Taq DNA polymerase variants with increased A addition efficiency

Abstract

The invention provides a Taq DNA polymerase variant, the Taq DNA polymerase variant has improved polymerization activity and A adding efficiency, and when the variant enzyme is used for library construction, the library yield and the library next-machine sequencing data quality can be improved.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biotechnology, and particularly relates to a Taq DNA polymerase mutant with improved A-tailing efficiency. BACKGROUND

[0002] As one of the most important molecular biology analysis methods, DNA and RNA sequencing not only provides important data for basic biological research such as genetic information revelation and gene expression regulation, but also plays an important role in application research such as genetic diagnosis and gene therapy. With the development of science, the second-generation sequencing technology can determine the sequences of hundreds of thousands to millions of DNA molecules in parallel at one time. In the application fields of medical diagnosis, biotechnology development, forensic biology, systems biology and microbiology, high-throughput gene sequencing has become one of the core technologies.

[0003] Taking the Illumina platform as an example, the general steps for constructing a transcriptome library include mRNA enrichment, fragmentation of RNA, single-strand synthesis, double-strand synthesis, end repair and A-tailing, adapter ligation, and PCR amplification. Since the adapter ligation step mainly uses T / A ligation method, end repair and dA-tailing are very important in this process. The A-tailing efficiency directly affects the efficiency of the subsequent library construction. Due to the limitation of A-tailing efficiency, the ends may appear as blunt ends and partially G-tailed, but these abnormal structures may still be connected to the adapters by T4 ligase, and eventually mixed into the normal library through PCR amplification. When reading Read 2, these abnormal libraries cannot efficiently perform base synthesis in the sequencing reaction because the last base of the 3' end of the sequencing primer cannot be normally paired, resulting in inconsistent travel speeds of molecules in the same cluster in the subsequent cycles, with both red and green signals. Therefore, the error reading becomes A base, and the proportion of A base in Read 2 of the sequencing library is significantly increased, resulting in the phenomenon of base imbalance (such as Figure 1 the region indicated by the arrow in the middle).

[0004] At present, the A-tailing enzyme used in most kits is Taq DNA polymerase, which belongs to the DNA polymerase I family and has 5'→3' polymerase activity and 5'→3' exonuclease activity. The polymerization product usually forms a sticky end with A at the 3' end, but the A-tailing efficiency is low. Therefore, it is necessary to modify the wild-type Taq enzyme to make it more suitable for application in high-throughput sequencing. SUMMARY

[0005] The present disclosure aims to provide a Taq DNA polymerase variant with higher A-tailing efficiency, which can improve the yield of library and the sequencing quality of library off-machine data when used in A-tailing reaction in library construction.

[0006] The present disclosure provides a Taq DNA polymerase variant, which has the following mutations relative to the reference sequence: the amino acid residue corresponding to the amino acid residue H at position 443 of SEQ ID NO: 1 is replaced with the amino acid residue R, the amino acid residue corresponding to the amino acid residue V at position 449 of SEQ ID NO: 1 is replaced with the amino acid residue L, the amino acid residue corresponding to the amino acid residue E at position 491 of SEQ ID NO: 1 is replaced with the amino acid residue S, the amino acid residue corresponding to the amino acid residue R at position 512 of SEQ ID NO: 1 is replaced with the amino acid residue H, the amino acid residue corresponding to the amino acid residue I at position 553 of SEQ ID NO: 1 is replaced with the amino acid residue L, the amino acid residue corresponding to the amino acid residue H at position 561 of SEQ ID NO: 1 is replaced with the amino acid residue Y, the amino acid residue corresponding to the amino acid residue F at position 564 of SEQ ID NO: 1 is replaced with the amino acid residue L, and the amino acid residue corresponding to the amino acid residue F at position 598 of SEQ ID NO: 1 is replaced with the amino acid residue S. The amino acid residue positions are numbered in accordance with the order of SEQ ID NO: 1.

[0007] The present disclosure provides another Taq DNA polymerase variant, which has the following mutations relative to the reference sequence: the amino acid residue corresponding to the amino acid residue S at position 486 of SEQ ID NO: 1 is replaced with the amino acid residue R, the amino acid residue corresponding to the amino acid residue V at position 518 of SEQ ID NO: 1 is replaced with the amino acid residue E, and the amino acid residue corresponding to the amino acid residue R at position 595 of SEQ ID NO: 1 is replaced with the amino acid residue C. The amino acid residue positions are numbered in accordance with the order of SEQ ID NO: 1.

