AU2020103472A4 - A PCR Method for Obtaining the Product with High Fidelity and 3'-end Addition of "A" - Google Patents
A PCR Method for Obtaining the Product with High Fidelity and 3'-end Addition of "A" Download PDFInfo
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Abstract
The invention discloses a PCR method for obtaining the product with high fidelity and 3'
end addition of "A", which comprises the following steps: First, each reaction component and
high fidelity Taq DNA polymerase is added into the PCR reaction system, then the PCR
amplification is carried out according to the following procedures: (1) Pre-denaturation at 95°C
for 2mins; (2) 95°C, 15sec; annealing temperature, 30sec; 72°C, elongation time; 28 cycles; (3)
The common Taq DNA polymerase is added directly into the reaction PCR tube and mixed
uniformly; (4) 95°C, 15sec; annealing temperature, 30sec; 72°C, elongation time; 3 cycles; (5)
72°C, 10 min. The common Taq DNA polymerase is added in the last 3 cycles in the PCR reaction
when applying the high fidelity Taq DNA polymerase in the invention. After the PCR is
completed, the PCR product with high fidelity and 3'-end addition of "A" is obtained, overcoming
the problem that current high fidelity PCR process is unable to add "A" directly at 3'-end of the
product, eliminating the procedure of high fidelity PCR product purification required for the
addition of "A", and simplifying the process steps before the high fidelity PCR product TA is
cloned.
Description
A PCR Method for Obtaining the Product with High Fidelity and 3'-end Addition of "A"
[0001] The invention relates to a PCR method for obtaining the product with high fidelity and 3'-end addition of "A", belonging to the technical field of PCR method.
[0002]PCR technology is one of the most classical and commonly used methods in molecular biology. It is mainly used for gene detection and cloning. Because of its simple process, short experiment cycle, good reproducibility and relatively low cost of reagents, it has become one of the most popular methods in molecular biology. Although there are many methods for gene acquisition, such as chemical synthesis, PCR has irreplaceable advantages and is still the most important method.
[0003] The basic process of PCR technology for gene cloning is to firstly obtain the DNA template or cDNA template of the target gene, then design and synthesize the upstream and downstream primers of the target gene, select the appropriate Taq DNA polymerase and PCR program to obtain the PCR product by high efficiency amplification, that is, the target gene DNA, the product after purification and proper treatment is cloned into the cloning vector, and the target gene is obtained by bacterial transformation, screening and identification.
[0004] The importing of PCR product into the cloning vector varies due to different products, which is closely related to the Taq DNA polymerase selected during the PCR process. If the common Taq DNA polymerase is selected, the 3' end of the PCR product has a built-in protruding "A", which can be directly connected with the TA cloning vector on the market by T-A pairing to obtain the cloning vector with the target gene efficiently. However, due to the characteristics of the common Taq DNA polymerase, its PCR products are often accompanied by certain fixeTaq base mutation, so it is unable to clone the target gene. In order to overcome this defect, it is commonly used in molecular biology to replace the conventional Taq DNA polymerase with high fidelity Taq DNA polymerase, such as pfu DNA polymerase, platinum Taq DNA polymerase, High-Fidelity DNA polymerase and so on. However, no matter which one of the high-fidelity Taq DNA polymerase, the end of the PCR product is flat, there is no 3' with protruding "A". In order to make sure that the PCR product can be cloned into the cloning vector, the bio-reagent company. Has introduced the flat-end cloning vector, but its low cloning efficiency limits the promotion and application of the flat-end vector. In view of this problem, the bio-reagent company also introduces the A-tailing enzyme which added "A" to the flat end product of PCR, and realizes T-A cloning by adding "A" to the 3' end of the flat end product of the purified PCR, which is more efficient and popular among researchers, but the process needs the steps of electrophoresis identification, purification and adding "A" to the PCR product in order to carry out TA cloning.
