CN102757960A - Semi-random polymerase chain reaction (PCR) technique for amplifying unknown sequence and primer and kit used for technique - Google Patents
Semi-random polymerase chain reaction (PCR) technique for amplifying unknown sequence and primer and kit used for technique Download PDFInfo
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Abstract
The invention discloses a semi-random polymerase chain reaction (PCR) technique for amplifying an unknown sequence and a primer and a kit used for the technique. The semi-random primer sequence comprises the following components in sequence from 5' end to 3' end: 12-30 fixed basic groups, 3-8 degenerate basic groups N and 3-7 fixed basic groups, wherein N is A or G or C or T. The sequence of the semi-random primer is as shown in SEQ ID NO.1 or SEQ ID NO.2 in a sequence list. The process of the PCR reaction containing the primer preferably comprises the following steps of: (1) degenerating for 5 minutes at 95 DEG C; (2) degenerating for 30 seconds at 94 DEG C; (3) annealing for 30 seconds at 56 DEG C; (4) extending for 2minutes at 72 DEG C, repeating the step (2)-(3) for 30 cycles; (5) extending for 300 seconds at 72 DEG C; and (6) preserving the product at 4 DEG C. The semi-random primer is strong in generality, and is particularly suitable for amplification of unknown gene sequences. The PCR reaction process is simple, and is strong in specificity for amplifying target sequences of nucleic acid.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of half random PCR technology and primer and test kit of amplify unknown sequence.
Background technology
Polymerase chain reaction technology (PCR) is the important technical in the molecular biology, but must design primer in the round pcr, and this just need know the partial sequence of target gene.Up to the present, the genome sequence of most species is still unknown, and the gene order variation is big, the design of primers difficulty.People design various technical schemes, detect unknown nucleotide sequence.It is more accurate wherein to build the storehouse order-checking, but trivial operations, experimental period is long, and funds are high; Existing arbitrarily primed PCR technology, though operation is simpler, expense is lower, because annealing temperature is relatively low in the pcr amplification program, so the nucleic acid aim sequence specificity that amplifies is relatively poor.
Summary of the invention
Therefore, the technical problem that the present invention will solve is exactly lower to existing arbitrarily primed PCR technology annealing temperature, and the relatively poor defective of nucleic acid aim sequence specificity that amplifies provides a kind of half random PCR technology and primer and test kit of amplify unknown sequence.
For solving the problems of the technologies described above; One of technical scheme that the present invention takes is: a kind of oligonucleotide; Wherein said oligonucleotide sequence holds 3 ' end to be followed successively by from 5 ': 12～30 fixing bases; The base N of 3～8 full degeneracys and 3～7 are base fixedly, and wherein said N is: A or G or C or T.
Oligonucleotide according to the invention is the conventional oligonucleotide in this area.Wherein said oligonucleotide preferably comprises one or more in the polynucleotide of polydeoxyribonucleotide, polybribonucleotide and other types; Like pyrimidine or the purine bases after purine or the modification of pyrimidine bases process.Do not have the difference on the length between " oligonucleotide " and " nucleic acid ", can exchange use.Oligonucleotide of the present invention is originated and is conventional source, this area, and the source of said oligonucleotide preferably comprises the artificial synthetic oligonucleotide or from naturally occurring genome, extracts the described oligonucleotide of acquisition.The artificial synthesis of wherein said oligonucleotide preferably comprises: one or more in phosphotriester method, phosphodiester method, diethylammonium phosphoamide method and the solid phase vector.
The sequence preference ground of wherein said oligonucleotide is: shown in SEQ ID NO.1 in the sequence table or SEQ ID NO.2.The Nucleotide of the N representative in the wherein said oligonucleotide comprises that preferably length is the Nucleotide of 3～8 full degeneracy bases, and wherein said full degenerate core thuja acid is preferably the Nucleotide by 6 full degeneracy based compositions.Wherein said full degeneracy base is the conventional nucleotide base in this area, and said full degeneracy nucleotide base preferably comprises: VITAMIN B4 (A), guanine (G), cytosine(Cyt) (C) or thymus pyrimidine (T).
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: a kind of PCR step is moved the amplification gene test kit, and the wherein said PCR step moves the amplification gene test kit and comprises aforesaid oligonucleotide, dNTP, MgCl
2, 10 * PCR damping fluid and TaqDNA polysaccharase.
