CN102757960B - 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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- CN102757960B CN102757960B CN201210266240.7A CN201210266240A CN102757960B CN 102757960 B CN102757960 B CN 102757960B CN 201210266240 A CN201210266240 A CN 201210266240A CN 102757960 B CN102757960 B CN 102757960B
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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 molecular biology, but must design primer in round pcr, and this just need to 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 large, the design of primers difficulty.People design various technical schemes, detect unknown nucleotide sequence.Wherein build the storehouse order-checking more accurate, but trivial operations, experimental period is long, and funds are high; Existing arbitrarily primed PCR technology, although operation is simpler, expense is lower, because in the pcr amplification program, annealing temperature is relatively low, so the nucleic acid aim sequence specificity amplified is poor.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly lower for existing arbitrarily primed PCR technology annealing temperature, and the poor defect of nucleic acid aim sequence specificity amplified 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 is followed successively by 3 ' end from 5 ' end: 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.
The oligonucleotide that oligonucleotide of the present invention is this area routine.Wherein said oligonucleotide preferably comprises one or more in the polynucleotide of polydeoxyribonucleotide, polybribonucleotide and other types; Pyrimidine or the purine bases after modifying as purine or pyrimidine bases.There is no the difference on length between " oligonucleotide " and " nucleic acid ", can Alternate.Oligonucleotide of the present invention is originated as conventional source, this area, and the source of described oligonucleotide preferably comprises the artificial synthetic oligonucleotide or extract the described oligonucleotide of acquisition from naturally occurring genome.The artificial synthesis of wherein said oligonucleotide preferably comprises: one or more in phosphotriester method, phosphodiester method, diethyl phosphoamide method and solid phase vector.
The sequence preference ground of wherein said oligonucleotide is: as shown in SEQ ID NO.1 in sequence table or SEQ ID NO.2.The Nucleotide of the N representative in wherein said oligonucleotide preferably comprises the Nucleotide that length is 3～8 full degeneracy bases, and wherein said full degenerate core thuja acid is preferably the Nucleotide by 6 full degeneracy based compositions.The nucleotide base that wherein said full degeneracy base is this area routine, described 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 wherein said PCR step is moved the amplification gene test kit and comprised oligonucleotide as above, dNTP, MgCl
2, 10 * PCR damping fluid and TaqDNA polysaccharase.
Wherein said PCR step is moved the amplification gene test kit and is preferably comprised the conventional PCR reagent in this area.Described 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: a kind of method that PCR step is moved amplification gene, the method that wherein said PCR step is moved amplification gene be take aforesaid oligonucleotide as primer, take target dna as template, the reaction system that described 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 described pcr amplification comprises:
(1) 92～95 ℃, sex change 3～6 minutes;
(2) 92～94 ℃, sex change 30～45 seconds;
(3) 52 ℃～64 ℃, anneal 30 seconds;
(4) 72 ℃ are extended 2 minutes, and wherein 30～45 circulations are repeated in step (2)～(4);
(5) 70～75 ℃ are extended 120～300 seconds;
(6) product is in 4 ℃ of preservations.
The method that PCR step of the present invention is moved amplification gene is the conventional nucleic acid aim sequence amplification method used in this area.The amplification method of described nucleic acid aim sequence preferably refers to polymerase chain reaction (PCR).Described polymerase chain reaction (PCR) refers to a kind of method of the special purpose fragment of external enzymatic nucleic acid, the method program preferably comprises: a few step reaction composition one-period such as high-temperature denatured, low-temperature annealing (renaturation) and thermophilic extension, loop, thereby make nucleic acid purpose fragment be able to rapid amplification.
Polymerase chain reaction (PCR) system that wherein said polymerase chain reaction system is this area routine, described PCR reaction system preferably comprises: oligonucleotide (primer), nucleic acid polymerase, dNTP, template nucleotide and Mg
2+.PCR reaction system of the present invention preferably comprises: described oligonucleotide content is preferably 0.2～3 μ mol/L, is preferably 0.5 μ mol/L, and the content of described dNTP is preferably 0.2～1mmol/L, and content is preferably 0.2mmol/L, Mg
2+concentration is preferably 1.5～3mmol/L, concentration is preferably 1.5mmol/L, and Taq archaeal dna polymerase addition is preferably 0.02～1U/ μ L, and addition is preferably 0.02U/ μ L, the template nucleotide addition is preferably 20～1000ng, and addition is preferably 50ng.
