Mutant Taq polymerases providing faster amplification

Abstract

The present invention includes a mutant Taq polymerase that can significantly extend and amplify a target sequence wherein the time of the extension condition is limited to be as short as 1 second. The mutant Taq polymerase or the bioactive fragment of the mutant Taq polymerase has one or more substitutions different from those of a wild type, and is shown in a table I;

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on February 27, 2020, is named Abclonal-Fast_SL.txt and is 2,224,491 bytes in size. Background Art

[0003] During the polymerase chain reaction (PCR) for DNA amplification, the target: primer: polymerase: dNTP: buffer mixture is subjected to successive rounds of heating at different temperatures to promote target DNA strand deannealing (usually performed at about 90°C-99°C), primer: target DNA strand annealing (usually performed at about 40°C-70°C), and DNA polymerase-mediated primer extension (usually performed at about 50°C-72°C) to produce new complementary amplicon strands. The reaction can include up to 25-45 cycles to produce sufficient amplification.

[0004] PCR is typically performed using a thermostable DNA polymerase that can withstand the high temperatures associated with deannealing without becoming inactivated by heat-induced protein denaturation.

[0005] Increasing the speed of PCR can be a significant commercial advantage because amplified samples can be generated more quickly with fewer heating and cooling cycles. Existing Taq polymerases extend primers at a limited rate.

[0006] Therefore, there is a clear need for a thermostable DNA polymerase that can perform PCR amplification at an increased rate. Summary of the invention

[0007] The present invention includes mutant Taq polymerases having one or more point mutations different from the wild type (SEQ ID NO: 4 shows the nucleotide wild type sequence with a C-terminal His tag; SEQ ID NO: 5 shows the wild type amino acid sequence), having one or more amino acid point substitutions (at one or more of the following positions) as shown in Table 1 below:

[0008] Table I – Mutations and corresponding sequence ID numbers

[0009] D144R (SEQ ID NO:6-7), D578S (SEQ ID NO:8-9)

[0010] D732C(SEQ ID NO:10-11),D732G(SEQ ID NO:12-13),D732H(SEQ ID NO:14-15),D732N(SEQ ID NO:16-17),D732P(SEQ ID NO:18-19),D732Q(SEQ ID NO:20-21),D732S(SEQ ID NO:22-23),D732T(SEQ ID NO:24-25)

[0011] E39K(SEQ ID NO:26-27)

[0012] E39K / E230K(SEQ ID NO:28-29),E39K / D320R(SEQ ID NO:30-31),E39K / E507K(SEQ ID NO:32-33),E39K / E520K(SEQ ID NO:34-35),E39K / E537K(SEQ ID NO:36-37),E39K / D578R(SEQ ID NO:38-39),E39K / D732R(SEQ ID NO:40-41),E39K / E742K(SEQ ID NO:42-43),E39K / E189K(SEQ ID NO:44-45)

[0013] E101K(SEQ ID NO:46-47)

[0014] E117K(SEQ ID NO:48-49)

[0015] E130K(SEQ ID NO:50-51)

[0016] K131E(SEQ ID NO:52-53)

[0017] E132K(SEQ ID NO:54-55)

[0018] E159K(SEQ ID NO:56-57)

[0019] E189C(SEQ ID NO:58-59),E189D(SEQ ID NO:60-61),E189K(SEQ ID NO:62-63)

[0020] E189K / E230K(SEQ ID NO:64-65),E189K / E507K(SEQ ID NO:66-67),E189K / E537K(SEQ ID NO:68-69),E189K / D578R(SEQ ID NO:70-71),E189K / D732R(SEQ ID NO:72-73),E189K / E742K(SEQ ID NO:74-75)

[0021] E189K / E230K / E537K(SEQ ID NO:76-77),E189K / E507K / E537K(SEQ ID NO:78-79),E189K / E520K / E537K(SEQ ID NO:80-81),E189K / E230K / E507K(SEQ ID NO:82-83),E189K / E230K / E520K(SEQ ID NO:84-85),E189K / E230K / D578R(SEQ ID NO:86-87),E189K / E507K / D578R(SEQ ID NO:88-89),E189K / E230K / D732R(SEQ ID NO:90-91),E189K / E537K / D732R(SEQ ID NO:92-93),E189K / E507K / E520K(SEQ ID NO:94-95),E189K / E537K / D578R(SEQ ID NO:96-97),E189K / E537K / E742K(SEQ ID NO:98-99),E189K / D732R / E742K(SEQ IDNO:100-101)

[0022] E189L(SEQ ID NO:102-103),E189M(SEQ ID NO:104-105),E189P(SEQ ID NO:106-107),E189Q(SEQ ID NO:108-109),E189R(SEQ ID NO:110-111),E189T(SEQ ID NO:112-113),E189W(SEQ ID NO:114-115),

[0023] E201K(SEQ ID NO:116-117)

[0024] E209K(SEQ ID NO:118-119)

[0025] E230A(SEQ ID NO:120-121),E230C(SEQ ID NO:122-123),E230F(SEQ ID NO:124-125),E230H(SEQ ID NO:126-127),E230K(SEQ ID NO:128-129)

[0026] E230K / E507K(SEQ ID NO:130-131),E230K / E520K(SEQ ID NO:132-133),E230K / E537K(SEQ ID NO:134-135),E230K / D578R(SEQ ID NO:136-137),E230K / D732R(SEQ IDNO:138-139),E230K / E742K(SEQ ID NO:140-141)

[0027] E230K / E507K / E520K(SEQ ID NO:142-143),E230K / E507K / E537K(SEQ ID NO:144-145),E230K / E520K / D578R(SEQ ID NO:146-147),E230K / E537K / D578R(SEQ ID NO:148-149),E230K / E537K / D732R(SEQ ID NO:150-151),E230K / E507K / E742K(SEQ ID NO:152-153),E230K / E520K / E742K(SEQ ID NO:154-155),E230K / E537K / E742K(SEQ ID NO:156-157),E230K / D578R / E742K(SEQ ID NO:158-159),E230K / D732R / E742K(SEQ ID NO:160-161)

[0028] E230M(SEQ ID NO:162-163),E230N(SEQ ID NO:164-165),E230P(SEQ ID NO:166-167),E230Q(SEQ ID NO:168-169),E230R(SEQ ID NO:170-171),E230S(SEQ ID NO:172-173),E230T(SEQ ID NO:174-175),E230V(SEQ ID NO:176-177),E230W(SEQ ID NO:178-179),E230Y(SEQ ID NO:180-181)

[0029] E315K(SEQ ID NO:182-183)

[0030] D320R(SEQ ID NO:184-185)

[0031] L322S(SEQ ID NO:186-187)

[0032] A323F(SEQ ID NO:188-189)