The wherein said PCR step moves the amplification gene test kit and preferably comprises the conventional PCR reagent in this area.Said PCR reagent preferably comprises: dNTP, MgCl
2Solution, PCR damping fluid and TaqDNA polysaccharase.
For solving the problems of the technologies described above; Three of the technical scheme that the present invention takes is: the method that a kind of PCR step is moved amplification gene; The method that the wherein said PCR step is moved amplification gene is a primer with aforesaid oligonucleotide, is template with the target dna, and the reaction system that the said PCR step is moved the method for amplification gene comprises: foregoing oligonucleotide 0.2～3 μ mol/L; 0.2～1mmol/L dNTP, the Mg of 1.5～3mmol/L
2+, Taq archaeal dna polymerase 0.02～1U/ μ L, the addition of template nucleotide is 20～1000ng; The response procedures of said pcr amplification comprises:
(1) 92～95 ℃, sex change 3～6 minutes;
(2) 92～94 ℃, sex change 30～45 seconds;
(3) 52 ℃～64 ℃, annealed 30 seconds;
(4) 72 ℃ were extended 2 minutes, and wherein 30～45 circulations are repeated in step (2)～(4);
(5) 70～75 ℃ were extended 120～300 seconds;
(6) product is in 4 ℃ of preservations.
The method that the PCR step of the present invention is moved amplification gene is the conventional nucleic acid aim sequence amplification method that uses in this area.The amplification method of said nucleic acid aim sequence preferably refers to polymerase chain reaction (PCR).Said polymerase chain reaction (PCR) is meant the segmental a kind of method of the special purpose of external enzymatic nucleic acid; This method program preferably comprises: one-period are formed in several steps reactions such as high-temperature denatured, low-temperature annealing (renaturation) and thermophilic extension; Circulation is carried out, thereby makes nucleic acid purpose fragment be able to rapid amplification.
Wherein said polymerase chain reaction system is conventional polymerase chain reaction (PCR) system in this area, and said PCR reaction system preferably comprises: oligonucleotide (primer), nucleic acid polymerase, dNTP, template nucleotide and Mg
2+PCR reaction system according to the invention preferably comprises: described oligonucleotide content preferably is 0.2～3 μ mol/L, is preferably 0.5 μ mol/L, and the content of said dNTP is 0.2～1mmol/L preferably, and content is preferably 0.2mmol/L, Mg
2+Concentration preferably is 1.5～3mmol/L; Concentration is preferably 1.5mmol/L, and Taq archaeal dna polymerase addition preferably is 0.02～1U/ μ L, and addition is preferably 0.02U/ μ L; The template nucleotide addition is 20～1000ng preferably, and addition is preferably 50ng.
Wherein said polymerase chain reaction condition is the conventional PCR reaction conditions in this area, and said PCR response procedures preferably comprises:
(1) denaturation temperature is 92～95 ℃ preferably, is preferably 94 ℃, and the sex change time is preferably 5 minutes for being 3～6min preferably;
(2) denaturation temperature is 92～94 ℃ preferably, is preferably 94 ℃, and the sex change time is 30～45 seconds preferably, is preferably 30 seconds;
(3) annealing temperature is 52～60 ℃ preferably, is preferably 56 ℃, and annealing time preferably is 30 seconds;
(4) elongating temperature is preferably 72 ℃, and the extension time is preferably 2 minutes, and wherein step (2)～(4) cycle index is 30～45 preferably, is preferably 30 circulations;
(5) elongating temperature is 70～75 ℃ preferably, is preferably 72 ℃, and the extension time is 120～300 seconds preferably, is preferably 300 seconds;
(6) product is in 4 ℃ of preservations.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
Than prior art; Beneficial effect of the present invention is following: utilize half random PCR technology provided by the invention and primer thereof and test kit to carry out the polymerase chain reaction; Need not to design the primer target gene sequence that can increase, this method highly versatile is particularly suitable for the amplification of unknown gene sequence.PCR response procedures according to the invention is simple; Annealing temperature is higher than 50 ℃, and the extension increasing sequence specificity is stronger, derives from the unknown sample of gene orders such as mikrobe, animal, plant and all can use this method; Therefore this method highly versatile has very wide application prospect.
Description of drawings
Below in conjunction with description of drawings characteristic of the present invention and beneficial effect.