[0005] To sum up, although the target DNA fragments amplified with common Taq DNA polymerase has the protruding "A" the 3' end, it often contains internal mutation sites; The PCR product amplified by high-fidelity Taq DNA polymerase whose 3' end is flat cannot be directly TA cloned, and the PCR product requires special adding "A" kit to be added to the 3' end for TA cloning; Direct combination of common Taq DNA Polymerase and high-fidelity Taq DNA polymerase (such as a biological company's Taq Platinum DNA Polymerase), although has solved some of the above problems, however, the high fidelity of PCR products and the efficiency of adding "A" at the 3' end decrease obviously.
[0006] Therefore, it is of great value to explore a simple and rapid method for obtaining high fidelity PCR products that adds "A" at the 3' end.
[0007] The technical problem to be solved in the invention is to provide a PCR method for obtaining the product with high fidelity and 3' end addition of "A". The high-fidelity PCR product with 3' end addition of "A" by single PCR not only ensures the high fidelity of the PCR product, but also makes the 3' end of the PCR product have protruding "A" base, without adding additional "A" to the PCR product, so the problems existing in the above-mentioned techniques could be solved.
[0008] The technical scheme of the invention is that: A PCR method for obtaining the product with high fidelity and 3' end addition of "A". The method comprises the following steps: Firstly adding PCR buffer, dNTP, upstream and downstream primers, template and high fidelity Taq DNA polymerase in the PCR reaction system, and then PCR amplification is carried out according to the following reaction procedures:
[0009] (1) Pre-denaturation at 95°C for 2 min;
[0010] (2) 95°C, 15 sec; annealing temperature, 30 sec; 72°C, elongation time; 28 cycles;
[0011] (3) The common Taq DNA polymerase (1.25U/50jL reaction system) is directly added into the PCR tube and mixed uniformly.
[0012] (4) 95°C, 15 see; annealing temperature, 30 sec; 72°C, elongation time; 3 cycles;
[0013] (5) 72°C, 10 min.
[0014] The final concentration of PCR buffer is "1x", the dNTPs is 200 pM, the upstream and downstream primers are 0.2 pM, the template is 500 ng, the high-fidelity Taq DNA polymerase is 1.25 U/50 pL, and the common Taq DNA polymerase is 1.25 U/50 pL.
[0015] The beneficial effect of the invention is that: Compared with the prior art, the common Taq DNA polymerase is added in the last 3 cycles in the PCR reaction when applying the high fidelity Taq DNA polymerase in the invention. After the PCR is completed, the PCR product with high fidelity and 3'-end addition of "A" is obtained, overcoming the problem that current high fidelity PCR process is unable to add "A" directly at 3'-end of the product, eliminating the procedure of high fidelity PCR product purification required for the addition of "A", simplifying the process steps before the high fidelity PCR product TA is cloned, and simplifying the intermediate steps; It can also reduce reagent consumption, release the need of adding "A" kit and saves the experimental cost.
[0016] FIGURES
[0017] Figure 1 shows the PCR amplification schematic diagram for the reaction program;
[0018] Figure 2 shows the DNA fragments of PKC& gene coding region of PCR products detected by agarose gel electrophoresis.
[0019] Figure 3 shows the TA cloning of the purified product of PCR.
[0020] Figure 4 shows the monoclonal colony of TA cloning identified by Xho I enzyme digestion.
[0021] Figure 5 shows the results of plasmid sequencing of the monoclonal strain obtained from TA cloning of LA Taq DNA polymerase product.
[0022] Sequence comparison results;
[0023] Figure 6 shows the results of plasmid sequencing of A monoclonal strain obtained from TA cloning of LA Taq DNA polymerase product compared with the sequencing of PKC6 gene coding region (Note: The results of comparison shows that there are 6 mutation sites, which are at 223 A> G, 497 A> G, 950 G> A, 1257 T> C, 1357 A> G,
1379 T> C, 2126 T> C, respectively).
[0024] Figure 7 shows the result of plasmid sequencing of the monoclonal strain obtained from TA cloning of G X L Taq DNA polymerase product with 3'-end addition of "A".
[0025] Figure 8 shows the result of plasmid sequencing of the monoclonal strain obtained from the TA cloning of the PCR product of G X L Taq DNA polymerase combined with Z-Taq DNA polymerase
(Note: The product has no mutation in comparison of the sequencing).
