CN1278558A - Asynchronous chain-extending polymerase chain reaction - Google Patents
Asynchronous chain-extending polymerase chain reaction Download PDFInfo
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- CN1278558A CN1278558A CN 99114949 CN99114949A CN1278558A CN 1278558 A CN1278558 A CN 1278558A CN 99114949 CN99114949 CN 99114949 CN 99114949 A CN99114949 A CN 99114949A CN 1278558 A CN1278558 A CN 1278558A
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- necrosis factor
- tumor
- factor glycoproteins
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Abstract
The present invention relates to a kind of two chains asynchronously-elongated DNA amplification technology in vitro, which is characterized by that firstly, a pair of specific primers is synthesized according to target DNA sequence to be amplified, then a repetitive sequence complementary oligo-repetitive sequence of 3'target DNA chain whose tail end is modified and elongation vitality is lost, then said oligo-repetitive sequence, chain primer, heat-resisting DNA polymerase, dNTP substrate, template DNA, magnesium ion, PCR buffer solution and ultra-pure water are mixed uniformly and made into reaction system, then said reaction system is undergone the processes of high-temp., low-temp., medium-low temp., medium-temp, and repeated heat circulation treatment in the heat-circulating instrument to obtain million copies of specific target DNA fragments. Said invention adopts multiple repeated heat circulation process, so that is can product lots of target DNA fragments.
Description
The present invention relates to a kind of positive and negative two the asynchronous synthetic DNA of chain amplification in vitro technology.
In existing DNA amplification in vitro technology, polymerase chain reaction (polymeras chain reaction, PCR) be main also be otherwise effective technique.The reaction system that hot resistant DNA polymerase, Auele Specific Primer, dNTP substrate, template DNA, magnesium ion etc. will be contained in this technology system carries out repeatedly that the sex change of high temperature template, low temperature primer combine with template, the thermal cycling of middle temperature primer extension, and target dna fragment is increased external obtaining.But when the tumor-necrosis factor glycoproteins of the big copy number of amplification, because the tumor-necrosis factor glycoproteins template is easy to anneal, conventional PCR is difficult to obtain satisfied amplification.At first be that amplification efficiency is low, general detection means such as ethidium bromide staining or silver dye and are difficult to detect, and secondly are to occur many shadow bands easily, influence the judgement of amplification.We think that this mainly is because tumor-necrosis factor glycoproteins is easy to mutual annealing when low temperature and middle temperature, have influenced that the building-up process of new chain and combined coefficient cause.Have not yet to see the way that fundamentally solves this difficult problem.
It is a kind of simple to operate that the object of the invention is to provide, the DNA amplification in vitro technology of the tumor-necrosis factor glycoproteins of the big copy number that can effectively increase.
Asynchronous chain-extending polymerase chain reaction of the present invention (asynchronous strand-extension polymerase chain reaction, ASE-PCR) at first according to the synthetic a pair of specific strand primer of target DNA sequence to be amplified, this a pair of primer defines the target dna fragment that will increase, the tumor-necrosis factor glycoproteins that synthesizes one section 15-20 base again according to the tumor-necrosis factor glycoproteins that will increase, and add dideoxy nucleotide or phosphorylation modification at its 3 '-end, make it lose the extension ability, the note do few tumor-necrosis factor glycoproteins (oligo-repeat sequence, ORS).ORS, primer, hot resistant DNA polymerase, dNTP substrate, template DNA, magnesium ion, PCR damping fluid, ultrapure water etc. are mixed into reaction system, on thermal cycler, carry out the thermal cycling repeatedly of high temperature, low temperature, middle low temperature, middle temperature.When reaction system is in high temperature, the template DNA sex change; When system dropped to low temperature and middle low temperature, primer combined the synthetic of the new chain of guiding with special target DNA, simultaneously, the annealing of the tumor-necrosis factor glycoproteins of ORS and its complementary strand, thus the tumor-necrosis factor glycoproteins between the inhibition template is annealed mutually.In can at first not instruct the synthetic new chain of complete complementation as template during low temperature with ORS bonded chain; Then be obstructed with ORS bonded chain, can not synthesize the new chain of complete complementation at the new chain extension in tumor-necrosis factor glycoproteins district.In when temperature ORS from separating with its bonded template strand, this template then can instruct the synthetic new chain of complete complementation this moment.Through after 25-35 the thermal cycling, just can obtain the special target dna fragment that contains tumor-necrosis factor glycoproteins of copies up to a million like this.
(fragile X mental retardation 1,5 ' non-translational region FMR1) is that example is elaborated with No. 1 gene of amplification fragile X mental retardation below.
At first according to sequence (GenBanK S74494 and the Nature Genet of FMR1,8 (1) 88-94,1994) synthetic special positive primers F the F:5 '-GTTTCGGTTTCACCTTCCGGTGGAGG-3 ' of a pair of FMR1, FR:5 ' CCTCCATCTTCTCTTCAGCCCTGC-3 '; The few tumor-necrosis factor glycoproteins of resynthesis also carries out phosphorylation or add dideoxy nucleotide modifying at its 3 '-end: 5 '-CGGCGGCGGCGGCGGCGG-3 ', note is made ORS
Mix the 50ul reaction system, include each 20pmol of two primers, few tumor-necrosis factor glycoproteins ORS100pmol normal people DNA 50-100 ng, the 1XPCR damping fluid, Taq archaeal dna polymerase 2u,
Mg++Final concentration 2mmol/L, 96 ℃ of following sex change 5 minutes, then on PE9600 by 96 ℃ 20 seconds, 56 ℃ 30 seconds, 65 ℃ 30 seconds, 75 ℃ of down circulations 35 times in 1 minute 30 seconds, 2% agarose gel electrophoresis, ethidium bromide staining, ultraviolet lamp are observed down, can find to expect the DNA band of size.