The PCR reaction conditions that wherein said polymerase chain reaction condition is this area routine, described PCR response procedures preferably comprises:
(1) denaturation temperature is preferably 92～95 ℃, is preferably 94 ℃, and the sex change time, for being preferably 3～6min, is preferably 5 minutes;
(2) denaturation temperature is preferably 92～94 ℃, is preferably 94 ℃, and the sex change time is preferably 30～45 seconds, is preferably 30 seconds;
(3) annealing temperature is preferably 52～60 ℃, is preferably 56 ℃, and annealing time is preferably 30 seconds;
(4) elongating temperature is preferably 72 ℃, and the extension time is preferably 2 minutes, and wherein step (2)～(4) cycle index is preferably 30～45, is preferably 30 circulations;
(5) elongating temperature is preferably 70～75 ℃, is preferably 72 ℃, and the extension time is preferably 120～300 seconds, is preferably 300 seconds;
(6) product is in 4 ℃ of preservations.
The raw material that the present invention is used or reagent except special instruction, equal commercially available obtaining.
Than prior art, beneficial effect of the present invention is as follows: utilize half random PCR technology provided by the invention and primer thereof and test kit to carry out polymerase chain reaction, without the design primer target gene sequence that can increase, the method highly versatile, be particularly suitable for the amplification of unknown gene sequence.PCR response procedures of the present invention is simple, annealing temperature is higher than 50 ℃, and the extension increasing sequence specificity is stronger, and the sample that derives from the gene order the unknowns such as microorganism, animal, plant all can be used the method, therefore the method highly versatile, have very wide application prospect.
The accompanying drawing explanation
Below in conjunction with accompanying drawing, feature of the present invention and beneficial effect are described.
Fig. 1 is XA bacterial strain Semi-random primer PCR result.The 1st, PCR is electrophoretogram as a result, and M is DL2,000DNA marker.
Fig. 2 is BX Animal genome Semi-random primer PCR result.The 1st, PCR is electrophoretogram as a result, and M is DL2,000DNA marker.
Embodiment
Below with embodiment, further illustrate the present invention, 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.
1.1 template preparation
Separation of bacterial from pollute dairy products: use 3ml LB substratum, add the pollution dairy products of 0.1ml, cultivate 48 hours for 37 ℃, screening obtains polluting the unknown bacterium in dairy products.
Extract bacterial genomes.The test kit used is: TaKaRa minibest bacterial genomic dna extraction kit ver.2.0(Takara Biotechnology (Dalian) Co., Ltd.), the bacterial genomes DNA that extracting separates from pollute dairy products.Described 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., Ltd., and article number is DR001A.
PCR product electrophoresis result is shown in Fig. 1, and in figure, numbering " 1 " is XA Semi-random primer PCR result, and " M " is DL2, and 000DNA Marker, purchased from Takara Biotechnology (Dalian) Co, Ltd..The all purpose bands that are greater than 500bp in swimming lane 1 are reclaimed to 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 is greater than 99% with the subtilis homology, low with other bacterium homology.Identify that thus XA is 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 purchased 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 ℃, extend 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 is DR001A.
PCR product electrophoresis result is shown in Fig. 2, and in figure, numbering " 1 " is Animal genome PCR result, and " M " is DL2, and 000DNA Marker, purchased from Takara Biotechnology (Dalian) Co, Ltd..
The all purpose bands that are greater than 500bp in swimming lane 1 are reclaimed to purifying 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%, low with other species homology.Identify thus the tissue that BX is ox.
Embodiment 3 random primer Identifying micro-organisms kinds
3.1 template preparation
From pollute dairy products, the method for separation of bacterial is: use 3ml LB substratum, add the pollution dairy products of 0.1ml, cultivate 48 hours for 37 ℃, screening obtains polluting the unknown bacterium in dairy products.