[0026] The present invention is further introduced in combination with specific embodiments.
[0027] Embodiment 1: A PCR method for obtaining the product with high fidelity and 3' end addition of "A", which is: The PCR buffer with a final concentration of "1x", 200 pM dNTP, 0.2 pM upstream and downstream primers, 500 ng template and 1.25 U/50 pL high fidelity Taq DNA polymerase were firstly added into the PCR reaction system. Then PCR amplification was carried out according to the following procedure:
[0028] (1) Pre-denaturation at 95C for 2 min;
[0029] (2) 95°C, 15 sec (here indicating second or not) denaturation; annealing temperature, see; 72°C, elongation time; 28 cycles;
[0030] (3) The common Taq DNA polymerase (1.25 U/50 pL reaction system) was directly added into the PCR tube and mixed uniformly.
[0031] (4) Denaturation at 95°C, 15 sec; annealing temperature, 30 sec; 72°C, elongation time; 3 cycles;
[0032] (5) 72°C, 10 min.
[0033]In order to illustrate the beneficial effect of the invention, the following tests were carried out:
[0034] Taking in vitro amplification of PKCc gene in human hepatocellular carcinoma cells as an example:
[0035] (I) Reagent materials and main instruments
[0036] 1. Reagent materials
[0037] RPMI-1640 cell culture medium was from GIBCO, newborn bovine serum was from
Hangzhou Sijiing, SMMC-7721 was from Shanghai Cell Bank of CAS, Trizol was from Invitrogen, various organic chemical reagents were from Shanghai Sinopharm, DNA-Tailing Kit, Z-Taq DNA polymerase, TaKaRa LA Taq8with GC Buffer, and reverse transcription kit were from Takarabiomed.
[0038] 2. Main Instruments
[0039] Thermo PCR instrument, Beijing Liuyi electrophoretic apparatus, Sigma high-speed refrigerating centrifuge.
[0040] 3. DNA sequencing
[0041] Primer synthesis and DNA sequencing were commissioned by Invitrogen Company.
[0042] (II) Operation Process
[0043] 1. Human hepatocellular carcinoma cell line SMMC-7721 culture
[0044] Human hepatocellular carcinoma cell SMMC-7721 was cultured in RPMI-1640 cell culture medium supplemented with 10% of newborn bovine serum at 37°C and 5% C02.
[0045] 2. Total RNA extracted from cells by Trizol method
[0046] When the fusion growth of the cells reached to 80%-90% in the culture dish with the diameter of 6 cm, the cells in the culture dish were cleaned with the sterileaseptic PBS treated by with DEPC for 3 times. Then the 1 ml RNA extractant, Trizol, was added and repeatedly blown to the bottom of the culture dish until the cells were all cracked. The lysate was placed in the 1.5 ml centrifuge tube of RNase-free, then 200 pl chloroform was added. After 15 see of severe shake, it was centrifuged at 4°C and 10000 g for 10 min. The water phase solution was collected, 500 pl isopropanol was added, and it was centrifuged at 7500 g for 5 min after inversion and uniform mixing, and then the supernatant was removed. The white sediment was cleaned with 75% of ethanol for twice, each centrifuged at 7500 g for 5 min and the supernatant was removed. It was dried naturally at room temperature for 3 min, and the white RNA sediment was dissolved with 100pl sterilized RNase-free ddH 20.
[0047] 3. Reverse transcription of mRNA into cDNA
[0048] Taking 2pg RNA and referring to the instruction of Takara reverse transcription Kit, Prime Script II1st Strand cDNA Synthesis Kit, mRNA was reverse transcribed into cDNA.
[0049] 4. Using cDNA as the template, the coding region of PKCs gene protein in human hepatocellular carcinoma cells was amplified.
