CN1201015C - Quantitative polymerase chain reaction process applying high density primer - Google Patents
Quantitative polymerase chain reaction process applying high density primer Download PDFInfo
- Publication number
- CN1201015C CN1201015C CN 02125686 CN02125686A CN1201015C CN 1201015 C CN1201015 C CN 1201015C CN 02125686 CN02125686 CN 02125686 CN 02125686 A CN02125686 A CN 02125686A CN 1201015 C CN1201015 C CN 1201015C
- Authority
- CN
- China
- Prior art keywords
- primer
- pcr
- polymerase chain
- chain reaction
- quantitative
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a technology of molecular biology, i.e., a quantitative polymerase chain reaction method with improved PCR reaction parameters and a detecting program of target product electrophoretic bands. The present invention is characterized in that the final concentration of the positive primer and the negative primer of the polymerase chain reaction are from 1 to 100 mu M, the unwinding temperature of the positive primer and the negative primer in the reaction is from 85 to 92 DEG C, and the length of the primers of the PCR reaction is from 25 to 50 bases. The PCR reaction comprises high-temperature template denaturation and primer and template annealing extension. The temperature of the annealing extension is from 72 to 82 DEG C, a coloring agent of SYBR gold and a PCR sample are mixed together for electrophoresis, and then, the strength of PCR product bands on electrophoretic gel is detected quantitatively. The weight percentage concentration of the SYBR gold in a sample loading buffer is from 0.01% to 1%. The PCR reaction is more stable, the occurrence of a PCR horizontal slope is delayed, and the accumulating period of a straight line of a target product is prolonged. Therefore, the classical PCR has the advantages of simplicity, high speed and specificity, and simultaneously, a quantitative function is realized. The method for mixing the SYBR gold and the sample together for electrophoresis not only improves the sensitivity of quantitative dyeing and enlarges the linear range of the quantitative detection, but also omits dyeing steps and reduces the consumption of the coloring agent.
Description
Technical field
The present invention relates to Protocols in Molecular Biology, promptly a kind of polymerase chain reaction method.
Background technology
Polymerase chain reaction, promptly PCR is a kind of simple, quick, single-minded, sensitive gene amplification method.Each circulation of existing typical PCR was made up of sex change, annealing and three steps of extension, and sex change and annealed temperature head reach 30-50 ℃, and only be about 30 seconds the perdurability of each step reaction of round-robin, this just causes the temperature course between inner reaction tube liquid and the tube and tube also inconsistent, makes reaction efficiency that difference be arranged.Though this difference is small, circulation is the accumulation of exponential manner through 25-30, just brings the error of can not ignore.And under the parameter condition of classical PCR, the PCR flat slope occurs early, and the semi-invariant that enters the preceding amplified production of flat slope is too low.This all has influence on its target product band is carried out detection by quantitative.So far, the classical PCR method adopts gel electrophoresis to separate usually and detects the target product band with the ethidium bromide staining method.The ethidium bromide staining method detects the inspection limit of DNA band on the gel between 0.1-1ng, and that still measures in this scope is repeated very poor, and the linear relationship between band concentration and the staining power is also very poor, and does not meet the requirement of quantitative analysis.Measurement result shows that it is 10-100ng that the ethidium bromide staining standard measure is measured the linearity range of gel DNA band.The molconcentration of amplified production was lower than 1.0nM when reaction entered flat slope under the reaction parameter condition of classical PCR, supposed that amplified production length is 500 alkali bases, and then the weight concentration of amplified production is 0.3ng/ul.Usually get the 10ul reaction solution and do electrophoretic analysis, the dna content of target amplification product band is 3ng.These data show the electrophoretic band with ethidium bromide staining method energy qualitative detection PCR reaction product, but can not carry out quantitative assay accurately.So, under classical PCR reaction conditions parameter because the PCR flat slope occurs early and bromine second staining quantitative analysis sensitivity is low, cause classical PCR be difficult to realize to its amplified production carry out simply, detection by quantitative accurately.