[0033] R328D(SEQ ID NO:190-191)

[0034] G330P(SEQ ID NO:192-193)

[0035] R334D(SEQ ID NO:194-195)

[0036] P336G(SEQ ID NO:196-197)

[0037] E337K(SEQ ID NO:198-199)

[0038] P338G(SEQ ID NO:200-201)

[0039] Y339A(SEQ ID NO:202-203)

[0040] D344R(SEQ ID NO:204-205)

[0041] E347K(SEQ ID NO:206-207)

[0042] A348F(SEQ ID NO:208-209)

[0043] R349D(SEQ ID NO:210-211)

[0044] L351S(SEQ ID NO:212-213)

[0045] L361S(SEQ ID NO:214-215)

[0046] L365S(SEQ ID NO:216-217)

[0047] G366P(SEQ ID NO:218-219)

[0048] P368G(SEQ ID NO:220-221)

[0049] M374S(SEQ ID NO:222-223)

[0050] L380S(SEQ ID NO:224-225)

[0051] D381R(SEQ ID NO:226-227)

[0052] P382G(SEQ ID NO:228-229)

[0053] S383I(SEQ ID NO:230-231)

[0054] N384R(SEQ ID NO:232-233)

[0055] A391F(SEQ ID NO:234-235)

[0056] E397K(SEQ ID NO:236-237)

[0057] E401K(SEQ ID NO:238-239)

[0058] A407F(SEQ ID NO:240-241)

[0059] A414F(SEQ ID NO:242-243)

[0060] E507A(SEQ ID NO:244-245)

[0061] E507K / E520K(SEQ ID NO:246-247),E507K / E537K(SEQ ID NO:248-249),E507K / D578R(SEQ ID NO:250-251),E507K / D732R(SEQ ID NO:252-253),E507K / E742K(SEQ IDNO:254-255)

[0062] E507K / E537K / D578R(SEQ ID NO:256-257),E507K / D578R / D732R(SEQ ID NO:258-259),E507K / E520K / E537K(SEQ ID NO:260-261),E507K / E520K / D578R(SEQ ID NO:262-263),E507K / E520K / E742K(SEQ ID NO:264-265),E507K / E537K / E742K(SEQ ID NO:266-267),E507K / D732R / E742K(SEQ ID NO:268-269)

[0063] E507Q(SEQ ID NO:270-271),E507R(SEQ ID NO:272-273),E507S(SEQ ID NO:274-275),E507T(SEQ ID NO:276-277),E507Y(SEQ ID NO:278-279)

[0064] V518A(SEQ ID NO:280-281)

[0065] E520V(SEQ ID NO:282-283)

[0066] E520K / E537K(SEQ ID NO:284-285)

[0067] E537A(SEQ ID NO:286-287),E537F(SEQ ID NO:288-289),E537I(SEQ ID NO:290-291),E537K(SEQ ID NO:292-293)

[0068] E537K / D732R(SEQ ID NO:294-295),E537K / E742K(SEQ ID NO:296-297)

[0069] E537K / D578R / D732R(SEQ ID NO:298-299)

[0070] E520K / E537K / E742K(SEQ ID NO:300-301)

[0071] E537R(SEQ ID NO:302-303),E537Y(SEQ ID NO:304-305)

[0072] L541S(SEQ ID NO:306-307)

[0073] N565R(SEQ ID NO:308-309)

[0074] T569A(SEQ ID NO:310-311)

[0075] A570F(SEQ ID NO:312-313)

[0076] S575I(SEQ ID NO:314-315)

[0077] S577I(SEQ ID NO:316-317)

[0078] D578F(SEQ ID NO:318-319),D578H(SEQ ID NO:320-321),D578R(SEQ ID NO:322-323)

[0079] D578R / D732R(SEQ ID NO:324-325),D578R / E742K(SEQ ID NO:326-327)

[0080] D578V(SEQ ID NO:328-329)

[0081] I584S(SEQ ID NO:330-331)

[0082] E626K(SEQ ID NO:332-333)

[0083] N627R(SEQ ID NO:334-335)

[0084] V631S(SEQ ID NO:336-337)

[0085] H639E(SEQ ID NO:338-339)

[0086] W645A(SEQ ID NO:340-341)

[0087] T664I(SEQ ID NO:342-343)

[0088] A675F(SEQ ID NO:344-345)

[0089] E694K(SEQ ID NO:346-347)

[0090] Q698R(SEQ ID NO:348-349)

[0091] S699I(SEQ ID NO:350-351)

[0092] E708K(SEQ ID NO:352-353)

[0093] D732A(SEQ ID NO:354-355),D732K(SEQ ID NO:356-357),D732M(SEQ ID NO:358-359),D732R(SEQ ID NO:360-361)

[0094] D732R / E742K(SEQ ID NO:362-363),D732V(SEQ ID NO:364-365),D732Y(SEQ IDNO:366-367)

[0095] E742A(SEQ ID NO:368-369),E742D(SEQ ID NO:370-371),E742F(SEQ ID NO:372-373),E742H(SEQ ID NO:374-375),E742I(SEQ ID NO:376-377),E742K(SEQ ID NO:378-379),E742M(SEQ ID NO:380-381),E742N(SEQ ID NO:382-383),E742P(SEQ ID NO:384-385),E742T(SEQ ID NO:386-387),E742V(SEQ ID NO:388-389),E742Y(SEQ ID NO:390-391)

[0096] E745K(SEQ ID NO:392-393)

[0097] M751S(SEQ ID NO:394-395)

[0098] R801D (SEQ ID NO:396-397)

[0099] K804E (SEQ ID NO:398-399)

[0100] E805K (SEQ ID NO:400-401)

[0101] G32P (SEQ ID NO:402-403)

[0102] I163S (SEQ ID NO:404-405)

[0103] Q42R (SEQ ID NO:406-407)

[0104] T34I (SEQ ID NO:408-409)

[0105] V103S (SEQ ID NO:410-411)

[0106] Y116A (SEQ ID NO:412-413)

[0107] The present invention also includes mutant Taq polymerases having one or more amino acid substitutions as shown above, and wherein the remainder of the Taq polymerase sequence is at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to the wild-type Taq polymerase.

[0108] The present invention also includes a nucleic acid sequence encoding any one of the above mutant Taq polymerases, including the corresponding sequence listed in the sequence listing, and all degenerate nucleic acid sequences encoding the amino acid sequence of any one of the mutant Taq polymerases shown above or listed in the sequence listing; as well as vectors incorporating such nucleic acid sequences and cells transformed with such nucleic acid sequences and capable of expressing any one of the above mutant Taq polymerases.