[0050] PCR primers were designed according to the sequence number of PKCs gene (NM_005400) in NCBI. The sequences are as follow:
[0051] Upstream primer: 5'-TCTCGAGCGACCATGGTAGTGTTCAATGGCCTTC-3'
[0052] Downstream primer: 5'-TGGATCCCAGGGCATCAGGTCTTCACCAAAGTAG-3'
[0053] The first tube of PCR reaction solution was prepared with Takara LA Taq DNA polymerase according to the instruction; The second and third tube of PCR reaction solution was prepared with Takara high-fidelity PCR kit PrimeSTAR* G X L DNA Polymerase according to the instruction; And the PCR product was
obtained by 28 cycles after annealing at 56°C by three-step method. Then, Takara Z-Taq was added to the second tube of reaction solution. DNA polymerase. The third tube of reaction solution continued to react for 3 cycles. The specific dosage of the reagent and the experimental procedure are shown in Table 1 below:
[0054]
Table 1. Specific dosage of reagents and experimental procedures
Reaction tube 1 Reaction tube 2 Reaction tube 3
Reagents Dosage ( ii L) Reagents Dosage (pL) Reagents Dosage (pL)
2 X GC buffer 1 25 5 XGXL buffer 10 5 XGXL buffer 10
dNTPs (2.5 mM) 4 dNTPs (2.5 mM) 4 dNTPs (2.5 mM) 4
Primers (10 kM) 1, respectively Primers (10 M) 1, respectively Primers (10 M) 1, respectively
cDNA 1 cDNA 1 cDNA 1
LA Taq DNA 0.25 GXL Taq DNA 1 GXL Taq DNA 1 polymerase polymerase polymerase
Sterile water 17.75 Sterile water 32 Sterile water 32
94 °C, 30 sec; 56 °C, 30 sec; 72 °C, 3 min; 28 cycles
--- --- Z-Taq DNA 0.5 --- -- polymerase
94 °C, 30 sec; 56 °C, 30 sec; 72 °C, 3 min; 3 cycles
[0056] 5. The PCR product was subject to TA cloning
[0057] The third tube of PCR products was precipitated with acidified ethanol of twice the volume, centrifuged at 7500g for 5 min, then the supernatant was removed, the DNA sediment was washed with % ethanol for twice, the DNA was re-dissolved with aseptic water, and the volume was fixed to 1
Itg/ILL.
[0058] 5 g of the PCR purified product of reaction tube 3 was taken out, the adding "A" reaction solution was prepared according to Takara DNA A-Tailing Kit's specification and it was prepared at 72°C for 30 min.
[0059] Each PCR product was subject to TA cloning, and the reaction solution was prepared as follows:
Purified products in reaction tube 1 and 2 Product in reaction tube 3
Reagent name Dosage(pL) Reagent name Dosage(pL)
pMD18-T vector 1 pMD18-T vector 1
PCR product 0.4 (0.4 Vg) PCR product 4 (0.4 Vg)
Sterile water 3.6 Sterile water 0
Solution 1 5 Solution 1 5
[0060]
[0061] Reaction at 4°C overnight.
[0062]6. Bacterial transformation and identification
[0063] 100 L competent bacteria were put into ice bath, and the coupling reaction solution was added to the competent bacteria, 30 min in ice bath, heat shocked for 60 s at 42°C, 2 min in ice bath, then 900 L LB bacteria culture medium was added, recovery for 1 h at 37°C, and 200 L bacteria solution was coated on the solid culture plate with ampicillin, and 37°C for overnight culture. The clone colony was selected for expansion culture, the plasmid was extracted and identified by restriction nucleic acid endonuclease digestion (Xho I/BamH I). The correctly identified plasmid by enzyme digestion was then subject to DNA sequencing and identification.
[0064] (III) Results of the experiment
[0065] 1. Acquisition of DNA fragments in the encoding region of PKC gene
[0066] As shown in Figure 2, PKCc gene coding region DNA was amplified and obtained by PCR method with different Taq DNA polymerase, and the length was the same as expected.
[0067] In Figure 2: 1, DNA molecular weight standard DL5000 (takara); 2, PKC gene coding region DNA amplified by LA Taq DNA polymerase; 3, PKC gene coding region DNAjointly amplified by G X L Taq/Z-Taq DNA polymerase; 4, PKCc gene coding region DNA amplified by G X L Taq DNA
polymerase.