Need for detecting, people set up on classical PCR method basis and have developed some kinds of gene quantification methods, comprise PCR and molecular hybridization coupling, labeled primer PCR, competition quantitative PCR and real-time fluorescence quantitative PCR.Though these methods are skillfully constructed, characteristics are respectively arranged, separately all limitations and deficiency are also arranged.Molecular hybridization method and labeled primer method need increase a series of detection steps behind PCR; Competition quantitative PCR and real-time fluorescence probe quantitative PCR then need be before PCR design at each determined template and prepare in mark or molecular beacon.This all situation not only makes the workload of mensuration be multiplied and has brought new, perhaps bigger than PCR itself source of error for whole mensuration.In addition, existing various quantivative approach also exists some common shortcoming and problems.The first, existing various quantivative approachs are all continued to use the general reaction parameter of classical PCR, cause reacting between unstable and pipe reasons of error and are not all overcome and improve.The second, existing various quantivative approachs all do not have the reason at the appearance of PCR flat slope, find out and postpone the countermeasure that flat slope occurs.Existing various quantivative approach all is to avoid the puzzlement of flat slope appearance to quantitative analysis with the means of controlling table by increasing determination step or reagent.The 3rd, because mark in adding, fluorescence dye or molecular beacon etc. and template play the reagent of single-minded or non-exclusive reaction, make the reaction system of PCR complicated artificially, thereby influenced the stable and repeated of reaction, be unfavorable for obtaining believable, accurate quantitative analysis result.
The primary process of PCR reaction is that the sex change at high temperature of double-stranded DNA template is unwind and is single stranded DNA, when temperature primer and template annealing when denaturation temperature is reduced to annealing temperature, the Taq archaeal dna polymerase begins primer by extending with template order complementary mode, so the annealing of primer and template is the successful basis of each circulation of PCR at 3 ' end of primer and template complementary portions then.The common primer concentration scope of classical PCR method is 0.1-1.0uM.The too low needs that can not satisfy synthetic a large amount of amplified productions of primer concentration, according to calculating and practice, primer concentration is that 0.2uM guarantees that enough pcr amplification product is detected by qualitative, and a large amount of tests show the primer that adopts higher concentration often for the formation of non-single-minded amplified production and primer dipolymer is strengthened greatly, so general for a long time with the optimum concentration range of 0.2-0.5uM as Standard PC R.But this just is to consider the way that the thinking of standard and the worry that non-single-minded amplified production and primer dipolymer are formed and shortage overcome with qualitative detection, and causing for a long time, the PCR primer concentration is limited to above-mentioned narrower and small scope.Also make the quantitative function of PCR delay to realize.With some typical PCR response datas the relation that primer concentration and PCR flat slope occur is described below.In PCR when beginning reaction,, every 100ul reaction solution contains 105 copy templates usually, and promptly its molconcentration is 1fM, and the starting point concentration of primer is between 0.1-1uM, and promptly primer concentration is hundred million times of the 1-10 of template concentrations.Template concentrations is quite low during PCR reaction beginning, and the annealing of template self needs a few hours to a couple of days just can finish, and primer can rely on its concentration advantage to finish annealing with template in several seconds to tens seconds.Constantly increase along with the reaction cycle number increases template concentrations, template is finished the self-annealing required time and is constantly reduced.In general, template concentrations is also lower before 20 circulations, needed tens minutes just can finish self-annealing, though primer is consumed with reaction process, but proportion is also very little with respect to initial value, the concentration of primer almost remains unchanged and is still several ten thousand of template concentrations and arrives millions of times, and primer and template annealing are smoothly linearly accumulated amplified production.But PCR circulation through about 25, the amplification of template can reach 1,0,000,000 times, this moment, the consumption of primer was about 1-10%, the concentration of primer still is original 90-99%, but the ratio of primer concentration and template drops to 10-100 doubly for hundred million times from the 1-10 of beginning, and primer annealing reduces greatly to the competitive edge of template self-annealing.On the other hand, from the initial 1fM 10nM that increases, template self is finished 50% annealing required time and was foreshortened to about 10 seconds from several days through 1,000 ten thousand times of concentration of amplifying rear pattern plate.Because the temperature of template self-annealing exceeds about 20 degree usually than primer annealing temperature, and PCR appliance requires 10-20 could drop to common primer annealing temperature from denaturation temperature second, in this ramp of tens of seconds in the time, the template of some finished the annealing of self, thereby stoped combining of primer and template.So, along with the process primer of PCR not only slowly loses competitive edge on concentration, and also having lost the chance that is at war with in time, these 2 is the major cause that causes the PCR flat slope to occur.When template continues amplification, when concentration reaches 100nM, template is finished self 50% annealing and was only needed for 1 second, finishing self all anneals also only needs about 5 seconds, in this case when PCR instrument temperature also during the annealing temperature of no show primer, template has been finished the Full Annealing of self in the time of this section ramp, the annealing of primer and template is blocked fully, and PCR enters the period of saturation.