[0008] The present disclosure provides another Taq DNA polymerase variant, which has the following mutations relative to the reference sequence: the amino acid residue corresponding to the amino acid residue E at position 471 of SEQ ID NO: 1 is replaced with the amino acid residue D, the amino acid residue corresponding to the amino acid residue G at position 504 of SEQ ID NO: 1 is replaced with the amino acid residue S, and the amino acid residue corresponding to the amino acid residue Q at position 566 of SEQ ID NO: 1 is replaced with the amino acid residue K. The amino acid residue positions are numbered in accordance with the order of SEQ ID NO: 1.

[0009] In one embodiment, the reference sequence is a wild-type sequence of Taq DNA polymerase. In one embodiment, the wild-type sequence is set forth in SEQ ID NO: 1.

[0010] In one embodiment, the reference sequence is set forth in SEQ ID NO: 1.

[0011] In one embodiment, the Taq DNA polymerase variant is set forth in SEQ ID NO: 2.

[0012] In one embodiment, the Taq DNA polymerase variant is set forth in SEQ ID NO: 3.

[0013] In one embodiment, the Taq DNA polymerase variant is set forth in SEQ ID NO: 4.

[0014] The second aspect of the present disclosure provides a nucleic acid encoding the Taq DNA polymerase variant of the first aspect of the present disclosure.

[0015] The third aspect of the present disclosure provides a vector comprising the nucleic acid of the second aspect of the present disclosure.

[0016] The fourth aspect of the present disclosure provides a cell comprising the nucleic acid of the second aspect of the present disclosure or the vector of the third aspect of the present disclosure.

[0017] The fifth aspect of the present disclosure provides a method of producing a Taq DNA polymerase variant, comprising: culturing the cell of the fourth aspect of the present disclosure, such that the Taq DNA polymerase variant of the first aspect of the present disclosure is expressed.

[0018] In one embodiment, the method further comprises: recovering the Taq DNA polymerase variant. In one embodiment, the method further comprises: purifying the Taq DNA polymerase variant.

[0019] The sixth aspect of the present disclosure provides a Taq DNA polymerase variant produced by the method of the fifth aspect of the present disclosure.

[0020] The seventh aspect of the present disclosure provides a composition comprising the Taq DNA polymerase variant of any one of the first and sixth aspects of the present disclosure.

[0021] The eighth aspect of the present disclosure provides a kit comprising the Taq DNA polymerase variant of any one of the first and sixth aspects of the present disclosure.

[0022] The ninth aspect of the present disclosure provides use of the Taq DNA polymerase variant of the first aspect of the present disclosure for reducing the extent of library A / T separation.

[0023] In one embodiment, the Taq DNA polymerase variants of the present disclosure have increased DNA polymerase activity. In one embodiment, the Taq DNA polymerase variants of the present disclosure have increased DNA polymerase activity compared to a Taq DNA polymerase having the reference sequence. In one embodiment, the Taq DNA polymerase variants of the present disclosure have at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% increased DNA polymerase activity compared to a Taq DNA polymerase having the reference sequence. In one embodiment, the DNA polymerase activity of the Taq DNA polymerase (variant) is measured by the assay described in Example 2.

[0024] In one embodiment, the Taq DNA polymerase variants of the present disclosure have increased A-tailing efficiency. In one embodiment, the Taq DNA polymerase variants of the present disclosure have increased A-tailing efficiency compared to a Taq DNA polymerase having the reference sequence. In one embodiment, the Taq DNA polymerase variants of the present disclosure have at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% increased A-tailing efficiency compared to a Taq DNA polymerase having the reference sequence. In one embodiment, the A-tailing efficiency of the Taq DNA polymerase (variant) is measured by the assay described in Example 3.

[0025] In one of the above embodiments, the Taq DNA polymerase variants of the present disclosure have increased library yield after library construction. In one embodiment, the Taq DNA polymerase variants of the present disclosure have increased library yield compared to a Taq DNA polymerase having the reference sequence. In one embodiment, the Taq DNA polymerase variants of the present disclosure have at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% increased library yield compared to a Taq DNA polymerase having the reference sequence. In one embodiment, the library yield of the Taq DNA polymerase (variant) is measured by the assay described in Example 4.