[0068] 2. Cloning formation of colonies and identification of plasmid pMD18T-PKCc with enzyme digestion
[0069] The DNA fragment of target PKCc gene encoding region was separated and purified with the agarose gel, and the DNA of PKCc gene encoding region amplified by G X L Taq DNA was partially
taken and added "A" at the 3' end, then the same amount of DNA was subject to TA cloning, of which the results were shown in Figure 3. The TA cloning of the product amplified by adding "A" at the 3' end with LA-Taq DNA polymerase could be directly achieved (Figure 3A), while the TA cloning of the product that could not be amplified by adding "A" at the 3' end with G X L Taq DNA polymerase failed
(Figure 3D). The product amplified with high fidelity DNA polymerase could be subject to TA cloning after adding "A" at the 3' end (Figure 3B), while the product obtained by highfidelity DNA polymerase together with the common DNA polymerase that could be added "A" at the end could be directly subject to TA cloning (Figure 3C). These monoclonal colony was taken and subject to expansion culture and plasmid extraction, and was identified with Xho I enzyme digestion. The results were shown in Figure 4. The PCR products were all cloned into the clone vector pMD18-T effectively.
[0070] In Figure 3: A, The TA cloning of the PCR product amplified by LA Taq DNA polymerase; B, The TA cloning of the PCR product amplified by B, G X L Taq DNA polymerase after3'-end addition
of "A"; C, The TA cloning of the PCR product amplified by G X L Taq DNA polymerase combined with
Z-Taq DNA polymerase; D, The TA cloning of the PCP product amplified by G X L Taq DNA
polymerase.
[0071] In Figure 4: 1, The individual colony plasmid subject to TA cloning of the PCR product amplified with LA-Taq DNA polymerase; 2. The individual colony plasmid subject to TA cloning of the PCR product amplified by G X L Taq DNA polymerase with addition of "A" at 3' end; and the
individual colony plasmid subject to TA cloning of the PCR product jointly amplified by G X L Taq
DNA polymerase combined with Z-Taq DNA polymerase.
[0072] 3, PCR Sequence Analysis Result and Sequence Comparison
[0073] The results of DNA sequencing and splicing showed that the TA monoclonal strains obtained by three different methods successfully cloned the PCR product, namely PKCc gene protein coding region, into the T vector pMD18-T (Figure 5, Figure 7, Figure 8). However, there were 6 mutations in PCR products amplified by LA TaqDNA polymerase. They were 223A> G, 497A> G, 950G> A, 1257T> C, 1357A> G, 1379T> C, 2126T> C (Figure 5, Figure 6). No mutation occurred in PCR products amplified with G X L Taq DNA polymerase.
[0074] (IV) Experimental conclusions
[0075] The target fragment amplified with common Taq DNA polymerase can be directly subject to TA cloning, but its fidelity is obviously insufficient. The target fragment could be amplified with fidelity by high-fidelity Taq DNA polymerase, but because of the lack of protruding "A" at the 3' end of the product, additional adding "A" kit is needed for TA cloning. In this experiment, high-fidelity Taq DNA polymerase was to amplify the target fragment, and then the common TA DNA polymerase was directly added into the reaction tube in the last three cycles of PCR reaction to achieve reaction. The obtained product not only has high fidelity, but also adds "A" at the 3' end. The PCR product could be directly subject to TA cloning. This method is easy and convenient, and can solve the current problem of adding additional "A" for high-fidelity PCR.
[0076] The above mentioned is only the specific implementation of the invention, but the protection scope of the invention is not limited to this. Any technical personnel familiar with the technical field in the invention within the technical scope, may easily figure out the change or replacement, which should be covered in the protection scope of the invention. Therefore, the protection scope of the invention should be based on the protection scope of the Claims.