Summary of the invention
For the straight line accumulated phase that prolongs the PCR product with quantitatively detect PCR target amplification product ahead of time, thereby realize that the classical PCR function is by the qualitative amplification quantitative analysis of marching toward, the appearance of PCR flat slope is postponed, the invention provides a kind of quantitative polymerase chain reaction process of applying high density primer, the ultimate density that promptly makes positive and negative primer in polymerase chain reaction is 1-100uM; The melting temperature(Tm) of positive and negative primer can be 85-92 ℃ in the reaction; Primer can be that length is the extraordinary primer of 25-50 base, and primer length is preferably the 30-40 base; And the step of PCR reaction also can be simplified and merges into the sex change of high temperature template, primer and template annealing and extended for two steps, and the annealing elongating temperature is 72-82 ℃, and the best elongating temperature of annealing is 75-80 ℃.And can adopt after staining agent SYBR gold and the PCR sample mix, carry out electrophoresis together, the intensity of PCR product band on the quantitative assay running gel.The weight percent concentration of SYBR gold in sample loading buffer be ten thousand/to one of percentage.
Polymerase chain reaction method of the present invention has kept advantages such as simple, single-minded, the quick and sensitivity of classical PCR fully.In the PCR reaction system, do not increase any new reagent, do not need special detecting instrument and analysis software, still adopt traditional electrophoretic separation and detection of fluorescent dyes method, just substitute the sensitivity that ethidium bromide improves DNA band quantitative assay on the gel with the SYBR gold.The SYBR gold is a kind of new DNA staining agent that U.S. MolecularProbe company released in 1999.SYBA gold and ethidium bromide show the parallel check experiment that the DNA band carries out quantitative analysis on running gel, high ten times of the quantitative analysis remolding sensitivity ethidium bromides of SYBR gold, thereby can before the PCR flat slope occurs, carry out quantitative analysis to the product band.And the present invention carries out the electrophoretic practice with the SYBR gold with sample mix, has then not only omitted staining procedure, has reduced the staining agent consumption, and has expanded the linearity range of quantitative assay.In the present invention, owing to strengthen the competition of primer annealing and template self-annealing with the high density primer of 1.0-100uM, thus postponed the appearance of PCR flat slope.But shown that as a large amount of tests high density primer can cause more serious non-single-minded product and primer dipolymer, thereby the accuracy and the stability of quantitative assay are brought disadvantageous effect.Therefore the present invention also uses the primer of melting temperature(Tm) extra-high-speed, this primer can with template self-annealing temperature very near or higher temperature under anneal, thereby improved the competitive power of primer and template self-annealing opportunity from annealed, occur to postpone flat slope.PCR can annealed under the high-temperature very much, thereby weaken or eliminate fully the formation of non-single-minded product and primer dipolymer greatly, overcome in quantitative PCR owing to use the problem that high density primer brought.The 3rd effect using the primer of melting temperature(Tm) extra-high-speed is to make the annealing of reaction become a step with the extension process by two step degeneracys, and the temperature spread between annealing and the sex change is shortened greatly, thereby improves the stability of reaction.
Description of drawings
Fig. 1 is the comparison diagram that common staining and dyestuff add electrophoresis sample method.
Fig. 2 is the influence figure of primer concentration to the appearance of PCR flat slope.
Fig. 3 is the influence figure of the general primer of concentration 0.1 and 20uM to the flat slope appearance.
Fig. 4 is high density primer 5uM quantitative PCR figure.
Fig. 5 is high density primer 30uM quantitative PCR figure.
Fig. 6 is a ultra-long primer influences one of figure to what the PCR flat slope occurred.
Fig. 7 is a ultra-long primer influences two of figure to what the PCR flat slope occurred.
Fig. 8 is high density ultra-long primer quantitative PCR figure.