[0109] The present invention also includes a composition or kit comprising any of the above mutant Taq polymerases, nucleic acid sequences encoding them, or vectors incorporating such nucleic acid sequences. The present invention also includes a method for amplifying a target nucleic acid, wherein any of the above mutant Taq polymerases is employed in a reaction mixture designed to amplify the target nucleic acid, and the reagent mixture is subjected to conditions for amplifying the target nucleic acid.

[0110] The above mutant Taq polymerase can amplify the target DNA sequence faster than the wild type, as demonstrated by the results in the following examples and figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 A merged image of multiple gels is shown, each gel representing the results of PCR using a mutant Taq polymerase. "WT" means wild type (SEQ ID NO: 4 and 5). Each such mutant is indicated by a number or a number and a letter corresponding to one of the mutation sites and sequences listed in Table 1. All mutation sites and corresponding sequences are listed in Table 1. Figure 1 The order in which they appear is listed below. For each gel, after amplifying the target nucleic acid having the sequence of SEQ ID NO: 1, the PCR amplification products were subjected to gel electrophoresis separation. Each gel has two lanes because each mutant was tested in duplicate, and the results were then combined into Figure 1 of the combined gel.

[0112] A391F(SEQ ID NO:234-235);E397K(SEQ ID NO:236-237);E401K(SEQ ID NO:238-239);A407F(SEQ ID NO:240-241);A414F(SEQ ID NO:242-243);E507Q(SEQ ID NO:270-271);E507R(SEQ ID NO:272-273);E507T(SEQ ID NO:276-277);E520V(SEQ ID NO:282-283);E537K(SEQ ID NO:292-293);E537A(SEQ ID NO:286-287);E537F(SEQ ID NO:288-289);E537I(SEQ ID NO:290-291);E537R(SEQ ID NO:302-303);E537Y(SEQ ID NO:304-305);N565R(SEQ ID NO:308-309);A570F(SEQ ID NO:312-313);S575I(SEQ ID NO:314-315);S577I(SEQ ID NO:316-317);D578R(SEQ ID NO:322-323);D578F(SEQ ID NO:318-319);E337K(SEQ ID NO:198-199);P338G(SEQ ID NO:200-201);Y339A(SEQ ID NO:202-203);D344R(SEQ ID NO:204-205);E347K(SEQ ID NO:206-207);A348F(SEQ ID NO:208-209);R349D(SEQ ID NO:210-211);L351S(SEQ ID NO:212-213);L361S(SEQ ID NO:214-215);L365S(SEQ ID NO:216-217);G366P(SEQ ID NO:218-219);P368G(SEQ ID NO:220-221);M374S(SEQ ID NO:222-223);L380S(SEQ ID NO:224-225);D381R(SEQ ID NO:226-227);P382G(SEQ ID NO:228-229);S383I(SEQ ID NO:230-231);N384R(SEQ ID NO:232-233);E201K(SEQ ID NO:116-117);E209K (SEQ ID NO:118-119); E230K (SEQ ID NO:128-129); E230A (SEQ ID NO:120-121); E230C (SEQ ID NO:122-123); E230F (SEQ ID NO:124-125); E230H (SEQ ID NO:126-127); E230N (SEQ ID NO:164-165); E230P (SEQ ID NO:166-167); E315K (SEQ ID NO:182-183); D320R (SEQ ID NO:184-185); L322S (SEQ ID NO:182-183); NO:186-187); A323F (SEQ ID NO:188-189); R328D (SEQ ID NO:190-191); G330P (SEQ ID NO:192-193); R334D (SEQ ID NO:194-195); P336G (SEQ ID NO:196-197); D578R (SEQ ID NO:322-323); G32P (SEQ ID NO:402-403); T34I (SEQ ID NO:408-409); E39K (SEQ ID NO:26-27); Q42R (SEQ ID NO:406-407); E101K (SEQ ID NO:46-47); V103S (SEQ ID NO:410-411); Y116A (SEQ ID NO:412-413); E117K (SEQ ID NO:410-411); NO:48-49); E130K (SEQ ID NO:50-51); K131E (SEQ ID NO:52-53); E132K (SEQ ID NO:54-55); D144R (SEQ ID NO:6-7), E159K (SEQ ID NO:56-57); I163S (SEQ ID NO:404-405); E189K (SEQ ID NO:62-63), E189T (SEQ ID NO:112-113); E189W (SEQ ID NO:114-115). ;

[0113] Figure 2 A merged image of multiple gels is shown, each gel representing the results of PCR using one of the mutant Taq polymerases shown, with the target and conditions being the same as Figure 1 in which each mutant is classified according to their Figure 2 The order of appearance is as follows Figure 1"WT" means wild type (SEQ ID NOs: 4 and 5).

[0114] D732N (SEQ ID NO: 16-17); D732Y (SEQ ID NO: 366-367); E742K (SEQ ID NO: 378-379); E742D (SEQ ID NO: 370-371); E742H (SEQ ID NO: 374-375); E742N (SEQ ID NO: 378-379); NO:382-383); E742P (SEQ ID NO:384-385); E742T (SEQ ID NO:386-387); E742V (SEQ ID NO:388-389); E742Y (SEQ ID NO:390-391); E745K (SEQ ID NO:392-393); R801D (SEQ ID NO:396-397); K804E (SEQ ID NO:398-399); E805K (SEQ ID NO:400-401); "1518" is V518A (SEQ ID NO:280-281); "1569" is T569A (SEQ ID NO:310-311); D578R (SEQ ID NO:322-323); D578H (SEQ ID NO:320-321); D578V (SEQ ID NO:328-329); I584S (SEQ ID NO:330-331); E626K (SEQ ID NO:332-333); N627R (SEQ ID NO:334-335); V631S (SEQ ID NO:332-333); NO:336-337); W645A (SEQ ID NO:340-341); T664I (SEQ ID NO:342-343); A675F (SEQ ID NO:344-345); E694K (SEQ ID NO:346-347); Q698R (SEQ ID NO:348-349); S699I (SEQ ID NO:350-351); E708K (SEQ ID NO:352-353); D732A (SEQ ID NO:354-355); D732K (SEQ ID NO:356-357); D732M (SEQ ID NO:358-359).

[0115] Figure 3 A merged image of multiple gels is shown, each gel representing the results of PCR using one of the mutant Taq polymerases shown, with the target and conditions being the same as Figure 1wherein each mutant has mutations at two sites and the sequence of this mutant is represented in Table 1 by Figure 3 "WT" means wild type (SEQ ID NOs: 4 and 5).

[0116] Figure 4 A merged image of multiple gels is shown, each gel representing the results of PCR using one of the mutant Taq polymerases shown, with the target and conditions being the same as Figure 1 Some of the mutants have mutations at one site, some have mutations at two sites, and others have mutations at three sites, and the sequences of all the mutants are represented in Table 1 by Figure 4 "WT" means wild type (SEQ ID NOs: 4 and 5).