Sequence_0 ATGGTAGTGTTCAATGGCCTTCTTAAGATCAAAATCTGCGAGGCCGTGAGCTTGAAGC cc
Sequence_1 ATGGTAGTGTTCAATGGCCTTCTTAAGATCAAAATCTGCGAGGCCGTGAGCTTGAAGC cc
Sequence_0 ACAGCCTGGTCGCTGCGCCATGCGGTGGGACCCCGGCCGCAGACTTTCCTTCTCGACCC C
Sequence_1 ACAGCCTGGTCGCTGCGCCATGCGGTGGGACCCCGGCCGCAGACTTTCCTTCTCGACCC C
Sequence_0 TACATTGCCCTCAATGTGGACGACTCGCGCATCGGCCAAACGGCCACCAAGCAGAAGA cc
Sequence_1 TACATTGCCCTCAATGTGGACGACTCGCGCATCGGCCAAACGGCCACCAAGCAGAAGA cc
Sequence_0 AACAGCCCGGCCTGGCACGACGAGTTCGTCACCGATGTGTGCGACGGACGCAAGATCG AG
Sequence_1 AACAGCCCGGCCTGGCACGACGAGTTCGTCACCGATGTGTGCAACGGACGCAAGATCG AG
Sequence_0 CTGGCTGTCTTTCACGATGCCCCCATAGGCTACGACGACTTCGTGGCCAACTGCACCAT
Sequence_1 CTGGCTGTCTTTCACGATGCCCCCATAGGCTACGACGACTTCGTGGCCAACTGCACCAT C
Sequence_0 CAGTTTGAGGAGCTGCTGCAGAACGGGAGCCGCCACTTCGAGGACTGGATTGATCTGG AG
sequence_1 CAGTTTGAGGAGCTGCTGCAGAACGGGAGCCGCCACTTCGAGGACTGGATTGATCTGG AG
Sequence_0 CCAGAAGGAAGAGTGTATGTGATCATCGATCTCTCAGGGTCGTCGGGTGAAGCCCCTA AA
Sequence_1 CCAGAAGGAAGAGTGTATGTGATCATCGATCTCTCAGGGTCGTCGGGTGAAGCCCCTA AA
Sequence_0 GACAGGAGGGGGGGGGGGGGGGGGCCGGGGACAATGAAGAGCGTGTGTTCAGGGAA CGCATGCGGCCGAGGAAGCGGCAGGGGGCCGTCGTTCAGGGAACGCATGCGGCCGAG GAAGCGGCAGGGGGCCGTC
Sequence_1 GAATGAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
Sequence_0 AGGCGCAGGGTCCATCAGGTCAACGGCCACAAGTTCATGGCCACCTATCTTCGGCAGC CC
Sequence_1 AGGCGCAGGGTCCATCGGGTCAACGGCCACAAGTTCATGGCCACCTATCTTCGGCAGC CC
Sequence_0 ACCTACTGCTCCCATTGCAGAGACTTCATCTGGGGTGTCATAGGAAAGCAGGGATACC AGCCAG
Sequence_1 ACCTACTGCTCCCATTGCAGAGACTTCATCTGGGGTGTCATAGGAAAGCAGGGATACC AG
Sequence_0 TGTCAAGTCTGCACCTGCGTGGTCCACAAGCGGTGCCACGAGCTCATAATCACAAAGT GT
Sequence_1 TGTCAAGTCTGCACCTGCGTGGTCCACAAGCGGTGCCACGAGCTCATAATCACAAAGT GT
Sequence_0 GCTGGGTTAAAGAAGCAGGAGACCCCCGACCAGGTGGGCTCCCAGCGGTTCAGCGTCA AC
Sequence_1 GCTGGGTTAAAGAAGCAGGAGACCCCCGACCAGGTGGGCTCCCAGCGGTTCAGCGTCA AC
Sequence_0 ATGCCCCACAAGTTCGGTATCCACAACTACAAGGTCCCTACCTTCTGCGATCACTGTGG G sequence_1 ATGCCCAGTGGGGGG
Sequence_1 ATGCCCCACAAGTTCGGTATCCACAACTACAAGGTCCCTACCTTCTGCGATCACTGTGG
Sequence_0 TCCCTGCTCTGGGGACTCTTGCGGCAGGGTTTGCAGTGTAAAGTCTGCAAAATGAATGT T
Sequence_1 TCCCTGCTCTGGGGACTCTTGCGGCAGGGTTTGCAGTGTAAAGTCTGCAAAATGAATGT T
Sequence_0 CACCGTCGATGTGAGACCAACGTGGCTCCCAACTGTGGAGTGGATGCCAGAGGAATCG cc