[0026] In one embodiment, the Taq DNA polymerase variant of the present disclosure is used for RNA library construction. In one embodiment, the RNA library constructed by the Taq DNA polymerase variant of the present disclosure has a better quality compared to the RNA library constructed by the Taq DNA polymerase with the reference sequence. In one embodiment, the Q30 value of the RNA library constructed by the Taq DNA polymerase variant of the present disclosure is increased compared to the RNA library constructed by the Taq DNA polymerase with the reference sequence. In one embodiment, the Q30 value of the RNA library constructed by the Taq DNA polymerase variant of the present disclosure is increased by at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% compared to the RNA library constructed by the Taq DNA polymerase with the reference sequence. In one embodiment, the redundancy rate of the RNA library constructed by the Taq DNA polymerase variant of the present disclosure is decreased compared to the RNA library constructed by the Taq DNA polymerase with the reference sequence. In one embodiment, the redundancy rate of the RNA library constructed by the Taq DNA polymerase variant of the present disclosure is decreased by at least about 0.1%, 0.3%, 0.5%, 0.7%, 1%, 2%, 3%, 4%, or 5% compared to the RNA library constructed by the Taq DNA polymerase with the reference sequence. In one embodiment, the A / T bias of the RNA library constructed by the Taq DNA polymerase variant of the present disclosure is decreased compared to the RNA library constructed by the Taq DNA polymerase with the reference sequence. In one embodiment, the A / T bias of the RNA library constructed by the Taq DNA polymerase variant of the present disclosure is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% compared to the RNA library constructed by the Taq DNA polymerase with the reference sequence. In one embodiment, the quality and A / T bias of the RNA library constructed by the Taq DNA polymerase (variant) is measured by the assay as described in Example 5. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 : Base percentage in abnormal library dual-channel sequencing;

[0028] Figure 2 : Results of Qubit assay of the library in Example 4;

[0029] Figure 3Qubit assay results of the library in Example 5;

[0030] Figure 4A Peak shape plot of the library constructed with wild type Taq enzyme in Example 5;

[0031] Figure 4B Peak shape plot of the library constructed with Taqm1 variant enzyme in Example 5;

[0032] Figure 4C Peak shape plot of the library constructed with Taqm2 variant enzyme in Example 5;

[0033] Figure 4D Peak shape plot of the library constructed with Taqm3 variant enzyme in Example 5. DETAILED DESCRIPTION

[0034] The technical solutions of the present application are further illustrated in the following specific embodiments in conjunction with the accompanying drawings, but the following examples are only simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0035] In the following examples, the reagents and consumables used are purchased from conventional reagent manufacturers in the art, and the experimental methods and technical means used are conventional methods and means in the art, unless otherwise specified.

[0036] Example 1: Construction and purification of Taq DNA polymerase mutants

[0037] Wild type Taq DNA polymerase and recombinant mutants were expressed and purified using a conventional E. coli expression system, including:

[0038] (1) Wild type Taq DNA polymerase (Taq-WT), the protein sequence is shown in SEQ ID NO: 1;

[0039] (2) Taq DNA polymerase mutant Taqm1, the mutation sites are H443R, V449L, E491S, R512H, I553L, H561Y, F564L, F598S, and the protein sequence is shown in SEQ ID NO: 2;

[0040] (3) Taq DNA polymerase mutant Taqm2, the mutation sites are S486R, V518E, R595C, and the protein sequence is shown in SEQ ID NO: 3;

[0041] (4) Taq DNA polymerase mutant Taqm3, the mutation sites are E471D / G504S / Q566K, and the protein sequence is shown in SEQ ID NO: 4.

[0042] Example 2: Activity test of Taq DNA polymerase mutant compared to wild type enzyme

[0043] The 5'→3' polymerization activity of Taq DNA polymerase variants and wild type enzyme was determined by primer extension reaction. The M13 ssDNA-primer complex with primer binding sequence was used as template in the activity test, and DNA strand extension occurred under the action of wild type or Taq DNA polymerase variants, resulting in elongated double-stranded DNA product. Fluorescent molecules were added to the reaction product, and the amount of double-stranded DNA product was detected to calculate the activity of Taq DNA polymerase. The primer sequence used for amplification was: 5'-agcgaacctcccgacttgcgggagg-3'(SEQ ID NO:5). The 10x PCR buffer formula used for PCR amplification was: 100 mM Tris, 500 mM KCl, 15 mM MgCl2, 25% glycerol, 0.5 mg / mL BSA. The reaction system was prepared according to Table 1, and then placed in a PCR instrument, 72°C for 5 min, then 0.5 μL of 0.5 M EDTA was added to terminate the reaction, and then the amount of double-stranded DNA product was detected by Qubit, and the polymerization activity of mutant and wild type Taq DNA polymerase was calculated.