Fig. 1 is XA bacterial strain half arbitrarily primed PCR result.The 1st, PCR is electrophoretogram as a result, and M is DL2,000DNA marker.
Fig. 2 is BX animal gene group half arbitrarily primed PCR result.The 1st, PCR is electrophoretogram as a result, and M is DL2,000DNA marker.
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1 random primer Identifying micro-organisms kind
1.1 template preparation
Separation of bacterial from pollute dairy products: use 3ml LB substratum, add the pollution dairy products of 0.1ml, cultivated 48 hours for 37 ℃, screening obtains polluting the unknown bacterium in the dairy products.
Extract bacterial genomes.The test kit that uses is: (Takara Biotechnology (Dalian) Co., Ltd.), extracting is the bacterial isolated genomic dna from pollute dairy products for TaKaRa minibest bacterial genomic dna extraction kit ver.2.0.Said bacterial genomes DNA is numbered XA.
1.2PCR amplification
Use half random primer AP1:5 '-ggc gtt tat tca gaa g (n)
6Cgc c-3 ' does pcr amplification.The PCR program is:
(1) 94 ℃, sex change time 5min;
(2) 94 ℃, sex change 30S;
(3) 52 ℃, annealing 30S;
(4) 70 ℃, extend 30S; 30 circulations are repeated in step (2)～(4);
(5) 72 ℃ are extended 5min
(6) product is in 4 ℃ of preservations.
PCR reaction system volume is 20 μ l, and wherein each component final concentration is: 1 μ mol/L, half random primer, 0.2mmol/L dNTP, 1 * PCR damping fluid, the Mg of 1.5mM
2+, Taq archaeal dna polymerase 0.02U/ μ L, template 20ng.Wherein the manufacturer of TaqDNA polysaccharase is TakaraBiotechnology (Dalian) Co., and Ltd., article number are DR001A.
PCR product electrophoresis result is seen Fig. 1, and numbering " 1 " is XA half arbitrarily primed PCR result among the figure, and " M " is DL2, and 000DNA Marker is available from Takara Biotechnology (Dalian) Co, Ltd..All purpose bands greater than 500bp in the swimming lane 1 are reclaimed purifying, cloning and sequencing.Select 6 clone son order-checkings, check order and be listed in the NCBI website and be BLAST, its result all only with the subtilis homology greater than 99%, low with other bacterium homology.Identify that thus XA is a subtilis.
Embodiment 2 random primers are identified animal species
2.1 template preparation
Extract animal tissues's genomic dna.Used kit is: DNA isolation reagent for meat and meat products, and available from Takara Biotechnology (Dalian) Co, Ltd..Animal tissues's genome of gained is numbered BX.
2.2PCR amplification
With half random primer AP2:5 '-gtc ggc gtt tat tca gaa g (n)
6Acg cc-3 ' does pcr amplification.The PCR program is:
(1) 94 ℃, sex change 5min;
(2) 94 ℃, sex change 30S;
(3) 58 ℃, annealing 30S;
(4) 72 ℃, extended two minutes; 30 circulations are repeated in step (2)～(4);
(5) 72 ℃ are extended 5min
(6) product is in 4 ℃ of preservations.
PCR reaction system volume is 20 μ l, and wherein each component final concentration is: 1 μ mol/L, half random primer, 0.2mmol/L dNTP, 1 * PCR damping fluid, the Mg of 1.5mM
2+, TaqDNA polysaccharase 0.02U/ μ L, template DNA 200ng, wherein the manufacturer of TaqDNA polysaccharase is Takara Biotechnology (Dalian) Co., Ltd., article number are DR001A.
PCR product electrophoresis result is seen Fig. 2, and numbering " 1 " is animal gene group PCR result among the figure, and " M " is DL2, and 000DNA Marker is available from Takara Biotechnology (Dalian) Co, Ltd..
All purpose bands greater than 500bp in the swimming lane 1 are reclaimed purifying and cloning and sequencing.Check order and be listed in the NCBI website and be BLAST, its result and cow genome homology are greater than 99%, and be low with other species homology.Identify that thus BX is the tissue of ox.
Embodiment 3 random primer Identifying micro-organisms kinds
3.1 template preparation
The method of separation of bacterial is from pollute dairy products: use 3ml LB substratum, add the pollution dairy products of 0.1ml, cultivated 48 hours for 37 ℃, screening obtains polluting the unknown bacterium in the dairy products.
