A mutant Taq DNA polymerase and its kit and application

Abstract

A mutant Taq DNA polymerase and a kit and application thereof. The mutant Taq DNA polymerase comprises one or more of the following mutation sites compared to the wild-type Taq DNA polymerase SEQ ID NO:1: mutation sites at positions 230, 519, 580, and 754 compared to the SEQ IND NO:1 sequence. The mutant Taq DNA polymerase has at least one mutation site in the 175-261 amino acid region with 5'-3' exonuclease activity and in the 410-832 amino acid region with 5'-3' polymerase activity, respectively. The mutant Taq DNA polymerase provided in the present application has higher enzyme activity and stability.

Description

Technical Field

[0001] The present invention relates to biotechnology, in particular to a mutant Taq DNA polymerase, its related kits and its application. Background Art

[0002] In the polymerase chain reaction (PCR) of DNA amplification, the activity, stability, sensitivity and other properties of DNA polymerase are closely related to the specificity, efficiency and fidelity of the PCR reaction. However, conventional Taq DNA polymerase is easily affected by inhibitors, ion concentration, amplification template concentration, temperature and other factors in the amplification reaction system, which can have adverse effects on the amplification results, for example, the template concentration is low or too low to amplify the positive fragment.

[0003] Therefore, there is a need for Taq DNA polymerase that can improve the specificity, efficiency and fidelity of PCR reactions. Summary of the invention

[0004] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0005] The present application provides a mutant Taq DNA polymerase, a related kit and an application thereof. The mutant Taq DNA polymerase provided in the present application has higher enzyme activity and stability.

[0006] In an embodiment of the present application, a mutant Taq DNA polymerase is provided, wherein the mutant Taq DNA polymerase comprises one or more of the following mutation sites compared to the wild-type Taq DNA polymerase SEQ ID NO:1: mutation sites at positions 230, 519, 580, and 754 compared to the SEQ IND NO:1 sequence.

[0007] In some embodiments of the present application, the mutant Taq DNA polymerase comprises one of the following mutation sites: mutation sites at positions 230, 519, 580, and 754 compared with the sequence of SEQ IND NO:1.

[0008] In some embodiments of the present application, the mutant Taq DNA polymerase comprises three of the following mutation sites: mutation sites at positions 230, 519, 580, and 754 compared with the sequence of SEQ IND NO:1.

[0009] In some embodiments of the present application, the mutant Taq DNA polymerase comprises four of the following mutation sites: sites at positions 230, 519, 580, and 754 compared with the sequence of SEQ IND NO:1.

[0010] In some embodiments of the present application, compared with the sequence of SEQ IND NO: 1, the sites at positions 230, 519, 580, and 754 are E230K, L519R, N580R, and Q754K, respectively.

[0011] In some embodiments of the present application, the mutation at position 230 is located in the 175-261 amino acid region having 5'-3' exonuclease activity of Taq DNA polymerase.

[0012] In some embodiments of the present application, the mutations at positions 519, 580, and 754 are located in the 410-832 amino acid region of Taq DNA polymerase having 5'-3' polymerase activity.

[0013] In some embodiments of the present application, there is at least one mutation site in the 175-261 amino acid region with 5'-3' exonuclease activity of Taq DNA polymerase and in the 410-832 amino acid region with 5'-3' polymerase activity of Taq DNA polymerase.

[0014] In some embodiments of the present application, the mutant Taq DNA polymerase comprises the sequence of SEQ IND NO:2.

[0015] In an embodiment of the present application, a DNA amplification kit is provided, wherein the DNA amplification kit comprises any of the mutant Taq DNA polymerases described above, a treatment solution, and dNTPs.

[0016] In an embodiment of the present application, the use of the mutant Taq DNA polymerase in PCR is provided.

[0017] Compared with the related art, this application has the following advantages:

[0018] The present application provides a Taq DNA polymerase mutant with improved thermal stability; provides a Taq DNA polymerase mutant with higher enzyme activity and amplification efficiency; provides a Taq DNA polymerase mutant with better performance and a wider range of use in practical applications of qPCR.

[0019] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0021] Figure 1 This is a PCR comparison diagram of wild-type Taq enzyme and mutant Taq enzyme after heat treatment;

[0022] Figure 2 It is the qPCR curve graph of wild-type Taq enzyme and mutant Taq enzyme in FAM channel;

[0023] Figure 3 It is the qPCR curve graph of wild-type Taq enzyme and mutant Taq enzyme in HEX channel;

[0024] Figure 4 It is the qPCR curve graph of wild-type Taq enzyme and mutant Taq enzyme of ROX channel;

[0025] Figure 5 It is the qPCR curve graph of wild-type Taq enzyme and mutant Taq enzyme in Cy5 channel; DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of this application more clear, the embodiments of this disclosure will be described in detail below. It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of this disclosure can be combined with each other arbitrarily.