[0044] Table 1: Reaction system for enzyme activity determination

[0045] Component Volume 10 x PCR buffer 2 μl M13 ssDNA-primer complex template 2 μl dNTP 1 μl Wild type or mutant Taq DNA polymerase 1 μl ddH2O Upto 20 μl

[0046] By calculation, the activity of Taq DNA polymerase mutant and wild type Taq DNA polymerase was obtained, and the results are shown in Table 2. From the calculation results, the activity of the mutant is better than that of the wild type Taq DNA polymerase.

[0047] Table 2: Calculation results of wild type and mutant Taq DNA polymerase activity

[0048] Taq DNA polymerase name 5'→3' polymerization activity (fold) Wild type Taq DNA polymerase 1.0 Taqml 1.5 Taqm2 1.21 Taqm3 1.4

[0049] Example 3: Comparison of A addition efficiency of mutant Taq DNA polymerase and wild type enzyme

[0050] A blunt-ended double-stranded DNA with known sequence (as the substrate for A addition reaction) was obtained by amplification, purified and diluted with water. 10 ng was taken and subjected to A addition reaction with equal amounts of mutant Taq DNA polymerase and wild type enzyme, and the reaction system is shown in Table 3.

[0051] Table 3: A addition reaction system

[0052] Component Volume 10 x dA tailing buffer 2 μl Blunt-ended double-stranded template 10 ng dATP 1 μl Taq DNA polymerase mutant or wild type enzyme 5U ddH2O Upto 20 μl

[0053] The composition of the 10x dA tailing Buffer is as follows: 500 mM Tris, 200 mM MgCl2, 20 mM dATP, 2 mM dNTP.

[0054] The system was placed in a PCR instrument at 65 °C for 15 min, the product was purified using 3x magnetic beads, and the library was constructed using VAHTS ssDNA Library Prep Kit for Illumina (Vazyme # ND620). The end A addition was analyzed according to the sequencing data, and the results are shown in Table 4.

[0055] Table 4: A addition efficiency experiment results

[0056] Taq enzyme name A-adding efficiency Control without A-adding reaction 0％ Wild type Taq DNA polymerase 67.9％ Taqml 79.69％ Taqm2 73.72％ Taqm3 77.2％

[0057] The results in Table 4 show that the mutant Taq DNA polymerase has higher A addition efficiency than the wild-type Taq DNA polymerase.

[0058] Example 4: Performance comparison of mutant Taq DNA polymerase and wild-type enzyme

[0059] A known blunt-end double-stranded DNA (as a substrate for A tailing reaction) was obtained by PCR amplification using a primer modified with a phosphate group at the 5' end. 10 ng of the double-stranded DNA was used for A tailing reaction using equal amounts of mutant Taq DNA polymerase and wild-type Taq DNA polymerase, respectively. The reaction system was the same as that in Table 3. Then the system was placed in a PCR instrument at 65 °C for 15 min, followed by a linker ligation reaction using T4 DNA Ligase (Rapid) (Vazyme # N103). Finally, the library was amplified using VAHTS HiFi Universal Amplification Mix for Illumina (Vazyme # N618). The library concentration was determined using fluorescent dye Qubit, and the determination results are shown in Table 5. Figure 2 The results show that the mutant Taq DNA polymerase has higher yield than the wild-type Taq DNA polymerase in the A tailing reaction, indicating that the mutant Taq DNA polymerase has higher A addition efficiency.

[0060] Example 5: Application of Taq DNA polymerase mutant in RNA library construction

[0061] Take 1 μg of corn Total RNA as the starting template, after mRNA enrichment by VAHTS mRNA Capture Beads (Vazyme #N401), use VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme #NR605) to construct the library. Among them, Taq DNA polymerase mutant and wild type Taq DNA polymerase are used in the double-stranded synthesis & end repair & A reaction step.

[0062] According to the product instructions of N401 and NR605, including mRNA capture and fragmentation, double-stranded cDNA synthesis (different Taq enzymes are used in this step), adapter ligation, magnetic bead purification and sorting, library amplification, magnetic bead purification, finally different libraries are obtained, and Qubit determination is performed on the library yield, the results are shown in Figure 3 The constructed library is sequenced on the Illumina NovaSeq 6000 sequencing platform.

[0063] The off-machine data is shown in Table 5, and the peak shape of the library is shown in Figure 4A - Figure 4D From the experimental data, it can be seen that the mutant Taq DNA polymerase is obviously better than the wild type Taq DNA polymerase in library output, and does not affect the library peak type, and the sequencing quality Q30 and the number of gene detection are also obviously better.