Extract bacterial genomes.The test kit that uses is: TaKaRa minibest bacterial genomic dna extraction kit ver.2.0, and available from Takara Biotechnology (Dalian) Co, Ltd., extracting is the bacterial isolated genomic dna from pollute dairy products.Said bacterial genomes DNA is numbered XC.
3.2PCR amplification
Use half random primer AP1:5 '-ggcgtt tat tca gaa g (n)
6Cgc c-3 ' does pcr amplification.The PCR program is:
(1) 95 ℃, sex change 6 minutes;
(2) 94 ℃, sex change 45 seconds;
(3) 64 ℃, annealed 30 seconds;
(4) 72 ℃ were extended 2 minutes, and wherein 45 circulations are repeated in step (2)～(4);
(5) 75 ℃ were extended 180 seconds;
(6) product is in 4 ℃ of preservations.
PCR reaction system volume is 20 μ l, and wherein each component final concentration is: 3 μ mol/L, half random primer, 0.2mmol/L dNTP, 1 * PCR damping fluid, the Mg of 1.5mM
2+, Taq archaeal dna polymerase 0.02U/ μ L, template DNA 1000ng.Wherein the manufacturer of TaqDNA polysaccharase is Takara Biotechnology (Dalian) Co, and Ltd., article number are DR001A.
All purpose bands greater than 500bp in the swimming lane are reclaimed purifying, cloning and sequencing.Select 5 clone son order-checkings, check order and be listed in the NCBI website and be BLAST, the result all only with the subtilis homology greater than 99%, low with other bacterium homology.Identify that thus XC is a subtilis.
Embodiment 4 random primers are identified animal species
4.1 template preparation
Extract animal tissues's genomic dna.Used kit is: DNA isolation reagent for meat and meat products, and available from Takara Biotechnology (Dalian) Co, Ltd..Animal tissues's genome of gained is numbered DX.
4.2PCR amplification
With half random primer AP2:5 '-gtc ggc gtt tat tca gaa g (n)
6Acg cc-3 ' does pcr amplification.The PCR program is:
(1) 94 ℃, sex change 5 minutes;
(2) 93 ℃, sex change 40 seconds;
(3) 60 ℃, annealed 30 seconds;
(4) 72 ℃ were extended 2 minutes, and wherein 30～45 circulations are repeated in step (2)～(4);
(5) 72 ℃ were extended 120 seconds;
(6) product is in 4 ℃ of preservations.
PCR reaction system volume is 20 μ l, and wherein each component final concentration is: 0.2 μ mol/L, half random primer, 0.2mmol/LdNTP, 1 * PCR damping fluid, the Mg of 1.5mM
2+, TaqDNA polysaccharase 0.02U/ μ L, template DNA 500ng, wherein the manufacturer of TaqDNA polysaccharase is Takara Biotechnology (Dalian) Co, Ltd., article number are DR001A.
All purpose bands greater than 500bp in the swimming lane are reclaimed the purifying cloning and sequencing.Check order and be listed in the NCBI website and be BLAST, result and cow genome homology are greater than 99%, and be low with other species homology.Identify that thus DX is the tissue of ox.
Should be understood that after having read foregoing of the present invention those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (7)
1. an oligonucleotide is characterized in that, said oligonucleotide sequence holds 3 ' end to be followed successively by from 5 ': 12～30 fixing bases, and the base N of 3～8 full degeneracys and 3～7 are base fixedly, and wherein said N is: A or G or C or T.
2. oligonucleotide as claimed in claim 1 is characterized in that, the sequence of said oligonucleotide is shown in SEQ ID NO.1 in the sequence table or SEQ ID NO.2.
3. a PCR goes on foot the test kit that moves amplification gene, it is characterized in that, the test kit that the said PCR step is moved amplification gene comprises oligonucleotide as claimed in claim 1.
4. the PCR step as claimed in claim 3 is moved the test kit of amplification gene, it is characterized in that, the test kit that the said PCR step is moved amplification gene also comprises dNTP, MgCl
2, 10 * PCR damping fluid and TaqDNA polysaccharase.