[0027] The Taq enzyme protein consists of 832 amino acids, of which the 175-261 amino acid region has 5'-3' exonuclease activity and the 410-832 amino acid region has polymerase activity.

[0028] In order to facilitate the subsequent display effect, the wild-type Taq enzyme was named Taq0 (SEQ IND NO: 1), and multiple mutants were prepared, including 1 site E230K, L519R, N580K, Q754K (Taq 1, Taq2, Taq3, Taq4), 3 sites L519R, N580K, Q754K (Taq5), and 4 sites E230K, L519R, N580K, Q754K (Taq 6), a total of 6 Taq enzyme mutants. The amino acid sequences of Taq 1, Taq2, Taq3, Taq4, Taq5, and Taq6 are shown in SEQ IND NO: 2, SEQ IND NO: 3, SEQ IND NO: 4, SEQ IND NO: 5, SEQ IND NO: 6, and SEQ IND NO: 7, respectively.

[0029] The amino acid sequence of wild-type Taq0 SEQ IND NO:1 is as follows: MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERL FANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRYGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0030] Taq1's amino acid sequence SEQ IND NO:2 is as follows:

[0031] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAK

[0032] SLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIK

[0033] ELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLS

[0034] DRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIG

[0035] EKTARKLLEEWGSLEALLKNLDRLKPAIRKKILAHMDDLKLSWDLAKVRT

[0036] DLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPP

[0037] EGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGL

[0038] LAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEE

[0039] AGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVR

[0040] LDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLP

[0041] AIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIH

[0042] PRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLL

[0043] VALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPL

[0044] MRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIE

[0045] KTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTA

[0046] ADLMKLAMVKLFPRLEEMGARMLQVHDELVLEAPKERAEAVARLAKE

[0047] VMEGVYPLAVPLEVEVGIGEDWLSAKE

[0048] Taq2's amino acid sequence SEQ IND NO:3 is as follows:

[0049] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAK

[0050] SLLKALKEDDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIK

[0051] ELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLS

[0052] DRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIG

[0053] EKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRT

[0054] DLPLEVDFAKRRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPP

[0055] EGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGL

[0056] LAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEE

[0057] AGERAALSERLFANLWGRLEGEERLLLWLYREVERPLSAVLAHMEATGVR

[0058] LDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLP

[0059] AIGKTEKTGKRSTSAAVREALREAHPIVEKILQYRELTKLKSTYIDPLPDLIH

[0060] PRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLL

[0061] VALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPL

[0062] MRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIE

[0063] KTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTA

[0064] ADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKE

[0065] VMEGVYPLAVPLEVEVGIGEDWLSAKE

[0066] The amino acid sequence of Taq3 is SEQ ID NO:4 as follows:

[0067] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAK

[0068] SLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIK

[0069] ELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLS

[0070] DRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIG

[0071] EKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRT

[0072] DLPLEVDFAKRRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPP

[0073] EGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGL

[0074] LAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEE

[0075] AGERAALSERLFANLWGRLEGEERLLLWLYREVERPLSAVLAHMEATGVR

[0076] LDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLP

[0077] AIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIH

[0078] PRTGRLHTRFNQTATATGRLSSSDPRLQNIPVRTPLGQRIRRAFIAEEGWLL

[0079] VALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPL

[0080] MRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIE

[0081] KTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTA

[0082] ADLMKLAMVKLFPRLEEMGARMLQVHDELVLEAPKERAEAVARLAKE

[0083] VMEGVYPLAVPLEVEVGIGEDWLSAKE

[0084] Taq4's amino acid sequence SEQ IND NO:5 is as follows:

[0085] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAK

[0086] SLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIK

[0087] ELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLS

[0088] DRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIG

[0089] EKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRT

[0090] DLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPP

[0091] EGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGL

[0092] LAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEE

[0093] AGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVR

[0094] LDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLP

[0095] AIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIH

[0096] PRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLL

[0097] VALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPL

[0098] MRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIE

[0099] KTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVKGTA

[0100] ADLMKLAMVKLFPRLEEMGARMLQVHDELVLEAPKERAEAVARLAKE

[0101] VMEGVYPLAVPLEVEVGIGEDWLSAKE

[0102] Taq5's amino acid sequence SEQ IND NO:6 is as follows:

[0103] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAK

[0104] SLLKALKEDDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIK

[0105] ELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLS

[0106] DRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIG

[0107] EKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRT

[0108] DLPLEVDFAKRRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPP

[0109] EGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGL

[0110] LAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEE

[0111] AGERAALSERLFANLWGRLEGEERLLLWLYREVERPLSAVLAHMEATGVR

[0112] LDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLP

[0113] AIGKTEKTGKRSTSAAVREALREAHPIVEKILQYRELTKLKSTYIDPLPDLIH

[0114] PRTGRLHTRFNQTATATGRLSSSDPRLQNIPVRTPLGQRIRRAFIAEEGWLL

[0115] VALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPL

[0116] MRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIE

[0117] KTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVKGTA

[0118] ADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKE

[0119] VMEGVYPLAVPLEVEVGIGEDWLSAKE

[0120] The amino acid sequence of Taq6 is SEQ ID NO:7 as follows:

[0121] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAK

[0122] SLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIK

[0123] ELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLS

[0124] DRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIG

[0125] EKTARKLLEEWGSLEALLKNLDRLKPAIRKKILAHMDDLKLSWDLAKVRT

[0126] DLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPP

[0127] EGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGL

[0128] LAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEE

[0129] AGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVR

[0130] LDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLP

[0131] AIGKTEKTGKRSTSAAVREALREAHPIVEKILQYRELTKLKSTYIDPLPDLIH

[0132] PRTGRLHTRFNQTATATGRLSSSDPRLQNIPVRTPLGQRIRRAFIAEEGWLL

[0133] VALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPL

[0134] MRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIE

[0135] KTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVKGTA

[0136] ADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKE

[0137] VMEGVYPLAVPLEVEVGIGEDWLSAKE

[0138] Table 1 Mutation Sites of Mutants Compared with Wild Types

[0139] Location wild type Mutant 230 E K 519 L R 580 N R 745 Q K

[0140] Example 1: Site-directed mutagenesis of Taq enzyme plasmid

[0141] PCR amplification was performed using a plasmid containing a wild-type Taq enzyme gene (synthesized by GenScript). Forward and reverse mutation primers containing mutation sites were designed and synthesized, and amplified with the template plasmid using pfu polymerase (full gold, AP221-13). The amplification system is shown in Table 2:

[0142] Table 2 Amplification system

[0143]

[0144]

[0145] The primers for the mutants are as follows:

[0146] E230K Primer-F (SEQ IND NO:8): aaccggcaattcgtaagaaaattctggcgca

[0147] E230K Primer-R (SEQ IND NO:9): tgcgccagaattttcttacgaattgccggtt

[0148] L519R Primer-F(SEQ IND NO:10):ccagcgcggcggtccgtgaggcgctgagaga

[0149] L519R Primer-R(SEQ IND NO:11):tctctcagcgcctcacggaccgccgcgctgg

[0150] N580R Primer-F (SEQ IND NO: 12): caagcagcgacccaaatctgcagaacattcc

[0151] N580R Primer-R (SEQ IND NO: 13): ggaatgttctgcagacgtgggtcgctgcttg

[0152] Q754K Primer-F (SEQ IND NO: 14): tcaacatgccggtgaagggcaccgcagccga

[0153] Q754K Primer-R(SEQ IND NO:15):tcggctgcggtgcccttcaccggcatgttga

[0154] The PCR reaction procedure is as follows:

[0155]

[0156] After PCR amplification, place the reaction mixture on ice for several minutes, add 1 μL of restriction endonuclease DpnI (thermo Fisher, ER1701), digest at 37°C for 2 hours to eliminate the template plasmid, then heat inactivate the DpnI enzyme, directly heat-shock transform the treated PCR product into JM109 competent cells (Weidi Bio CAT#: EC1070), apply to the corresponding resistance plate and select monoclones to obtain the plasmid containing the mutant enzyme gene. According to the above scheme, at least one of the following mutations is performed in sequence: E230K, L519R, N580R, Q754K.