Extract bacterial genomes.The test kit used is: TaKaRa minibest bacterial genomic dna extraction kit ver.2.0, and purchased from Takara Biotechnology (Dalian) Co, Ltd., the bacterial genomes DNA that extracting separates from pollute dairy products.Described 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 ℃, anneal 30 seconds;
(4) 72 ℃ are extended 2 minutes, and wherein 45 circulations are repeated in step (2)～(4);
(5) 75 ℃ are 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 primers, 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, Ltd., and article number is DR001A.
The all purpose bands that are greater than 500bp in swimming lane are reclaimed to purifying, cloning and sequencing.Select 5 clone son order-checkings, check order and be listed in the NCBI website and be BLAST, result all only is greater than 99% with the subtilis homology, low with other bacterium homology.Identify that thus XC is 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 purchased 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 ℃, anneal 30 seconds;
(4) 72 ℃ are extended 2 minutes, and wherein 30～45 circulations are repeated in step (2)～(4);
(5) 72 ℃ are 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 is DR001A.
The all purpose bands that are greater than 500bp in swimming lane are reclaimed to 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%, low with other species homology.Identify thus the tissue that DX is ox.
Should be understood that, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (3)
1. the method for a pcr amplification gene, it is characterized in that, the method of described pcr amplification gene take as SEQ ID NO.1 in sequence table or the oligonucleotide as shown in SEQ ID NO.2 in sequence table be primer, take target dna as template, carry out pcr amplification reaction, the system of described pcr amplification reaction comprises: as SEQ ID NO.1 in sequence table or 0.2～3 μ mol/L of the oligonucleotide as shown in SEQ ID NO.2 in sequence table, 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 described pcr amplification comprises:
(1) 92～95 ℃, sex change 3～6 minutes;
(2) 92～94 ℃, sex change 30～45 seconds;
(3) 52～64 ℃, anneal 30 seconds;
(4) 72 ℃ are extended 2 minutes, and 30～45 circulations are repeated in step (2)～(4);
(5) 70～75 ℃ are extended 120～300 seconds;
(6) product is in 4 ℃ of preservations.
2. the method for pcr amplification gene as claimed in claim 1, it is characterized in that, the reaction system of the method for described pcr amplification gene comprises: 0.5 μ mol/L is as SEQ ID NO.1 in sequence table or the oligonucleotide as shown in SEQ ID NO.2 in sequence table, 0.2mmol/L dNTP, 1.5mmol/L Mg
2+, 0.02U/ μ L Taq archaeal dna polymerase and 20～500ng template.
3. the method for pcr amplification gene as claimed in claim 1, is characterized in that, the program of described pcr amplification gene comprises:
(1) 95 ℃ of sex change 5 minutes;
(2) 94 ℃ of sex change 30 seconds;
Anneal 30 seconds for (3) 56 ℃;
(4) 72 ℃ are extended 2 minutes, and 30 circulations are repeated in step (2)～(4);
(5) 72 ℃, extend 300 seconds;
(6) product is in 4 ℃ of preservations.
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| CN104560974B (en) * | 2014-12-26 | 2017-08-25 | 光明乳业股份有限公司 | The semi-random primer for obtaining the method for streptococcus thermophilus specific sequence and its using |
| 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 |
| CN104531695B (en) * | 2014-12-26 | 2017-06-06 | 光明乳业股份有限公司 | A kind of specificity molecular marker DNA sequence of Lactobacillus casei and application 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 |
| CN106701747B (en) * | 2015-08-28 | 2019-12-13 | 光明乳业股份有限公司 | Universal PCR primer pair and application thereof |
| CN112553299A (en) * | 2019-09-10 | 2021-03-26 | 北京大学第一医院 | NOTCH2NLC gene GGC repetitive sequence amplification method |
| CN112430653B (en) * | 2020-12-04 | 2024-07-05 | 南昌大学 | Differential annealing mediated stem-loop PCR technology for genome walking |
| CN113249507B (en) * | 2021-07-05 | 2021-12-10 | 湖南赛哲智造科技有限公司 | Co-detection method for existence and expression condition of pathogen drug resistance gene |
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