[0117] Figure 5 A merged image of multiple gels is shown, each gel representing the results of PCR using a mutant Taq polymerase with target SEQ ID NO: 1, the target and conditions being similar to Figure 1 Some of the mutants have mutations at two sites and others have mutations at three sites, and the sequences of all the mutants are represented in Table 1 by Figure 5 "WT" means wild type (SEQ ID NOs: 4 and 5).

[0118] Summary Description of Sequence Listing

[0119] SEQ ID NO:4 and 5 are the corresponding amino acid and nucleotide sequences of wild-type Taq polymerase labeled with histamine. The amino acid and DNA sequences of the various mutants listed in Table 1 above are listed in the attached sequence table in the same order as in Table 1, starting from the first mutant sequence in Table 1, i.e. D144R, SEQ ID NO:6 and 7 (which are their corresponding amino acid and nucleotide sequences). Each mutant Taq polymerase amino acid sequence (even numbering, starting from SEQ ID NO:6 and ending at SEQ ID NO:412) is followed by its unique nucleotide coding sequence (odd numbering, starting from SEQ ID NO:7 and ending at SEQ ID NO:413). DETAILED DESCRIPTION

[0120] The term "biologically active fragment" refers to any fragment, derivative, homolog or analog of a mutant Taq polymerase that has an in vivo or in vitro activity that is characteristic of this biological molecule. For example, a mutant Taq polymerase can be characterized by various biological activities, including DNA binding activity, nucleotide polymerization activity, primer extension activity, strand displacement activity, reverse transcriptase activity, nick-initiated polymerase activity, 3'-5' exonuclease (proofreading) activity, thermal stability, ionic stability, accuracy, processivity, etc. A "biologically active fragment" of a mutant Taq polymerase is any fragment, derivative, homolog or analog that can catalyze the polymerization of nucleotides (including homologs and analogs thereof) into a nucleic acid chain. In some embodiments, a biologically active fragment, derivative, homolog or analog of a mutant Taq polymerase has 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or greater of the biological activity of the mutant Taq polymerase in any in vivo or in vitro assay of interest, such as, for example, a DNA binding assay, a nucleotide polymerization assay (which can be template dependent or template independent), a primer extension assay, a strand displacement assay, a reverse transcriptase assay, a proofreading assay, an accuracy assay, a thermal stability assay, an ionic stability assay, etc.

[0121] The biological activity of the polymerase fragment can be determined by measuring any of the following: in vitro primer extension activity of the fragment under defined reaction conditions; in vitro polymerization activity of the fragment under defined reaction conditions; in vitro thermal stability of the fragment under defined reaction conditions; in vitro stability of the fragment under high ionic strength conditions; in vitro accuracy of the fragment under defined reaction conditions; in vitro processivity of the fragment under defined reaction conditions; in vitro chain displacement activity of the fragment under defined reaction conditions; in vitro read length activity of the fragment under defined reaction conditions; in vitro chain bias activity of the fragment under defined reaction conditions; in vitro proofreading activity of the fragment under defined reaction conditions; the output of the in vitro assay, such as the sequencing throughput or average read length performed by the polymerase fragment under defined reaction conditions; and the output of the in vitro nucleotide polymerization reaction, such as the raw accuracy of the polymerase fragment incorporating the correct nucleotide into the nucleotide polymerization reaction under defined reaction conditions.

[0122] In some embodiments, the biologically active fragment may include any part of the DNA binding domain of the mutant Taq polymerase or any part of the catalytic domain. In some embodiments, the biologically active fragment may optionally include any 25, 50, 75, 100, 150 or more continuous amino acid residues of the mutant Taq polymerase. The biologically active fragment of the modified polymerase may include at least 25 continuous amino acid residues, which have at least 80%, 85%, 90%, 95%, 98% or 99% identity with any one or more of the even-numbered sequences of SEQ ID NO:6 to SEQ ID NO:412. The present invention also includes a polynucleotide encoding any one of the aforementioned amino acid sequences (which is the coding portion of the odd-numbered sequences of SEQ ID NO:7 to SEQ ID NO:413, wherein each odd-numbered polynucleotide sequence encodes the previous even-numbered mutant Taq polymerase).

[0123] Biologically active fragments can be derived from translation of post-transcriptional processing or alternative splicing RNA, or alternatively can be produced by engineering, batch synthesis or other suitable manipulations. Biologically active fragments include fragments expressed in natural or endogenous cells, and fragments produced in expression systems (such as bacteria, yeast, plants, insects or mammalian cells).

[0124] As used herein, the phrase "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz (1979) Principles of Protein Structure, Springer-Verlag). According to such analysis, amino acid groups can be defined as groups in which amino acids are preferentially interchanged with each other and are therefore most similar to each other in terms of their effects on the overall protein structure (Schulz (1979) supra). Examples of amino acid groups defined in this manner may include: "charged / polar group", including Glu, Asp, Asn, Gln, Lys, Arg and His; "aromatic or cyclic group", including Pro, Phe, Tyr and Trp; and "aliphatic group", including Gly, Ala, Val, Leu, Ile, Met, Ser, Thr and Cys. Within each group, subgroups may also be identified. For example, the group of charged / polar amino acids can be subdivided into multiple subgroups, including: a "positively charged subgroup" including Lys, Arg, and His; a "negatively charged subgroup" including Glu and Asp; and a "polar subgroup" including Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into multiple subgroups, including: a "nitrogen ring subgroup" including Pro, His, and Trp; a "phenyl subgroup" including Phe and Tyr. In another further example, the aliphatic group can be subdivided into multiple subgroups, including: a "large aliphatic non-polar subgroup" including Val, Leu, and Ile; a "aliphatic weakly polar subgroup" including Met, Ser, Thr, and Cys; and a "small residue subgroup" including Gly and Ala. Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, such as, but not limited to: Lys for Arg, and vice versa, so that a positive charge can be maintained; Glu for Asp, and vice versa, so that a negative charge can be maintained; Ser for Thr, and vice versa, so that a free -OH can be maintained; and Gln for Asn, and vice versa, so that a free -NH2 can be maintained. A "conservative variant" is a polypeptide that includes one or more amino acids that have been substituted to replace one or more amino acids of a reference polypeptide (e.g., a polypeptide whose sequence is disclosed in a publication or sequence database, or whose sequence has been determined by nucleic acid sequencing) with an amino acid having common properties (e.g., belonging to the same amino acid group or subgroup as described above).