In the above-mentioned reaction system, when reaction system is in high temperature, the template DNA sex change; When system drops to 56 ℃ and 65 ℃, primer combines the synthetic of the new chain of guiding with special target DNA, simultaneously, ORS and (GCC) n tumor-necrosis factor glycoproteins annealing, thereby the tumor-necrosis factor glycoproteins between the inhibition template is annealed mutually, does not make and can at first instruct synthetic complete complementary new chain as template with ORS bonded (CGG) n chain; Then be obstructed with ORS bonded chain, can not synthesize the new chain of complete complementation at the new chain extension in tumor-necrosis factor glycoproteins district.ORS separates from (GCC) n template strand in the time of 75 ℃, makes this template can instruct the synthetic new chain of complete complementation this moment.Through after 25-35 the thermal cycling, just can obtain the special target dna fragment that contains tumor-necrosis factor glycoproteins of copies up to a million like this.
Claims (5)
1, a kind of reaction system that will contain few tumor-necrosis factor glycoproteins, primer, hot resistant DNA polymerase, dNTP substrate, template DNA, magnesium ion, PCR damping fluid, ultrapure water etc. is carried out high temperature, low temperature, middle low temperature, middle temperature thermal cycling and the DNA amplification in vitro technology of a large amount of amplifications of the dna fragmentation that achieves the goal repeatedly, few tumor-necrosis factor glycoproteins wherein is a bit of tumor-necrosis factor glycoproteins complementary oligonucleotide according to the tumor-necrosis factor glycoproteins synthetic of target DNA and one bar chain, and its 3 '-terminal process is modified the extension ability that lost; The low temperature of this widow's tumor-necrosis factor glycoproteins in the thermal cycling process and in during low thermophase, with the tumor-necrosis factor glycoproteins annealing of corresponding complementary template strand, thus the tumor-necrosis factor glycoproteins that suppresses between the template anneal mutually, make another template strand at first instruct synthetic complete complementary new chain; During middle thermophase in the thermal cycling process, from separating with its bonded template strand, this template instructs the synthetic new chain of complete complementation immediately; Through thermal cycling repeatedly, produce a large amount of target DNA fragments.
2, DNA amplification in vitro technology according to claim 1 is characterized in that containing in the reaction system few tumor-necrosis factor glycoproteins.
3, few tumor-necrosis factor glycoproteins according to claim 2, to it is characterized by be tumor-necrosis factor glycoproteins synthetic 3 ' according to target DNA-end loses the oligonucleotide extension ability and a tumor-necrosis factor glycoproteins complementary 15-25nt chain through modifying.
4, the 3 '-terminal oligonucleotide that loses the extension ability according to claim 3 through modifying, its 3 '-end carries out phosphorylation or adds dideoxy nucleotide modifying, or use other materials that can combine with positive primer specific but can not under the effect of hot resistant DNA polymerase, extend, as peptide nucleic acid(PNA) (PNA).
5, DNA amplification in vitro technology according to claim 1 is according to the DNA amplification in vitro test kit of this technology assembling.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99114949 CN1278558A (en) | 1999-06-22 | 1999-06-22 | Asynchronous chain-extending polymerase chain reaction |
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| CN 99114949 CN1278558A (en) | 1999-06-22 | 1999-06-22 | Asynchronous chain-extending polymerase chain reaction |
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| CN1278558A true CN1278558A (en) | 2001-01-03 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146447A (en) * | 2011-01-12 | 2011-08-10 | 山东博奥克生物科技有限公司 | Nest coamplific action polymerase chain reaction kit |
| CN102251027A (en) * | 2011-06-07 | 2011-11-23 | 刘钦松 | Suppression polymerase chain reaction kit |
| CN110358796A (en) * | 2019-07-06 | 2019-10-22 | 天津大学 | A kind of inexpensive DNA synthetic method of the archaeal dna polymerase based on template dependant |
| CN110527713A (en) * | 2019-07-02 | 2019-12-03 | 黄天谊 | A kind of PCR amplification method folding primer |
| WO2021114135A1 (en) * | 2019-12-11 | 2021-06-17 | 清华大学 | Blocking-type chain polymerization amplification reaction based in vitro fast synthesis of medium and high-copy dna repetitive sequence |
-
1999
- 1999-06-22 CN CN 99114949 patent/CN1278558A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146447A (en) * | 2011-01-12 | 2011-08-10 | 山东博奥克生物科技有限公司 | Nest coamplific action polymerase chain reaction kit |
| CN102146447B (en) * | 2011-01-12 | 2013-06-26 | 山东博奥克生物科技有限公司 | Nest coamplific action polymerase chain reaction kit |
| CN102251027A (en) * | 2011-06-07 | 2011-11-23 | 刘钦松 | Suppression polymerase chain reaction kit |
| CN102251027B (en) * | 2011-06-07 | 2013-06-26 | 刘钦松 | Suppression polymerase chain reaction kit |
| CN110527713A (en) * | 2019-07-02 | 2019-12-03 | 黄天谊 | A kind of PCR amplification method folding primer |
| CN110527713B (en) * | 2019-07-02 | 2024-01-30 | 黄天谊 | PCR amplification method of folding primer |
| CN110358796A (en) * | 2019-07-06 | 2019-10-22 | 天津大学 | A kind of inexpensive DNA synthetic method of the archaeal dna polymerase based on template dependant |
| WO2021114135A1 (en) * | 2019-12-11 | 2021-06-17 | 清华大学 | Blocking-type chain polymerization amplification reaction based in vitro fast synthesis of medium and high-copy dna repetitive sequence |
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