[0064] Table 5: Library data constructed by different Taq enzymes

[0065]

[0066] The average number of A and T proportions in the 30th-130th base of reads2 is calculated for each Taq enzyme constructed library, and the absolute value of A base proportion minus T base proportion is the separation degree of A / T base, the value closer to 0 represents better library quality, better base balance and more uniform library, and the larger the value represents worse base balance. Through calculation, the separation degree of A base and T base in the library is shown in Table 6.

[0067] Table 6: A / T separation degree of library constructed by different Taq enzymes

[0068]

[0069] Sequence

[0070] SEQ ID NO: 1 (wild type Taq enzyme)

[0071] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRIL TADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLE EWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFG SLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEA RGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLW GRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRD QLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRL HTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRV FQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFP KVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLF PRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0072] SEQ ID NO: 2 (Taqml)

[0073] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFA KSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLARMEATGLRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLSRVLFDELGLPAIGKTEKTGKHSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLLHPRTGRLYTRLNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRASIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0074] SEQ ID NO: 3 (Taqm2)

[0075] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRIL TADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLE EWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFG SLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEA RGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLW GRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNRRD QLERVLFDELGLPAIGKTEKTGKRSTSAAELEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGR LHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRICRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIR VFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLF PRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0076] SEQ ID NO: 4 (Taqm3)

[0077] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVR ILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTAR KLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLER LEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALR DLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERL FANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLDAEVFRLAGHPF NLNSRDQLERVLFDELGLPAISKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLI HPRTGRLHTRFNKTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLS GDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAF IERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLM KLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLS AKE

Claims

1. A Taq DNA polymerase variant, having one or more of the following mutations relative to a reference sequence: (1) the amino acid residue corresponding to the amino acid residue H at position 443 of SEQ ID NO: 1 is replaced with the amino acid residue R, the amino acid residue corresponding to the amino acid residue V at position 449 of SEQ ID NO: 1 is replaced with the amino acid residue L, the amino acid residue corresponding to the amino acid residue E at position 491 of SEQ ID NO: 1 is replaced with the amino acid residue S, the amino acid residue corresponding to the amino acid residue R at position 512 of SEQ ID NO: 1 is replaced with the amino acid residue H, the amino acid residue corresponding to the amino acid residue I at position 553 of SEQ ID NO: 1 is replaced with the amino acid residue L, the amino acid residue corresponding to the amino acid residue H at position 561 of SEQ ID NO: 1 is replaced with the amino acid residue Y, the amino acid residue corresponding to the amino acid residue F at position 564 of SEQ ID NO: 1 is replaced with the amino acid residue L, the amino acid residue corresponding to the amino acid residue F at position 598 of SEQ ID NO: 1 is replaced with the amino acid residue S; (2) the amino acid residue corresponding to the amino acid residue S at position 486 of SEQ ID NO: 1 is replaced with the amino acid residue R, the amino acid residue corresponding to the amino acid residue V at position 518 of SEQ ID NO: 1 is replaced with the amino acid residue E, the amino acid residue corresponding to the amino acid residue R at position 595 of SEQ ID NO: 1 is replaced with the amino acid residue C; or (3) the amino acid residue corresponding to the amino acid residue E at position 471 of SEQ ID NO: 1 is replaced with the amino acid residue D, the amino acid residue corresponding to the amino acid residue G at position 504 of SEQ ID NO: 1 is replaced with the amino acid residue S, the amino acid residue corresponding to the amino acid residue Q at position 566 of SEQ ID NO: 1 is replaced with the amino acid residue K, wherein the amino acid residue positions are numbered in order according to SEQ ID NO: 1, optionally, the reference sequence is the wild type sequence of Taq DNA polymerase, further optionally, the wild type sequence is set forth in SEQ ID NO:

1.

2. The Taq DNA polymerase variant of claim 1, wherein, the Taq DNA polymerase variant is set forth in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4. 3.A nucleic acid encoding the Taq DNA polymerase variant of claim 1 or 2. 4.A vector comprising the nucleic acid of claim 3. 5.A cell comprising the nucleic acid of claim 3 or the vector of claim 4.

6. A method of producing a variant of Taq DNA polymerase comprising: culturing the cell of claim 5 so that the Taq DNA polymerase variant of claim 1 or 2 is expressed.

7. The method of claim 6, further comprising: recovering the Taq DNA polymerase variant, and, optionally, purifying the Taq DNA polymerase variant. 8.A Taq DNA polymerase variant produced by the method of claim 6 or 7.

9. A composition comprising the Taq DNA polymerase variant of any one of claims 1, 2, and 8.

10. A kit comprising the Taq DNA polymerase variant of any one of claims 1, 2, and 8.

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