[0125] When referring to a gene, "mutant" means that the gene has at least one base (nucleotide) change, deletion or insertion relative to the native or wild-type gene. The mutation (change, deletion and / or insertion of one or more nucleotides) can be in the coding region of the gene, or can be in an intron, 3'UTR, 5'UTR or promoter region. As a non-limiting example, a mutant gene can be a gene with an insertion in the promoter region, which can increase or decrease the expression of the gene; it can be a gene with a deletion, resulting in the production of a non-functional protein, a truncated protein, a dominant negative protein or no protein; or it can be a gene with one or more point mutations, resulting in an amino acid change in the encoded protein or resulting in abnormal splicing of the gene transcript.

[0126] "Naturally occurring" or "wild type" refers to the form found in nature. For example, a naturally occurring or wild type polypeptide or polynucleotide sequence is a sequence that exists in an organism, like the Taq polymerase sequence, which has not been intentionally modified by human manipulation.

[0127] The term "percent identity" or "homology" about nucleic acid or peptide sequences is defined as the percentage of nucleotides or amino acid residues identical to known polypeptides in a candidate sequence after arranging the sequences to obtain the maximum percent identity and introducing a gap (if necessary) to achieve the maximum percent homology. N-terminal or C-terminal insertions or deletions should not be interpreted as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm (Altschul (1997), Nucleic Acids Res. 25, 3389-3402 and Karlin (1990), Proc. Natl. Acad. Sci. USA 87, 2264-2268) used by programs blastp, blastn, blastx, tblastn and tblastx, which are customized for sequence similarity searches. The method used by BLAST program is to first consider the similar fragments (with or without gaps) between the query sequence and the database sequence, then evaluate the statistical significance of all matches identified, and finally summarize only those matches that meet the pre-selected significance threshold. For discussion of basic issues in sequence database similarity search, see Altschul (1994), Nature Genetics 6, 119-129. The search parameters of histogram, description, comparison, expectation (that is, for reporting the statistical significance threshold matched with the database sequence), cut-off, matrix and filter (low complexity) can be default settings. The default scoring matrix used by blastp, blastx, tblastn and tblastx is the BLOSUM62 matrix (Henikoff (1992), Proc.Natl.Acad.Sci.USA89, 10915-10919), which is recommended for query sequences with a length exceeding 85 units (nucleotide bases or amino acids).

[0128] Using mutant Taq polymerase

[0129] In some embodiments, the present invention relates to a method (and related kits, systems, devices and compositions) for performing a nucleotide polymerization reaction, the method comprising or consisting of: contacting a mutant Taq polymerase or a biologically active fragment thereof with a nucleic acid template in the presence of one or more nucleotides, and polymerizing at least one of the one or more nucleotides using the modified polymerase or a biologically active fragment thereof. The mutant Taq polymerase or a biologically active fragment thereof comprises one or more amino acid modifications listed in Table 1 relative to the wild type, and the mutant Taq polymerase or a biologically active fragment thereof provides a faster amplification of a target sequence than the wild type.

[0130] In some embodiments, the method may further include polymerizing at least one nucleotide in a template-dependent manner. In some embodiments, the polymerization is performed under thermal cycling conditions. In some embodiments, the method may further include hybridizing a primer to a nucleic acid template before, during, or after contacting, and wherein the polymerization includes polymerizing at least one nucleotide to the end of the primer using a mutant Taq polymerase or a biologically active fragment thereof. In some embodiments, the polymerization is performed near a sensor or a biologically active fragment thereof that can detect polymerization. In some embodiments, the method may further include using a sensor to detect a signal indicating polymerization performed by a modified polymerase or a biologically active fragment thereof. In some embodiments, the sensor is an ISFET. In some embodiments, the sensor may include a detectable label or a detectable agent in the polymerization reaction.

[0131] In some embodiments, the method further comprises determining the identity of one or more nucleotides polymerized by the modified polymerase. In some embodiments, the method further comprises determining the number of nucleotides polymerized by the modified polymerase.

[0132] In some embodiments, the present invention relates to methods (and related kits, systems, devices, and compositions) for detecting nucleotide incorporation, the methods comprising or consisting of: performing a nucleotide incorporation reaction using a mutant Taq polymerase or a biologically active fragment thereof, a nucleic acid template, and one or more nucleotide triphosphates; generating nucleotide incorporation; and detecting nucleotide incorporation. Detecting nucleotide incorporation can be performed via any appropriate method, such as PAGE, fluorescence, dPCR quantification, nucleotide byproduct generation (e.g., hydrogen ion or pyrophosphate detection; suitable nucleotide byproduct detection systems include, but are not limited to, next generation sequencing platforms such as Rain Dance, Roche 454, and Ion Torrent systems) and nucleotide extension product detection (e.g., optical detection of extension products or detection of labeled nucleotide extension products). In some embodiments, methods (and related kits, systems, devices, and compositions) for detecting nucleotide incorporation include or consisting of: detecting nucleotide incorporation using a mutant Taq polymerase or a biologically active fragment thereof.

[0133] In some embodiments, the present invention relates to a method for amplifying nucleic acid (and related kits, systems, devices and compositions), the method being performed by: contacting the nucleic acid with a mutant Taq polymerase or a biologically active fragment thereof under suitable conditions to amplify the nucleic acid; using polymerase chain reaction, emulsion polymerase chain reaction, isothermal amplification reaction, recombinase polymerase amplification reaction, proximity ligation amplification, rolling circle amplification or strand displacement amplification to amplify the nucleic acid. Amplification includes cloning amplification nucleic acid in solution and cloning amplification nucleic acid on a solid support (such as nucleic acid beads, flow cells, nucleic acid arrays or wells present on the surface of a solid support).

[0134] In some embodiments, the method for amplifying nucleic acid includes amplifying the nucleic acid under bridge PCR conditions. Bridge PCR conditions include hybridizing one or more amplified nucleic acids to a solid support. The hybridized one or more amplified nucleic acids can be used as templates for further amplification.

[0135] In some embodiments, the present invention generally relates to methods (and related kits, systems, equipment and compositions) for synthesizing nucleic acids, the method being carried out by incorporating at least one nucleotide into the end of a primer using a mutant Taq polymerase or a biologically active fragment thereof. Optionally, the method also includes detecting that the at least one nucleotide is incorporated into the end of a primer. In some embodiments, the method also includes determining the identity of at least one of the at least one nucleotide incorporated into the end of a primer. In some embodiments, the method may include determining the identity of all nucleotides incorporated into the end of a primer. In some embodiments, the method includes synthesizing nucleic acids in a template-dependent manner. In some embodiments, the method may include synthesizing nucleic acids in a solution, on a solid support, or in an emulsion (such as emPCR).

