 ° C for 1 minute; the denaturation temperature for the 10 cycles after PCR was 85Ό15 seconds, and the fire and extension temperature were 62 ° C for 1 minute. . After the PCR was completed, transcription was performed according to the method of Example 9, and it was observed that the fluorescence increased with the transcription wing. Example 11. Two pairs of primers are divided into two tubes for PCR. The two reaction solutions are combined into one tube for transcription.

The two pairs of primers use the human beta actin gene (gene bank number BC016045) as a template. The primer sequences and properties are shown in Table 8. Table 8 Primer characteristics and sequence of Example 11

Bottom line in the table is the T ₇ DNA polymerase promoter sequence

The melting temperatures of F1 and F2 amplification products were 88.0 and 87.8 ° C, respectively, and the lengths of the amplification products were 652 and 619 base pairs, respectively. The RNA strands synthesized from the amplified products were 630 and 597 bases, respectively, of which the 588 bases of the two strands were complementary in sequence. F1 and F2 were used to perform PCR in two reaction tubes. The reaction conditions were the same as in Example 9. After the reaction was completed, the two reaction solutions were combined, and a transcription reagent and a fluorescent reagent were added. As a result, it was observed that the fluorescence intensity increased as the transcription time increased.

Claims

Rights request

1. A gene quantification method based on the polymerase chain reaction, which is characterized by including two steps of polymerase chain reaction and transcription reaction. The reaction steps are as follows:

(1) template denaturation;

(2) Primer annealing;

(3) DNA polymerase catalyzes the synthesis of complementary DNA strands;

(4) Perform the amplification reaction according to steps (1) — (3) in a cycle, for a total of 10-25 cycles;

(5) Add reagents for transcription reaction to synthesize RNA;

Among them, the cyclic reaction stops before the polymerase chain reaction enters non-exponential growth.

2. The method for quantifying genes according to claim 1, characterized in that the 5 'end of said at least one primer of the polymerase chain reaction contains a promoter sequence of RNA polymerase, and the sequence of 3, end and template is at least 20 — 30 bases complementary.

3. The method for quantifying genes according to claim 1, characterized in that the final concentration of the primers of the polymerase chain reaction is 0.01-1 μM, and the concentration of four kinds of deoxyribonucleotides is 0.01-0.2 mM, The amount of DNA polymerase used ranges from 0.5 to 5 units per 50 microliters.

4. The method for quantifying genes according to claim 1, wherein the volume of said PCR reaction solution is less than 10 microliters.

5. The method for quantifying genes according to claim 1, wherein the total volume of the transcription reaction solution is more than twice the volume of the PCR reaction solution.

6. The method for quantifying genes according to claim 1, characterized in that two or more pairs of primers are designed for two or more types of gene templates, the amplification products of each pair of primers have different lengths, and each pair of primers has At least one primer carries an RNA polymerase promoter sequence.

7. The method for quantifying genes according to claim 1, wherein the method for judging the progress of a transcription reaction is to add a fluorescent reagent that develops color with RNA to the transcription reagent.

8. The method for quantifying genes according to claim 1, characterized in that the positive and reverse primers of the PCR both carry a polymerase promoter sequence, and the obtained PCP amplification product can simultaneously synthesize complementary sense and antisense during transcription. Meaning RN chain.

9. The application of the gene quantification method according to claim 1 in improving the specificity of the PCR reaction, characterized in that two pairs of bow I objects are designed for the outer sleeve and the inner sleeve for the same template: The outer primer can be annealed at a temperature higher than 72 ° C, and the melting temperature of the amplified product is 2--10 higher than that of the inner amplified product. C;

One primer in the inner primer has an RNA polymerase promoter sequence, and the maximum matching annealing portion of the primer to the template matching sequence and the other inner primer is below 72 ° F.

10. The method of claim 1 for improving the specificity of a PCR reaction, characterized in that two primer pairs are designed for the same template:

The positive primer in the primer pair upstream of the template sequence carries the RNA polymerase promoter sequence; the reverse primer in the bow I pair downstream of the template sequence carries the RNA polymerase promoter sequence;