Sequence_1 CACCGTCGATGTGAGACCAACGTGGCTCCCAACTGTGGAGTGGATGCCAGAGGAATCG cc
Sequence_0 AAAGTACTGGCCGACCTGGGCGTTACCCCAGACAAAATCACCAACAGCGACCAGAGA AGG
Sequence_1 AAAGTACTGGCCGACCTGGGCGTTACCCCAGACAAAATCACCAACAGCGGCCAGAGA AGG
Sequence_0 AAAAAGCTCATTGCTGGTGCCGAGTCCCCGCAGCCTGCTTCTGGAAGCTCACCATCTGA G
Sequence_1 AAAAAGCTCATTGCTGGTGCCGAGTCCCCGCAGCCTGCTTCTGGAAGCTCACCATCTGA G
Sequence_0 GAAGATCGATCCAAGTCAGCACCCACCTCCCCTTGTGACCAGGAAATAAAAGAACTTG AG
Sequence_1 GAAGATCGATCCAAGTCAGCACCCACCTCCCCTTGTGACCAGGAAATAAAAGAACTTG AG
Sequence_0 AACAACATTCGGAAAGCCTTGTCATTTGACAACCGAGGAGAGGAGCACCGGGCAGCAT CG
Sequence_1 AACAACATTCGGAAAGCCTTGTCATTTGACAACCGAGGAGAGGAGCACCGGGCAGCAT CG
Sequence_0 TCTCCTGATGGCCAGCTGATGAGCCCCGGTGAGAATGGCGAAGTCCGGCAAGGCCAGG cc
Sequence_1 TCTCCTGATGGCCAGCTGATGAGCCCCGGTGAGAATGGCGAAGTCCGGCAAGGCCAGG cc
Sequence_0 AAGCGCCTGGGCCTGGATGAGTTCAACTTCATCAAGGTGTTGGGCAAAGGCAGCTTCG GC
Sequence_1 AAGCGCCTGGGCCTGGATGAGTTCAACTTCATCAAGGTGTTGGGCAAAGGCAGCTTTG GC
Sequence_0 AAGGTCATGTTGGCAGAACTCAAGGGCAAAGATGAAGTATATGCTGTGAAGGTCTTAA AG
Sequence_1 AAGGTCATGTTGGCAGAACTCAAGGGCAAAGATGAAGTATATGCTGTGAAGGTCTTAA AG
Sequence_0 AAGGACGTCATCCTTCAGGATGATGACGTGGACTGCACAATGACAGAGAAGAGGATTT TG
Sequence_1 AAGGACGTCATCCTTCAGGATGATGACGTGGACTGCGCAATGACAGAGAAGAGGATTT CG
* Sequence_0 GCTCTGGCACGGAAACACCCGTACCTTACCCAACTCTACTGCTGCTTCCAGACCAAGG AC
Sequence_1 GCTCTGGCACGGAAACACCCGTACCTTACCCAACTCTACTGCTGCTTCCAGACCAAGG AC
Sequence_0 CGCCTCTTTTTCGTCATGGAATATGTAAATGGTGGAGACCTCATGTTTCAGATTCAGCG C
Sequence_1 CGCCTCTTTTTCGTCATGGAATATGTAAATGGTGGAGACCTCATGTTTCAGATTCAGCG C
Sequence_0 TCCCGAAAATTCGACGAGCCTCGTTCACGGTTCTATGCTGCAGAGGTCACATCGGCCCT
Sequence_1 TCCCGAAAATTCGACGAGCCTCGTTCACGGTTCTATGCTGCAGAGGTCACATCGGCCCT C
Sequence_0 ATGTTCCTCCACCAGCATGGAGTCATCTACAGGGATTTGAAACTGGACAACATCCTTCT G
Sequence_1 ATGTTCCTCCACCAGCATGGAGTCATCTACAGGGATTTGAAACTGGACAACATCCTTCT G
Sequence_0 GATGCAGAAGGTCACTGCAAGCTGGCTGACTTCGGGATGTGCAAGGAAGGGATTCTGA AT
Sequence_1 GATGCAGAAGGTCACTGCAAGCTGGCTGACTTCGGGATGTGCAAGGAAGGGATTCTGA AT
Sequence_0 GGTGTGACGACCACCACGTTCTGTGGGACTCCTGACTACATAGCTCCTGAGATCCTGCA G
Sequence_1 GGTGTGACGACCACCACGTTCTGTGGGACTCCTGACTACATAGCTCCTGAGATCCTGCA G
Sequence_0 GAGTTGGAGTATGGCCCCTCCGTGGACTGGTGGGCCCTGGGGGTGCTGATGTACGAGA TG