[0136] Preparation of mutant Taq polymerase

[0137] In some embodiments, in order to provide a mutant Taq polymerase that can provide rapid polymerization, the amino acid substitution can be at one or more amino acids, 2 or more amino acids, 3 or more amino acids or more amino acids, including up to 30% of the total number of amino acids of the wild-type sequence being substituted. The embodiment of the mutant Taq polymerase can have 70% to 99.99% homology with the wild type. All embodiments of the mutant Taq polymerase include one or more substitutions shown in Table I, and can also include substitutions, insertions or modifications to the rest of the wild-type sequence. In some embodiments, in order to provide a mutant Taq polymerase that can provide rapid polymerization, several substitutions shown in Table I can be included. These additional substitutions are not limited to the combinations shown in Table I. The combinations in Table I and other combinations can be used together with other substitutions in Table I, or with mutant Taq polymerases that include substitutions, insertions or modifications to other parts of the wild-type sequence.

[0138] The mutant Taq polymerases of the invention can be expressed in any suitable host system, including bacterial, yeast, fungal, baculovirus, plant or mammalian host cells. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include promoters obtained from the Escherichia coli (E. coli) lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (Villa-Kamaroff et al., 1978, Proc. Natl Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80:21-25).

[0139] For filamentous fungal host cells, suitable promoters for directing transcription of the disclosed nucleic acid constructs include those obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase genes), and mutants, truncations, and hybrid promoters thereof.

[0140] In yeast hosts, useful promoters can be derived from genes for the following enzymes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0141] For baculovirus expression, insect cell lines from the order Lepidoptera (moths and butterflies), such as Spodoptera frugiperda, are used as hosts. Gene expression is controlled by a strong promoter (e.g., pPolh).

[0142] Plant expression vectors are based on the Ti plasmid of Agrobacterium tumefaciens, or on tobacco mosaic virus (TMV), potato virus X or cowpea mosaic virus. A commonly used constitutive promoter in plant expression vectors is the cauliflower mosaic virus (CaMV) 35S promoter.

[0143] For mammalian expression, cultured mammalian cell lines such as Chinese hamster ovary (CHO), COS, including human cell lines such as HEK and HeLa, can be used to produce mutant Taq polymerase. Examples of mammalian expression vectors include adenovirus vectors, pSV and pCMV series plasmid vectors, vaccinia virus and retroviral vectors, and baculovirus. Promoters of cytomegalovirus (CMV) and SV40 are commonly used in mammalian expression vectors to drive gene expression. Non-viral promoters, such as elongation factor (EF)-1 promoter are also known.

[0144] The control sequence for expression can also be a suitable transcription terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used.

[0145] For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0146] Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.

[0147] Terminators for insect, plant, and mammalian host cells are also well known.

[0148] The control sequence can also be a suitable leader sequence, i.e. the non-translational region of the mRNA that is very important to the translation performed by the host cell. The leader sequence can be operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any leader sequence with functionality in the selected host cell can be used. The exemplary leader sequence for filamentous fungal host cells is obtained from the gene of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. The suitable leader sequence for yeast host cells is obtained from the gene of following enzyme: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0149] The control sequence can also be a polyadenylation sequence, a sequence that is operably linked to the 3' end of the nucleic acid sequence and is recognized by the host cell as a signal for adding polyadenylic acid residues to the transcribed mRNA when transcribed. Any polyadenylation sequence that is functional in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells can be derived from the genes for the following enzymes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.

[0150] The control sequence may also be a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide to enter the secretory pathway of a cell. The 5' end of the coding sequence of the nucleic acid sequence may itself contain a signal peptide coding region that is naturally linked in the translation reading frame to the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding region, an exogenous signal peptide coding region may be required.

[0151] Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the selected host cell may be used.

[0152] Effective signal peptide coding regions for bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prs A. Further signal peptides are described by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

[0153] An effective signal peptide coding region for a filamentous fungal host cell may be that obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.

[0154] Useful signal peptides for yeast host cells can be derived from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase.Signal peptides for other host cell systems are also well known.

[0155] The control sequence can also be a propeptide coding region encoding an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases as a zymogen). Propolypeptides are typically inactive and can be converted into mature active polypeptides by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from the gene of the following enzyme: subtilis alkaline protease (aprE), subtilis neutral protease (nprT), saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic protease, and Myceliophthora thermophila lactase (WO 95 / 33836).

[0156] Where both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is located immediately adjacent to the amino terminus of the polypeptide and the signal peptide region is located immediately adjacent to the amino terminus of the propeptide region.

[0157] It may also be desirable to add regulatory sequences that allow expression of mutant Taq polymerases to be regulated relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). In prokaryotic host cells, suitable regulatory sequences include lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, for example, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter. Regulatory systems for other host cells are also well known.

[0158] Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these include dihydrofolate reductase genes that are amplified in the presence of methotrexate and metallothionein genes that are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the KRED polypeptide of the invention will be operably linked to the regulatory sequence.

[0159] Another embodiment comprises recombinant expression vector, it comprises the polynucleotide of coding engineered mutant Taq polymerase or its variant, and one or more expression regulatory regions such as promoter and terminator, and replication origin, this depends on the type of host that they will be introduced into.Above-mentioned various nucleic acid and control sequence can be linked together to produce recombinant expression vector, and described recombinant expression vector can comprise one or more convenient restriction sites to allow the insertion or replacement of the nucleotide sequence of encoding mutant Taq polymerase at such site.Alternately, can express the nucleotide sequence of mutant Taq polymerase by inserting nucleotide sequence or the nucleic acid construct comprising described sequence in the suitable vector for expression.When producing expression vector, coding sequence is located in carrier, makes coding sequence operably connected with the suitable control sequence for expression.

[0160] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the mutant Taq polymerase polynucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid.

[0161] The expression vector can be an autonomously replicating vector, i.e., a vector existing as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicated together with one or more chromosomes into which it has been integrated when it is introduced into the host cell. In addition, a single vector or plasmid or two or more vectors or plasmids containing the total DNA to be introduced into the host cell genome can be used, or a transposon can be used.

[0162] The expression vectors herein preferably contain one or more selective markers that allow for easy selection of transformed cells. A selective marker is a gene whose product provides biocide resistance or viral resistance, resistance to heavy metals, prototrophy to auxotrophic types, etc. Examples of bacterial selective markers are dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance (such as ampicillin, kanamycin, chloramphenicol (Example 1) or tetracycline resistance). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenylyltransferase), and trpC (anthranilate synthase), and equivalents thereof. Embodiments for use in Aspergillus cells include the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus. Selectable markers for use in insect, plant, and mammalian cells are also well known.

[0163] The expression vector of the present invention preferably contains one or more elements that allow the vector to be integrated into the genome of the host cell or the vector to replicate autonomously in the cell independently of the genome. For integration into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or non-homologous recombination.