Embodiment
As a result, in the test concentrations scope, all in 10%, this explanation test repeatability better for the relative error of six band intensity of same concentration.Simultaneously do parallel test with the ethidium bromide staining method, it is 3.75-250ng that the extent of dilution of adjustment criteria DNA and application of sample volume make the content of band.The result shows, when band dna content during greater than 15ng, the relative error of same six band intensity also is lower than 10%, illustrate that repeatability better, but when the band dna content is 3.75ng and 7.5ng, the relative error of six band intensity is about 1ng respectively up to 40% and 15% though this explanation ethidium bromide staining detects the qualitative detection sensitivity of DNA band on the gel, and the sensitivity of quantitative analysis only is about 10ng.So the painted quantitative analysis sensitivity of the SYBR that the present invention adopts gold is 1ng,, can shift to an earlier date the concentration that 3-4 circulation quantitative analysis goes out the PCR product than ethidium bromide method with the dyeing of SYBR gold than high about 10 times of ethidium bromide method.
The preparation of table one electrophoresis sample
| Sample number | The SYBR gold (ul) of 1% dilution | The sample DNA of 20ng/ul (ul) | The sample DNA of 1ng/ul (ul) | DNA content (ng) | 6X sample loading buffer (ul) | Deionized water (ul) |
| 1 | 2.5 | 1 | 1 | 5 | 16.5 | |
| 2 | 2.5 | 2 | 2 | 5 | 15.5 | |
| 3 | 2.5 | 5 | 5 | 5 | 12.5 | |
| 4 | 2.5 | 7.5 | 7.5 | 5 | 5 | |
| 5 | 2.5 | 10 | 10 | 5 | 2.5 | |
| 6 | 2.5 | 1.0 | 20 | 5 | 16.25 | |
| 7 | 2.5 | 2.5 | 50 | 5 | 15 | |
| 8 | 2.5 | 3.75 | 75 | 5 | 13.75 | |
| 9 | 2.5 | 5 | 100 | 5 | 12.5 | |
| 10 | 2.5 | 10 | 200 | 5 | 7.5 |
Test result as shown in Figure 1, usually the linearity range of dyeing process is narrower as can be seen from Fig. 1, when band DNA amount is good linear relationship, R during for 1-10ng
2=0.9962.But linear relationship was relatively poor when the DNA amount increased to 200ng, R
2Value=0.8377.And the method that SYBR adds the electrophoresis sample is had much wide linearity range, when the DNA amount is all to be good linear relationship when 1-100ng and 1-200ng, R by 1-10ng
2Value is respectively 0.9891,0.9953 and 0.9948.When adopting common staining to test, the little running gel in one 10 hole need be used the 5ulSYBR gold; And adopt the SYBR gold with the electrophoretic method of sample mix, 10 samples only need SYBR gold 0.25ul.The final concentration of SYBR gold in application of sample liquid is advisable with 0.01-1%, and staining agent can have a strong impact on the travelling speed of DNA and the shape of electrophoretic band during lower or greater concn.Embodiment 2 and embodiment 1 explanation adopt the SYBR Jin Dynasty for ethidium bromide and employing and the electrophoretic in the lump method of sample mixed together, omitted staining procedure, reduced whole mensuration required time, also saved reagent, the more important thing is, improved the sensitivity and the linearity range of DNA band on the quantitative analysis gel greatly.
Table two primer characteristic
| Primer | Primer length | The position | Melting temperature(Tm) | Order 5 ' to 3 ' |
| | 20 | 348-367 | 70.0℃ | CGC TGA GTA CGT GGA GT (lack and wait to mend) |
| Anti-primer | 17 | 772-788 | 70.4℃ | GCA GTG GGG ACA CGG AA |
The final concentration of table three PCR reaction major ingredient
| PCR reacts composition | Final concentration in the reaction solution |
| Tricine-KOH，pH8.7 | 40mM |
| KOAC | 15mM |
| Mg(OA) 2 | 3.5mM |
| The VITAMIN B4 deoxyribonucleotide | 0.2mM |
| The guanine deoxyribonucleotide | 0.2mM |
| The cytosine(Cyt) deoxyribonucleotide | 0.2mM |
| The cytosine(Cyt) deoxyribonucleotide | 0.2mM |
| The TaqDNA polysaccharase | Every microlitre 0.3 unit |
| c-DNA | The every 100ul reaction solution of 10ul/ |
95 ℃ of the sex change first of this PCR reaction, 1 minute, 95 ℃ of circulation sex change were annealed 65 ℃ for 30 seconds, 1 minute, extend 70 ℃, 1 minute.