5. a PCR goes on foot the method for moving amplification gene; It is characterized in that the method that the said PCR step is moved amplification gene is a primer with the described oligonucleotide of claim 1, is template with the target dna; Carry out pcr amplification reaction; The system of said pcr amplification reaction comprises: described oligonucleotide 0.2～3 μ mol/L of claim 1,0.2～1mmol/L dNTP, the Mg of 1.5～3mmol/L
2+, Taq archaeal dna polymerase 0.02～1U/ μ L, the addition of template nucleotide is 20～1000ng; The response procedures of said pcr amplification comprises:
(1) 92～95 ℃, sex change 3～6 minutes;
(2) 92～94 ℃, sex change 30～45 seconds;
(3) 52～64 ℃, annealed 30 seconds;
(4) 72 ℃ were extended 2 minutes, and 30～45 circulations are repeated in step (2)～(4);
(5) 70～75 ℃ were extended 120～300 seconds;
(6) product is in 4 ℃ of preservations.
6. the method for moving amplification gene like the said PCR step of claim 5 is characterized in that the reaction system that the said PCR step is moved the method for amplification gene comprises: the described oligonucleotide of 0.5 μ mol/L claim 1,0.2mmol/L dNTP, 1.5mmol/L Mg
2+, 0.02U/ μ L Taq archaeal dna polymerase and 20 ~ 500ng template.
7. the method for moving amplification gene like the said PCR step of claim 5 is characterized in that, the program that the said PCR step is moved amplification gene comprises:
(1) 95 ℃ of sex change 5 minutes;
(2) 94 ℃ of sex change 30 seconds;
Annealed 30 seconds for (3) 56 ℃;
(4) 72 ℃ were extended 2 minutes, and 30 circulations are repeated in step (2)～(3);
(5) 72 ℃, extended 300 seconds;
(6) product is in 4 ℃ of preservations.
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| CN104498492A (en) * | 2014-12-26 | 2015-04-08 | 光明乳业股份有限公司 | Method of acquiring specific sequence of lactobacillus casei and semi-random primer utilized by same |
| CN104531695A (en) * | 2014-12-26 | 2015-04-22 | 光明乳业股份有限公司 | Specific molecular marker DNA sequence of lactobacillus casei and application thereof |
| CN104560974A (en) * | 2014-12-26 | 2015-04-29 | 光明乳业股份有限公司 | Method for acquiring specific sequence of Streptococcus thermophiles and semi-random primer utilized by method |
| CN106701747A (en) * | 2015-08-28 | 2017-05-24 | 光明乳业股份有限公司 | Universal PCR (polymerase chain reaction) primer pair and application thereof |
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| CN113249507A (en) * | 2021-07-05 | 2021-08-13 | 广州赛哲生物科技股份有限公司 | Co-detection method for existence and expression condition of pathogen drug resistance gene |
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| CN104498492A (en) * | 2014-12-26 | 2015-04-08 | 光明乳业股份有限公司 | Method of acquiring specific sequence of lactobacillus casei and semi-random primer utilized by same |
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| CN104560974A (en) * | 2014-12-26 | 2015-04-29 | 光明乳业股份有限公司 | Method for acquiring specific sequence of Streptococcus thermophiles and semi-random primer utilized by method |
| CN104498492B (en) * | 2014-12-26 | 2017-05-17 | 光明乳业股份有限公司 | Method of acquiring specific sequence of lactobacillus casei and semi-random primer utilized by same |
| CN104498491A (en) * | 2014-12-26 | 2015-04-08 | 光明乳业股份有限公司 | DNA sequence of specific molecular marker of streptococcus thermophilus and use thereof |
| CN104498491B (en) * | 2014-12-26 | 2017-07-21 | 光明乳业股份有限公司 | A kind of specificity molecular marker DNA sequence of streptococcus thermophilus and application thereof |
| CN106701747A (en) * | 2015-08-28 | 2017-05-24 | 光明乳业股份有限公司 | Universal PCR (polymerase chain reaction) primer pair and application thereof |
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| CN112553299A (en) * | 2019-09-10 | 2021-03-26 | 北京大学第一医院 | NOTCH2NLC gene GGC repetitive sequence amplification method |
| CN112430653A (en) * | 2020-12-04 | 2021-03-02 | 南昌大学 | Differential annealing mediated stem-loop PCR technology for genome walking |
| CN113249507A (en) * | 2021-07-05 | 2021-08-13 | 广州赛哲生物科技股份有限公司 | Co-detection method for existence and expression condition of pathogen drug resistance gene |
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