Sequence_1 GAGTTGGAGTATGGCCCCTCCGTGGACTGGTGGGCCCTGGGGGTGCTGATGTACGAGA TG
Sequence_0 ATGGCTGGACAGCCTCCCTTTGAGGCCGACAATGAGGACGACCTATTTGAGTCCATCCT C
Sequence_1 ATGGCTGGACAGCCTCCCTTTGAGGCCGACAATGAGGACGACCTATTTGAGTCCATCCT C
Sequence_0 CATGACGACGTGCTGTACCCAGTCTGGCTCAGCAAGGAGGCTGTCAGCATCTTGAAAG CT
Sequence_1 CATGACGACGTGCTGTACCCAGTCTGGCTCAGCAAGGAGGCTGTCAGCATCTTGAAAG CT
Sequence_0 TTCATGACGAAGAATCCCCACAAGCGCCTGGGCTGTGTGGCATCGCAGAATGGCGAGG AC
Sequence_1 TTCATGACGAAGAATCCCCACAAGCGCCTGGGCTGTGTGGCATCGCAGAATGGCGAGG AC
Sequence_0 GCCATCAAGGATGATGATGATGATGATGATGATGATGTGTGCTGATGATGATGATGAT GATGAGGAGGAGAGAGAAG
Sequence_1 GCCATCAGCAGCAGCAGCATTTGATGGCCATCAAGCAGCACCCATTCTTCAAAGAGAT
Sequence_0 ATCAAGCCACCCTTCAAACCACGCATTAAAACCAAAAGAGACGTCAATAATTTTGACC AA
Sequence_1 ATCAAGCCACCCTTCAAACCACGCATTAAAACCAAAAGAGACGTCAATAATTTTGACC AA
Sequence_0 GACTTTACCCGGGAAGAGCCGGTACTCACCCTTGTGGACGAAGCAATTGTAAAGCAGA TC
Sequence_1 GACTTTACCCGGGAAGAGCCGGTACCCACCCTTGTGGACGAAGCAATTGTAAAGCAGA TC
Sequence_0 AACCAGGAGGAATTCAAAGGTTTCTCCTACTTTGGTGAAGACCTGATGCCCTGA
Sequence_1 AACCAGGAGGAATTCAAAGGTTTCTCCTACTTTGGTGAAGACCTGATGCCC---
Claims (2)
1. A PCR method for obtaining the product with high fidelity and 3'-end addition of "A", which is characterized in that: the method is that: the PCR buffer, dNTP, upstream and downstream primers, templates and high-fidelity Taq DNA polymerase are first added into the PCR reaction system and then PCR amplification is carried out according to the following procedures:
(1) Pre-denaturation at 95°C for 2 min;
(2) 95°C, 15 sec; annealing temperature, 30 sec; 72°C, elongation time; 28 cycles;
(3) The common Taq DNA polymerase is added directly into the reaction PCR tube and mixed uniformly ;
(4) 95°C, 15 sec; annealing temperature, 30 sec; 72°C, elongation time; 3 cycles;
(5) 72°C, 10 min.
2. A PCR method for obtaining the product with high fidelity and 3'-end addition of "A", as described in Claim 1, is characterized in that: the final concentration of PCR buffer is "1x ", the dNTP is 200 [M, the upstream and downstream primers are 0.2 [M, the template is 500 ng, the high-fidelity Taq DNA polymerase is 1.25 U/50 L, and the common Taq DNA polymerase is 1.25 U/50 L.
FIGURES 1/7
Figure 1
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