[0157] Example 2: Expression and purification of mutant Taq

[0158] 10 μL of the expression bacterial solution containing the mutant gene was inoculated into 10 ml of LB medium and cultured at 37°C, 180 rpm overnight. Then, it was transferred to 350 ml of LB liquid medium and cultured at 30°C, 200 rpm until the OD 600 To about 0.8, and then transferred to 42 ° C temperature to induce expression for 5h. The induced bacterial solution was centrifuged at 8000rpm in a 50mL centrifuge tube for 5min, the supernatant was discarded, the bacteria were collected, and the cells were resuspended in resuspension buffer (10mM Tris, pH 7.5, 100mM NaCl). The resuspended cells were sonicated for 1 minute at an amplitude setting of 60, and then placed on ice for 1 minute. The sonication was repeated in this way for a total of 5 times. The sample was incubated at 65 ° C for 10 minutes, then centrifuged at 9000rpm for 30min, the supernatant was recovered and further purified on the column. The purification methods include but are not limited to molecular size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and preparative gel electrophoresis. Place in Taq enzyme storage buffer (20mM Tris HCl (pH 8.0), 0.1mM EDTA, 1mM DTT, 0.1% TritonX-100and 50% glycerol) for 12h, change the solution once in the middle, and then concentrate and collect the final product. Take part of the product and add sample buffer and boil for 3 minutes, then use SDS-PAGE to detect protein purification, and its purity is ≥95%.

[0159] Example 3: Comparison of the activity of mutant Taq polymerase and homemade wild-type Taq polymerase

[0160] The enzyme solution prepared in Example 2 was used to prepare the mutant Taq polymerase at the same mass concentration as the wild-type Taq polymerase, and the EvaEZ TMFluorometric Polymerase Activity Assay Kit (Cat: 29051) was used to determine the enzyme activity. 10 μL of 2×EvaEZ Polymerase Activity Mix and 9 μL of ddH2O were added on ice, and then 1 μL of polymerase sample was added. The mixture was gently mixed and then placed in qPCR for testing. The results are shown in Table 3.

[0161] Table 3 Enzyme activity detection of mutant Taq polymerase and wild polymerase (enzyme activity unit: mU / μL)

[0162] Types of enzymes First measurement value Second measurement value average value Taq0 194 192 193 Taq1 192 196 194 Taq2 237 235 236 Taq3 220 222 221 Taq4 209 209 209 Taq5 254 250 252 Taq6 253 259 256

[0163] From the data in Table 3, it can be seen that the enzymatic activity of Taq polymerase with E230K mutation is equivalent to that of wild Taq polymerase, indicating that the mutation site in the 5'-3' nuclease activity region of Taq has no significant effect on the polymerization activity of the enzyme, while the mutations L519R, N580R, and Q754K in the polymerase activity region have different degrees of improvement in polymerization activity, suggesting that these mutations change the conformation of the enzyme and improve the enzyme's ability to bind to DNA.

[0164] Example 4: Comparison of rapid PCR between wild-type Taq enzyme and mutant Taq enzyme

[0165] The mouse genome was used as a template and primers were designed to amplify 500 kb DNA. The primer sequences were: F (SEQ IND NO: 16): tccaaaagataattggtattacatgttgaggac, R (SEQ IND NO: 17): gtttttgtattggaagggctcgtcgc. A rapid PCR experiment was then designed with extension times of 30 seconds, 10 seconds, 5 seconds, and 1 second, respectively.

[0166] Table 4 PCR system:

[0167]

[0168] PCR reaction conditions: 95°C, 5 min; 95°C, 15 s, 60°C, 15 s, 72°C, 30 / 10 s / 5 s / 1 s, 40 cycles; 72°C 5 min. Figure 1 The results showed that when the extension time was 1 second, the wild-type Taq0 did not amplify the PCR product, while Taq2, Taq3, Taq5 and Taq6 had relatively obvious target bands, among which Taq5 and Taq6 were the most obvious, indicating that these two mutants have strong rapid PCR capabilities.

[0169] Example 5: Amplification efficiency of mutant Taq enzyme

[0170] The wild-type Taq enzyme and the mutant Taq enzyme were subjected to the fluorescence quantitative method; the wild-type Taq enzyme and the mutant Taq enzyme were prepared into a 2×PCR mixture (Mix) according to the following formula, wherein the amount of the mutant Taq polymerase added was prepared according to the mass concentration of the wild-type Taq polymerase, thereby obtaining two PCR Mixes, as shown in the following Table 5:

[0171] Table 5

[0172]

[0173]

[0174] Add the two PCR mixtures separately according to the method in Table 6, and mix them together to make a reaction system of 20 μL.