[0164] Alternatively, the expression vector can contain other nucleotide sequences for guiding integration into the genome of the host cell by homologous recombination. Other nucleotide sequences enable the vector to be integrated into one or more precise locations of one or more chromosomes in the host cell genome. Integration elements can be any sequence homologous to the target sequence in the host cell genome. In addition, integration elements can be non-coding or encoding nucleic acid sequences. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0165] For autonomous replication, the vector can also include a replication origin that makes it possible for the vector to autonomously replicate in the host cell in question. The example of a bacterial replication origin is P15A ori, or the replication origin of the plasmid pBR322, pUC19, pACYC177 (the plasmid has P15Aori) or pACYC184 allowed to be replicated in Escherichia coli and the replication origin of the plasmid pUB110, pE194, pTA1060 or pAM31 allowed to be replicated in bacillus. The example of the replication origin used in yeast host cells is a combination of 2 microns of replication origin, ARS1, ARS4, ARS1 and CEN3 and ARS4 and CEN6. The replication origin can be a replication origin with a mutation that makes its function in the host cell sensitive to temperature (see, for example, Ehrlich, 1978, Proc Natl Acad Sci. USA 75:1433).

[0166] The nucleic acid sequence of more than one copy of the mutant Taq polymerase can be inserted into the host cell to increase the production of the gene product. The increased copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene together with the nucleic acid sequence, wherein cells containing amplified copies of the selectable marker gene and thus additional copies of the nucleic acid sequence can be selected by culturing the cells in the presence of an appropriate selective agent.

[0167] Expression vectors for mutant Taq polymerase polynucleotides are commercially available. Suitable commercial expression vectors include p3xFLAG from Sigma-Aldrich Chemicals, St. Louis Mo. TM Expression vectors including a CMV promoter and hGH polyadenylation site for expression in mammalian host cells, and a pBR322 origin of replication and an ampicillin resistance marker for amplification in E. coli. Other suitable expression vectors are pBluescriptII SK(-) and pBK-CMV, commercially available from Stratagene, La Jolla Calif., and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen), or pPoly (Lathe et al., 1987, Gene 57:193-201).

[0168] Suitable host cells for expressing polynucleotides encoding mutant Taq polymerases of the present disclosure are well known in the art, and include, but are not limited to, bacterial cells, such as Escherichia coli, Lactobacillus kefir, Lactobacillus brevis, Lactobacillus microti, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Suitable culture media and growth conditions for the above-mentioned host cells are well known in the art.

[0169] The polynucleotides for expressing the mutant Taq polymerase polypeptides can be introduced into cells by various methods known in the art. These techniques include electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are known to the skilled person.

[0170] The polynucleotide encoding mutant Taq polymerase can be prepared by a standard solid phase method according to known synthetic methods. In some embodiments, a fragment of up to about 100 bases can be synthesized separately, and then connected (for example, by enzymatic or chemical connection methods, or polymerase-mediated methods) to form any desired continuous sequence. For example, polynucleotides can be prepared by chemical synthesis, using, for example, the classical phosphoramidite method described by Beaucage et al., 1981, Tet Lett 22: 1859-69, or by Matthes et al., 1984, EMBO J.3: 801-05 method described, for example, as it is generally implemented in an automatic synthesis method. According to the phosphoramidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, annealed, connected and cloned into an appropriate vector. In addition, essentially any nucleic acid can be obtained from a variety of commercial sources, such as Midland Certified Reagent Company, Midland, Tex., The Great American Gene Company, Ramona, Calif., ExpressGen Inc. Chicago, Ill., and Operon Technologies Inc., Alameda, Calif.

[0171] Engineered mutant Taq polymerase expressed in host cells can be recovered from cells and / or culture medium using any one or more of the well-known protein purification techniques, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography. Suitable solutions for lysing and efficiently extracting proteins from bacteria such as E. coli are commercially available from Sigma-Aldrich, St. Louis, Missouri, under the trade name CelLytic B.TM.

[0172] Chromatographic techniques used to separate mutant Taq polymerase polypeptides include reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis and affinity chromatography. Purification conditions will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and will be apparent to those skilled in the art.

[0173] In some embodiments, affinity techniques can be used to separate mutant Taq polymerase. For affinity chromatography purification, any antibody that specifically binds to mutant Taq polymerase polypeptide can be used. In order to produce antibodies, various host animals (including but not limited to rabbits, mice, rats, etc.) can be immunized by injection of compounds. The compound can be attached to a suitable carrier, such as BSA, by a side chain functional group or a joint attached to a side chain functional group. Various adjuvants can be used to enhance immune response, depending on the host species, including but not limited to Freund's (Freund's) (completely and incompletely), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, complex polyols (pluronic polyols), polyanions, peptides, oil emulsions, keyhole limpet hemocyanin (keyhole limpet hemocyanin), dinitrophenol, and potentially useful human adjuvants such as BCG (BCG) and Corynebacterium brevis.

[0174] Example

[0175] Wild-type and mutant taq polymerases were used in PCR to amplify an exemplary target nucleic acid having the sequence of SEQ ID NO: 1:

[0176] ; using a forward primer with SEQ ID NO:2: CAGTGCTGCAATGATACC, and a reverse primer with SEQ ID NO:3: TCCTTGAGAGTTTTCGCC. PCR was performed one at a time using a Bio-Rad T100 thermal cycler under the following PCR conditions: 95°C for 3 min, 95°C for 1 s, 60°C for 1 s for 25 cycles. The resulting amplicon was then kept at 23°C. The remaining PCR conditions and reagent concentrations were as follows:

[0177] 4 μl of mutant (or wild-type) Taq polymerase solution, wherein the initial concentration of Taq polymerase is 50 ng / μl;

[0178] 1X PCR buffer (prepared as follows), 2 μl;

[0179] Forward primer, SEQ ID NO: 2 (100 uM) 0.1 μl;

[0180] Reverse primer, SEQ ID NO: 3 (100 uM) 0.1 μl;

[0181] dNTP (10 mM) 0.8 μl; and

[0182] pUC19 (1 ng / μl) 1 μl.

[0183] The total volume of the reaction mixture was brought to 20 μl by adding distilled water.

[0184] After PCR amplification of SEQ ID NO: 1, the amplicons in the reaction mixture were separated using gel electrophoresis under the following conditions: the gel used was 1.2% agarose (Agarose LE, Goldbio.com, catalog: A-2Q1-1000); 1x buffer consisted of: 20 mM Tris-HCl, 80 mM Tris-acetate, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 3 mM Mg-acetate, 0.1% pH 8.8, at 25°C (Research Products International, catalog: T22020-10.0); gel size was 12 x 14 cm; and electrophoresis was run at 200 V for 20 minutes.

[0185] Embodiment 1:

[0186] The electrophoresis results were Figures 1 to 5 An image of a single gel including many of the mutant Taq polymerases listed in Table 1 is shown.