When the primer concentration of PCR is 0.05,0.5 and 5uM, promptly be respectively 1/2nd of the common concentration limit of Standard PC R primer, the intermediate value and the upper limit five times.The PCR cycle number is 20-40, gets a pipe every four circulations and measures by embodiment 1 methods analyst.The result shows when primer concentration is 0.05uM that as shown in Figure 2 PCR hastens towards saturation in 28 circulations, and when 100 times of concentration raisings were 5uM, PCR began to enter flat slope in 28 circulations.When the intensity that primer concentration reaches the target product band of the period of saturation during for 5uM is 0.05uM nearly two times.
When the primer concentration of PCR is 0.1 and 20uM, promptly be respectively the lower bound of the common concentration of Standard PC R primer and 20 times of the upper limit.PCR cycle number 19-36 gets a pipe every one or two circulation and measures.The result shows when primer concentration is 0.1uM that as shown in Figure 3 PCR begins to enter flat slope in 27 circulations, and the PCR product linearly rises regressand value R in five circulations of 22-27
2=0.9873; PCR begins to enter flat slope in 32 circulations when primer concentration is 20uM, and the PCR product linearly rises regressand value R in eight circulations of 22-30
2=0.9856.When PCR product intensity was 0.1uM when primer concentration was 20uM more than three times.Present embodiment shows the appearance of high density primer deferrable flat slope.
The general primer of embodiment 4 usefulness high densitys carries out PCR by usual conditions, with the effect of explanation with the high density primer quantitative PCR.The primer is identical with embodiment 3 with the PCR reaction parameter.
When primer concentration is that 5uM is when being 5 times of the common primer concentration upper limit, every 20ul reaction solution adds template c-DNA1-4ul, and reaction cycle is several 23, and the result as shown in Figure 4, be presented at the scope inner formword concentration of trying and PCR target product band intensity be good linear relationship, regressand value R
2=0.996.
When primer concentration is that 30uM is 30 times of the common primer concentration upper limit, every 20ul reaction solution adds template c-DNA1-10ul, reaction cycle is several 23, and the result shows as shown in Figure 5 when the template relative concentration and doubly is good linear relationship, regressand value R with inner formword concentration and PCR target product band intensity at 1-5
2=0.9803.
Table four is positive and negative super.Primer is listed in the table below with the characteristic of the common long primer of using as parallel comparison test of homology
| Primer | Primer length | The position | Melting temperature(Tm) | PCR product length | Order 5 ' to 3 ' | |
| Overlength | Just | 39 | 553-591 | 89.1℃ | 388 | CTG GCC AAG GTC ATC CAT GAC AAC TTT GGT ATC GTG GAA |
| Instead | 37 | 904-940 | 88.9℃ | CGC TGT TGA AGT CAG AGG AGA CCA CCT GGT GCT CAG T | ||
| Common | Just | 19 | 573-591 | 63.0℃ | 348 | CAA CTT TGG TAT CGT GGA A |
| Instead | 17 | 904-920 | 63.2℃ | ACC ACC TGG TGC TCA GT | ||
Its primer concentration is that 0.5uM is the intermediate value of common primer concentration.General primer PCR and ultra-long primer PCR's is all identical with embodiment 3 with circulation denaturation temperature and terms and conditions first.General primer PCR condition 1 minute, is extended 72 ℃, 1 minute for 63 ℃ of annealing in the present embodiment; Ultra-long primer PCR annealing and extension are combined into a step, and condition is 75 ℃, 1 minute.
As PCR circulation 22-28, get a pipe every a circulation and measure by embodiment 1 methods analyst.The result as shown in Figure 6, general primer PCR enters flat slope in 25 circulations, the product accumulation linearly increases straight-line regression value R in 3 circulations of 22-25 round-robin
2=0.9959, the straight line accumulation of ultra-long primer PCR is extended to 28 circulations, straight-line regression value R
2=0.973.
When the PCR cycle number expands to 20-32, every next but two circulation is got a pipe and is measured.The result as shown in Figure 7, general primer product accumulation in 6 circulations of 20-26 round-robin linearly increases straight-line regression value R
2=0.9878, the straight line accumulation of ultra-long primer PCR is extended to 32 circulations, straight-line regression value R
2=0.983.
Claims (6)
1. the quantitative polymerase chain reaction process of applying high density primer, the ultimate density that it is characterized in that the positive and negative primer of said polymerase chain reaction is 1-100uM, after reaction finishes with after staining agent SYBR gold and the PCR sample mix, carry out electrophoresis together, the intensity of PCR product band on the quantitative assay running gel, the weight percent concentration of SYBR gold in sample loading buffer is ten thousand/to one of percentage.