[0175] Table 6 PCR system reaction components:

[0176] Components volume 2×PCR mixture 1 / 2 10μL Primer 1 (10uM) 0.4μL Primer 2 (10uM) 0.4μL TaqMan probe (10uM) 0.2μL Human 293T RNA (template) 10ng RNase inhibitors 2μL Reverse transcriptase MMLV (200U / μL) 0.2μL <![CDATA[DEPC H20]]> Up to 20 μL

[0177] The sources of primer 1, primer 2, Taqman probe, MMLV, RNase inhibitor, etc. are shown in Table 7 Table 7 Component sequences or sources

[0178] Test reagents Sequence or source Primer 1 (SEQ IND NO: 18) ccaaaagggtcatcatctctgc Primer 2 (SEQ IND NO: 19) gactgtggtcatgagtccttc TaqMan probe (SEQ IND NO: 20) 5-ROX-ctctgctgatgccccatgttcgtc-3-BHQ1 MMLV NEB:M0253 RNase inhibitors NEB:M0314

[0179] PCR reaction conditions: 90℃, 30s; 95℃, 15s; 95℃, 15s, 60℃, 10s, 72℃, 1min, 40 cycles; 72℃ 5min. Instrument: Bio-Rad CFX96 Touch, channel: ROX. The two enzymes were tested by qPCR under the same experimental conditions and the same template concentration.

[0180] Table 8: Amplification efficiency of wild-type Taq enzyme and mutant Taq enzyme

[0181]

[0182]

[0183] From the results, it can be seen that the Ct values ​​of Taq1, Taq2, and Taq6 are relatively small, and the Ct values ​​of the three differ by 2-4 from the wild type, and the Ct value of Taq6 is the smallest. Therefore, at the same template concentration, the amplification efficiency of the mutant Taq enzyme is greater than that of the wild-type Taq enzyme, which is consistent with the verification of enzyme activity; and Taq6 has the fastest amplification speed and better DNA polymerase activity, which is more obvious under conditions of low template amount.

[0184] Example 6: Comparison of performance of mutant Taq enzyme and wild-type Taq enzyme in qPCR reaction on multiple channels

[0185] According to the conditions of Example 4, qPCR reaction was performed on Taq0 and Taq6, and different fluorescence signals were detected for multiple channel-targets such as FAM-CCT7, HEX-GAPDH, ROX-ACTB, Cy5-HPRT1, etc., wherein the amplification primers and probe sequences corresponding to each channel-target are shown in the following table:

[0186] Table 9 Amplification primers and probe sequences corresponding to each channel-target

[0187]

[0188] The results showed that Taq6 could detect signals from multiple target channels, while the wild-type FAM channel could not detect them, indicating that the detection rate of the four amino acid mutant Taq enzyme was higher and the fluorescence signal intensity was greater ( Figures 2 to 5 ), indicating that the Taq6 mutant Taq enzyme has better performance and a wider range of applications in the practical application of qPCR.

Claims

1. A mutant Taq DNA polymerase, wherein: The amino acid differences of the mutant Taq DNA polymerase compared to the wild-type Taq DNA polymerase SEQ ID NO: 1 are the following mutation sites: the mutation site at position 519 compared to the sequence of SEQ ID NO: 1; Among them, the mutation at position 519 compared with the sequence of SEQ ID NO: 1 is L519R.

2. The mutant Taq DNA polymerase according to claim 1, wherein The amino acid sequence of the mutant Taq DNA polymerase is shown in SEQ ID NO:

3.

3. A mutant Taq DNA polymerase, wherein: The amino acid differences of the mutant Taq DNA polymerase compared to the wild-type Taq DNA polymerase SEQ ID NO: 1 are the following mutation sites: mutation sites at positions 519, 580 and 754 compared to the sequence of SEQ ID NO: 1; Among them, compared with the sequence of SEQ ID NO: 1, the mutations at positions 519, 580 and 754 are L519R, N580R and Q754K, respectively.

4. The mutant Taq DNA polymerase according to claim 3, wherein The amino acid sequence of the mutant Taq DNA polymerase is shown in SEQ ID NO:

6.

5. A mutant Taq DNA polymerase, wherein: The amino acid differences of the mutant Taq DNA polymerase compared to the wild-type Taq DNA polymerase SEQ ID NO: 1 are the following mutation sites: mutation sites at positions 230, 519, 580 and 754 compared to the sequence of SEQ ID NO: 1; Among them, compared with the sequence of SEQ ID NO: 1, the mutations at positions 230, 519, 580 and 754 are E230K, L519R, N580R and Q754K, respectively.

6. The mutant Taq DNA polymerase according to claim 5, wherein The amino acid sequence of the mutant Taq DNA polymerase is shown in SEQ ID NO:

7. 7 . A DNA amplification kit, wherein the DNA amplification kit comprises the mutant Taq DNA polymerase according to any one of claims 1 to 6 , a treatment solution, and dNTPs.

8. Use of the mutant Taq DNA polymerase according to any one of claims 1 to 6 in PCR.

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