[0187] In the test results of the experiments conducted in this paper (such as Figures 1 to 5 Each mutant was tested only during 1 s of extension time (after deannealing). Figures 1 to 5 The mutant in was able to extend the SEQ ID NO: 1 oligonucleotide by more than 600 bp, where the extension conditions lasted only 1 second. Under the same conditions, the wild-type Taq polymerase did not show significant extension.

[0188] Experiments performed herein demonstrate that the mutant Taq polymerases listed above and in Table I are capable of efficient extension even at extremely short extension times.

[0189] The specific methods and compositions described herein are representative of preferred embodiments, and are exemplary and are not intended to limit the scope of the invention. In view of this specification, other purposes, aspects and embodiments will occur to those skilled in the art, and are included in the spirit of the invention as defined by the scope of the claims. It will be apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described illustratively herein may be appropriately practiced in the absence of any one or more elements, or any one or more restrictions, that are not explicitly disclosed herein as necessary. Thus, for example, in each case herein, in an embodiment or example of the invention, any of the terms "comprising", "including", "containing", etc. should be read broadly and without restriction. The methods and processes exemplarily described herein may be appropriately implemented in different step orders, and they are not necessarily limited to the step order indicated herein or in the claims. It should also be noted that, unless the context clearly indicates otherwise, as used herein and in the appended claims, the singular forms "one / a kind" and "said" include plural indicators, and the plural includes the singular form. In any case, this patent application cannot be interpreted as being limited to the specific examples or embodiments or methods specifically disclosed herein.

[0190] The invention has been described broadly and generally herein. Each of the narrower classes and subgeneric classifications falling within the general disclosure also forms a part of the present invention. The terms and expressions that have been adopted are used as terms of description and are not restrictive, and it is not intended that any equivalents of the features shown and described or portions thereof be excluded using such terms and expressions, but it will be appreciated that different modifications within the scope of the invention claimed are possible. Therefore, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be taken by those of ordinary skill in the art, and such modifications or variations are considered to be within the scope of the invention as defined by the claims.

Claims

1. A mutant Taq polymerase comprising: one or more of the following amino acid mutations shown in the following even-numbered sequence, wherein the remainder of the mutant polymerase has at least 80% amino acid sequence identity with the wild-type Taq polymerase as shown in SEQ ID NO:4, but excluding the C-terminal 6-mer histidine tag and 6 adjacent glycine and serine amino acids as shown in SEQ ID NO:4: E189K / E230K / E537K(SEQ ID NO:76-77), E189K / E507K / E537K(SEQ ID NO:78-79), E189K / E520K / E537K(SEQ ID NO:80-81), E189K / E230K / E520K(SEQ ID NO:84-85), E189K / E230K / D578R (SEQ ID NO:86-87), E189K / E507K / D578R (SEQ ID NO:88-89), E189K / E230K / D732R (SEQ ID NO:90-91), E189K / E537K / D732R (SEQ ID NO:92-93), E189K / E507K / E520K (SEQ ID NO:94-95), E189K / E537K / D578R (SEQ ID NO:96-97), E189K / E537K / E742K (SEQ ID NO:98-99), E189K / D732R / E742K (SEQ ID NO:100-101).

2. The mutant Taq polymerase according to claim 1, wherein the remainder of the mutant polymerase has at least 95% amino acid sequence identity with the wild-type Taq polymerase.

3. The mutant Taq polymerase of claim 1, wherein the remainder of the mutant polymerase has at least 99% amino acid sequence identity with the wild-type Taq polymerase.

4. The mutant Taq polymerase according to claim 1, wherein the rest of the mutant polymerase is identical to the wild-type Taq polymerase.

5. A mutant Taq polymerase comprising: one or more of the following amino acid mutations shown in the following even-numbered sequence, wherein the remainder of the mutant polymerase has at least 80% amino acid sequence identity with the wild-type Taq polymerase as shown in SEQ ID NO:4, excluding the C-terminal 6-mer histidine tag and 6 adjacent glycine and serine amino acids as shown in SEQ ID NO:4: E189L (SEQ ID NO:102-103), E189M (SEQ ID NO:104-105), E189P (SEQ ID NO:106-107), E189Q (SEQ ID NO:108-109), E189R (SEQ ID NO:110-111), E189T (SEQ ID NO:112-113), E189W (SEQ ID NO:114-115), E201K (SEQ ID NO:116-117), E230C (SEQ ID NO:122-123), E230F (SEQ ID NO:124-125), E230H (SEQ ID NO:126-127), E230K / E520K (SEQ ID NO:132-133), E230K / E537K (SEQ ID NO:134-135), E230K / D578R (SEQ ID NO:136-137), E230K / D732R (SEQ ID NO:138-139), E230K / E742K (SEQ ID NO:140-141).

6. The mutant Taq polymerase of claim 5, wherein the remainder of the mutant polymerase has at least 95% amino acid sequence identity with the wild-type Taq polymerase.

7. The mutant Taq polymerase of claim 5, wherein the remainder of the mutant polymerase has at least 99% amino acid sequence identity with the wild-type Taq polymerase.

8. The mutant Taq polymerase according to claim 5, wherein the rest of the mutant polymerase is identical to the wild-type Taq polymerase.

9. A mutant Taq polymerase comprising: one or more of the following amino acid mutations shown in the following even-numbered sequence, wherein the remainder of the mutant polymerase has at least 80% amino acid sequence identity with the wild-type Taq polymerase as shown in SEQ ID NO:4, excluding the C-terminal 6-mer histidine tag and 6 adjacent glycine and serine amino acids as shown in SEQ ID NO:4: E230K / E507K / E520K(SEQ ID NO:142-143), E230K / E507K / E537K(SEQ ID NO:144-145), E230K / E520K / D578R(SEQ ID NO:146-147), E230K / E537K / D578R(SEQ ID NO:148-149), E230K / E537K / D732R (SEQ ID NO:150-151), E230K / E507K / E742K (SEQ ID NO:152-153), E230K / E520K / E742K (SEQ ID NO:154-155), E230K / E537K / E742K (SEQ ID NO:156-157), E230K / D578R / E742K (SEQ ID NO:158-159), E230K / D732R / E742K (SEQ ID NO:160-161).

10. The mutant Taq polymerase of claim 9, wherein the remainder of the mutant polymerase has at least 95% amino acid sequence identity with the wild-type Taq polymerase.

11. The mutant Taq polymerase of claim 9, wherein the remainder of the mutant polymerase has at least 99% amino acid sequence identity with the wild-type Taq polymerase.

12. The mutant Taq polymerase according to claim 9, wherein the rest of the mutant polymerase is identical to the wild-type Taq polymerase.

Images