2. the quantitative polymerase chain reaction process of applying high density primer according to claim 1, the melting temperature(Tm) that it is characterized in that positive and negative primer in the said polymerase chain reaction is 85-92 ℃.
3. according to the quantitative polymerase chain reaction process of claim 1 or 2 described applying high density primers, it is characterized in that the primer length of said PCR reaction is the 25-50 base.
4. according to the quantitative polymerase chain reaction process of claim 1 or 2 described applying high density primers, it is characterized in that the primer length of said PCR reaction is the 30-40 base.
5. according to the quantitative polymerase chain reaction process of the described applying high density primer of claim 1, it is characterized in that the step of said PCR reaction comprises the sex change of high temperature template, primer and template annealing extended for two steps, and the annealing elongating temperature is 72-82 ℃.
6. according to the quantitative polymerase chain reaction process of the described applying high density primer of claim 5, it is characterized in that the annealing elongating temperature of said PCR reaction is 75-80 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02125686 CN1201015C (en) | 2002-07-29 | 2002-07-29 | Quantitative polymerase chain reaction process applying high density primer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02125686 CN1201015C (en) | 2002-07-29 | 2002-07-29 | Quantitative polymerase chain reaction process applying high density primer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1388251A CN1388251A (en) | 2003-01-01 |
| CN1201015C true CN1201015C (en) | 2005-05-11 |
Family
ID=4745635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02125686 Expired - Fee Related CN1201015C (en) | 2002-07-29 | 2002-07-29 | Quantitative polymerase chain reaction process applying high density primer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1201015C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100355902C (en) * | 2003-04-11 | 2007-12-19 | 徐定邦 | PCR method using genome DNA as template and its reaction liquor |
| CN108334745B (en) * | 2018-03-19 | 2022-02-08 | 青岛理工大学 | Nonlinear hybrid system modeling method in polymerase chain reaction process |
-
2002
- 2002-07-29 CN CN 02125686 patent/CN1201015C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1388251A (en) | 2003-01-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7674582B2 (en) | Methods for amplification monitoring and gene expression profiling, involving real time amplification reaction sampling | |
| CN102344960B (en) | Quantification of gene expression | |
| Mansfield et al. | Nucleic acid detection using non-radioactive labelling methods | |
| US20210238652A1 (en) | Methods for analyzing samples | |
| CA2143857C (en) | Improved method for the quantification of nucleic acid | |
| CN102071251A (en) | PCR (polymerase chain reaction) nucleic acid detection method | |
| CN1201015C (en) | Quantitative polymerase chain reaction process applying high density primer | |
| EP1162278A2 (en) | Isometric primer extension method and kit for detection and quantification of specific nucleic acid | |
| US20090191562A1 (en) | METHOD FOR ASSAYING REG IV mRNA | |
| Eads et al. | [3] Troubleshooting Microarray Hybridizations | |
| WO2004050906A1 (en) | Methylation dna detecting method | |
| KR101606929B1 (en) | Method for modifying template to analyze multiple mRNA or 16S rRNA by capillary electrophoresis and detecting microorganism by using the same | |
| CN1661359B (en) | Method for measuring isothermal amplification of nucleic acid quantificationally | |
| CN118147373B (en) | Combined detection kit for influenza A viruses H1N1, H3N2, H5N1 and H7N9 | |
| CN1316036C (en) | Gene quantitation method by using PCR as basis | |
| JP4061459B2 (en) | Primers and probes for real-time detection PCR of HBV gene | |
| WO2023019671A1 (en) | Method and kit for constructing fluorescent oligonucleotide standard curve | |
| JP2005245310A (en) | Passive internal reference in real-time pcr | |
| US20040091921A1 (en) | Method for gene expression analysis and kit for gene expression analysis | |
| CN114317679A (en) | Activity detection method of terminal deoxynucleotidyl transferase | |
| WO2024027961A1 (en) | Method for the detection of sars-cov-2 by digital pcr | |
| CN117089596A (en) | Solid-phase probe and solid-phase multiplex fluorescence PCR detection method | |
| WO2004035835A2 (en) | Method for quantitative end-point pcr | |
| CN114544968A (en) | Method for detecting activity of single-stranded DNA binding protein | |
| EP0662156B1 (en) | Improved method for the quantification of nucleic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |