RU2779599C1 - Polymerases, compositions, and their application methods - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, and it is modified polymerase enzymes for improved inclusion of nucleotides and nucleotide analogs, in particular changed polymerases, which preserve high reliability in reduction in inclusion time, as well as methods and kits, in which they are used.

EFFECT: obtaining modified polymerase enzymes.

56 cl, 5 dwg, 2 tbl, 4 ex

Description

Cross-reference to related applications

[01] This application claims priority from U.S. Provisional Application No. 62/753,558, filed Oct. 31, 2018, the contents of which are incorporated herein by reference in their entirety.

Sequence listing

[02] This application contains a Sequence Listing electronically submitted via EFS-Web to the US Patent and Trademark Office as an ASCII text file called "IP-1546-PCT_ST25.txt" with a size of 224 kilobytes and created on October 31, 2019 d. The information contained in the Sequence Listing is incorporated herein by reference.

Field of invention

[03] The present disclosure relates, inter alia, to modified polymerases for use in conducting nucleotide incorporation reactions, especially in the context of nucleic acid sequencing by synthesis.

State of the art invention

[04] Next-generation sequencing (NGS) technology relies on DNA polymerases as an essential component of the sequencing process. Reducing the time for template sequencing while maintaining high confidence is desirable. Shortening each cycle of the sequencing by synthesis (SBS) process is an effective step to achieve shorter sequencing run times. One approach to shortening the cycle time is to shorten the turn-on time. However, while reducing turn-on time can greatly improve overall run time, this usually comes at the expense of reliability. For example, the phasing frequency, the pre-phasing frequency and/or the bypass frequency are increased and, as a consequence, the error rate is increased. With a low error rate during a sequencing run, most of the template molecules in the cluster end in the same labeled nucleotide and the signal is clear. In contrast, with reduced confidence during a sequencing run, more and more template molecules in the cluster end up with a mislabeled nucleotide, and the signal may have an increased noise level to accurately determine which nucleotide was included.

Description of the invention

[05] This application provides recombinant DNA polymerases. One example of a polymerase according to the present disclosure includes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 DNA polymerase 9°N.

[06] In one embodiment, the polymerase also includes an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least one amino acid substitution mutation at a position functionally equivalent to Phe152, Val278, Met329, Val471, Thr514, Leu631, or Glu734 in the amino acid sequence of the 9°N DNA polymerase, and optionally further includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of the 9°N DNA polymerase.

[07] In one embodiment, the polymerase also includes an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least one amino acid substitution mutation at a position functionally equivalent to Lys476, Lys477, Thr514, IIe521, or Thr590 in the amino acid sequence of the DNA -polymerase 9°N, and optionally further includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410 and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[08] In one embodiment, the polymerase also includes an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least one amino acid substitution mutation at a position functionally equivalent to Arg247, Glu599, Lys620, His633, or Val661 in the amino acid sequence of DNA -polymerase 9°N, and optionally further includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410 and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[09] In one embodiment, the polymerase also includes (i) an amino acid substitution mutation at a position functionally equivalent to Tyr497, (ii) at least one amino acid substitution mutation at a position functionally equivalent to Phe152, Val278, Met329, Val471, Leu631 or Glu734 at the amino acid sequence of DNA polymerase 9°N, and (Hi) at least one amino acid substitution type mutation at a position functionally equivalent to Lys476, Lys477, Thr514, IIe521, or Thr590 at the amino acid sequence of DNA polymerase 9°N, and optionally further includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[010] In one embodiment, the polymerase also comprises (i) an amino acid substitution mutation at a position functionally equivalent to Tyr497, (ii) at least one amino acid substitution mutation at a position functionally equivalent to Lys476, Lys477, Thr514, IIe521, or Thr590 in the amino acid sequence of DNA polymerase 9°N, and (iii) at least one amino acid substitution mutation at a position functionally equivalent to Arg247, Glu599, Lys620, His633, or Val661 in the amino acid sequence of DNA polymerase 9°N, and optionally additionally includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410 and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[011] In one embodiment, the polymerase also includes (i) an amino acid substitution mutation at a position functionally equivalent to Tyr497, (ii) at least one amino acid substitution mutation at a position functionally equivalent to Phe152, Val278, Met329, Val471, Leu631 or Glu734 in the amino acid sequence of DNA polymerase 9°N, (iii) at least one amino acid substitution mutation at a position functionally equivalent to Lys476, Lys477, Thr514, Ile521, or Thr590 in the amino acid sequence of DNA polymerase 9°N, and (iv) at least one amino acid substitution mutation at a position functionally equivalent to Arg247, Glu599, Lys620, His633, or Val661 in the amino acid sequence of DNA polymerase 9°N, and optionally further includes amino acid substitution mutations at functionally equivalent positions amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410 and Ala485 in the amino acid sequence of DNA-po polymerase 9°N.

[012] Another example of a polymerase according to the present disclosure includes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 8 DNA polymerase 9°N, and also includes (i) amino acid substitution mutations at positions functionally equivalent to Tyr497, Phe152, Val278, Met329, Val471 and Thr514 in the amino acid sequence of DNA polymerase 9°N; (ii) amino acid substitution mutations at positions functionally equivalent to Tyr497, Met329, Val471, and Glu734 in the amino acid sequence of DNA polymerase 9°; (iii) amino acid substitution mutations at positions functionally equivalent to Tyr497, Arg247, Glu599, and His633 in the amino acid sequence of DNA polymerase 9°N; (iv) amino acid substitution mutations at positions functionally equivalent to Tyr497, Arg247, Glu599, Lys620, and His633 in the amino acid sequence of DNA polymerase 9°N; (v) amino acid substitution mutations at positions functionally equivalent to Tyr497, Met 329, Thr514, Lys620, and Val661 in the amino acid sequence of DNA polymerase 9°N; or (vi) amino acid substitution mutations at positions functionally equivalent to Tyr497, Val278, Val471, Arg247, Glu599 and His633 in the amino acid sequence of DNA polymerase 9°N.

[013] This application also provides a recombinant DNA polymerase that includes an amino acid sequence according to any one of SEQ ID NO: 10-34, a nucleic acid molecule that encodes the polymerase disclosed in this application, an expression vector that includes a nucleic acid molecule, and a host cell that includes said vector.

[014] The present disclosure also includes methods. In one embodiment, the method is for incorporating modified nucleotides into a growing DNA strand. The method includes ensuring the interaction of the following components: (i) the polymerase described in this application; (ii) DNA templates; and (iii) a solution of nucleotides.

[015] The present application also proposes a kit. In one embodiment, the kit is for carrying out a nucleotide incorporation reaction. The kit may include, for example, described in this application, the polymerase and a solution of nucleotides.

Brief description of graphic materials

[016] FIG. 1 schematically shows the amino acid sequence alignment of polymerase from Thermococcus sp.9°N-7 (9°N, SEQ ID NO: 1), Thermococcus litoralis (Vent, SEQ ID NO: 2 and Deep Vent, SEQ ID NO: 3), Thermococcus waiotapuensis (Twa, SEQ ID NO: 7), Thermococcus kodakaraenis (KOD, SEQ ID NO: 5), Pyrococcus furiosus (Pfu, SEQ ID NO: 4), Pyrococcus abyssi (Pab, SEQ ID NO: 6). "*" (asterisk) indicates positions that have one completely conserved residue between all polymerases. ":" (colon) indicates the retention of highly similar properties between groups, as shown below, which is roughly equivalent to a score greater than 0.5 in the Gonnet PAM 250 matrix. "." (Point) indicates the retention of weakly similar properties between groups as shown below, which is roughly equivalent to a score of less than or equal to 0.5 and greater than 0 in the Gonnet PAM 250 matrix.

[017] FIG. 2 shows the reduction in phasing and cumulative errors at short turn-on times demonstrated by one of the modified polymerases of the present disclosure, Pol 1558 (SEQ ID NO: 11), compared to the control Pol 812 (SEQ ID NO: 8) (left panels) . The two enzymes show comparable phasing and error rates at standard turn-on times (right panels).

[018] FIG. 3A shows the reduction in R1 phasing error rates and E. coli cumulative errors at short on-time demonstrated by selected altered polymerases of the present disclosure, Pol 1558 (SEQ ID NO: 11), Pol 1671 (SEQ ID NO: 23), Pol 1682 (SEQ ID NO: 25) and Pol 1745 (SEQ ID NO: 28), no compared to controls Pol 812 (SEQ ID NO: 8) and Pol 963 (SEQ ID NO: 9). The dotted lines in the top and bottom panels show the E. coli cumulative error and R1 phasing frequency as demonstrated by Pol 812 at standard turn-on time. FIG. 3B compares the frequency of phasing and prephasing of the same altered polymerases versus controls Pol 812 and Pol 963.

[019] FIG. 4 compares PhiX cumulative error rates for Pol 1550 (SEQ ID NO: 10) and Pol 1558 (SEQ ID NO: 11) with that of control Pol 812 (SEQ ID NO: 8) at standard and short turn-on times during long reads. sequencing (2×250 cycles). Both mutants show a marked reduction in error rates after paired-end sequencing.

[020] FIG. 5A shows a comparison of NovaSeq™ sequencing performance for one of the modified polymerases of the present invention, Pol 1671 (SEQ ID NO: 23), demonstrated at short on-times, with those of control Pol 812 (SEQ ID NO: 8), demonstrated at standard and short turn-on time. The upper panels show the percentage of clusters that passed the filter ("Clusters PF"); the lower panels show the PhiX cumulative error rate. Light open circles indicate Pol 812 performance at standard on time, while dark open circles indicate Pol 812 performance at short on time. All Pol 1671 values indicated by solid circles have a short turn-on time. FIG. 5B summarizes the PhiX cumulative error rates, Q30 values, and phasing rates demonstrated by Pol 1671 versus Pol 812 control for NovaSeq™ reads 1 and 2 at standard and short turn-on times. A significant improvement in quality for both readings was observed using Pol 1671.

Detailed description of the invention

[021] The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[022] The words "preferred" and "preferably" refer to embodiments of the invention that may provide certain advantages under certain circumstances. However, other implementations may also be preferred under the same or different circumstances. In addition, the listing of one or more preferred embodiments is not intended to imply that other embodiments are not suitable, and is not intended to exclude other embodiments from the scope of the present invention.

[023] The terms "comprises" and variations thereof are not intended to be limiting where these terms appear in the specification and claims.

[024] It should be understood that wherever embodiments are described in this application using the wording "comprise", "includes" or "including" and the like, otherwise similar implementation options are also described by the terms "consisting of" and / or " consisting essentially of.

[025] Unless otherwise indicated, the definite and indefinite forms of the singular and "at least one" are used interchangeably and denote one or more.

[026] Conditions that are "suitable" for the occurrence of an event, or "suitable" conditions are conditions that do not prevent the occurrence of such events. Thus, these conditions allow, enhance, facilitate and/or facilitate the event.

[027] As used herein, "providing" in the context of a composition, article, nucleic acid, or core means obtaining a composition, article, nucleic acid, or core, acquiring a composition, article, nucleic acid, or core, or otherwise obtaining a compound, composition, article, or core .

[028] Also in this application, the enumeration of numerical ranges by endpoints includes all numbers included in this range (for example, from 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5 etc.).

[029] In this application, reference to "one implementation", "an implementation", "certain implementations", or "some implementations", etc. means that a particular feature, configuration, composition, or characteristic described in connection with a specified implementation option is included in at least one implementation option according to the present disclosure. Thus, the occurrence of such phrases in various places in this application does not necessarily refer to the same implementation variant according to the present disclosure. In addition, specific features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[030] A new generation of polymerases may contribute to maintaining or exceeding current levels of efficiency with faster on-time. The present application provides polymerase enzymes with greatly improved performance under fast cycle sequencing by synthesis (SBS) conditions. The present inventors have surprisingly identified some modified polymerases that exhibit improved performance, including improved accuracy over short turn-on times. Improved accuracy includes reduced error rates and reduced phasing. Altered polymerases have a number of other associated benefits, including lower prephasing, lower branching frequency, and improved performance in SBS reactions. This improvement is maintained even when polymerase is used at lower concentrations. Accordingly, in one embodiment, the concentration of DNA polymerase in the SBS reaction may be from 120 ng/µl to 80 ng/µl. In one embodiment, the concentration of DNA polymerase in the SBS reaction may be at most 120 ng/µl, at most 110 ng/µl, at most 100 ng/µl, or at most 90 ng/µl. In one embodiment, the concentration of DNA polymerase in the SBS reaction may be at least 80 ng/µl, at least 90 ng/µl, at least 100 ng/µl, or at least 110 ng/µl.

[031] Error rate refers to a measurement of the frequency of errors in identifying the correct base, ie, the complementary strand to the template sequence at a particular position, during a sequencing reaction. The confidence with which a sequenced library matches the original genome sequence can vary depending on the frequency of base mutations occurring at any stage from nucleic acid extraction to its sequencing on a sequencing platform. This frequency of occurrence sets an upper limit on the probability of correctness of the sequenced base. In some embodiments, the quality score is represented as a numeric value. For example, a quality score could be specified as QXX, where XX is a score and means that this particular query has an error probability of 10 ^-XX/10 . Thus, for example, Q30 corresponds to an error rate of 1 in 1000, or 0.1%, and Q40 corresponds to an error rate of 1 in 10,000, or 0.01%.

[032] Phasing and pre-phasing are terms known to those skilled in the art and are used to describe the loss of synchrony when copies of a cluster sequence are read. Phasing and pre-phasing cause the signal of the current cycle and noise from the previous and subsequent cycles to be included in the selected intensities of a certain cycle. Thus, in this application, the term "phasing" refers to the phenomenon in SBS that is caused by the incomplete incorporation of a nucleotide into some portion of the DNA strands within clusters by polymerases in a given sequencing run, and thus is a measure of the frequency with which individual molecules in a cluster lose synchronicity with each other. The phasing may be measured during the detection of the cluster signal in each cycle and may be represented as the percentage of the detected signal from the cluster that is not synchronous with the signal in the cluster. For example, a cluster is detected by a "green" fluorophore signal during cycle N. In the subsequent cycle (cycle N plus 1), 99.9% of the cluster signal is detected in the "red" channel, and 0.1% of the signal remains from the previous cycle and is detected in green channel. This result will indicate the occurrence of phasing, and can be represented as a numerical value, such as a phasing value of 0.1, indicating that 0.1% of the molecules in the cluster are shifted back in each cycle.

[033] As used herein, the term "prephasing" refers to a phenomenon in SBS that is caused by the incorporation of nucleotides without effective 3' terminators, whereby the incorporation event occurs one cycle ahead. As the number of cycles increases, the proportion of sequences in the cluster that are affected by phasing increases, making it difficult to identify the correct base. Pre-phasing can be detected by the sequencing instrument and represented as a numerical value, such as a pre-phasing value of 0.1, indicating that 0.1% of the molecules in the cluster moved forward in each cycle.

[034] Phasing and prephasing detection can be performed and recorded in accordance with any suitable methodology known in the art, for example, as described in US patent No. 8965076. For example, as described in the examples below, phasing is detected and regularly recorded during SBS sequencing runs on sequencing instruments such as HiSeq™, Genome Analyzer™, NextSeq™, NovaSeq™, iSeq™, MiniSeq™, or MiSeq™ sequencing platforms from IIIumina, Inc. (San Diego, CA) or any other suitable instrument known in the art.

[035] Reducing the cycle time can increase the frequency of occurrence of phasing, pre-phasing and/or forking, each of which contributes to the error rate. The discovery of altered polymerases that reduce the frequency of phasing, prephasing and/or branching, even when used under fast cycle conditions, is unexpected and provides a great advantage when used in SBS. For example, modified polymerases can provide faster SBS cycle times, lower phasing and prephasing values, and/or longer sequencing read lengths. The error rate and phasing characteristics for the modified polymerases as proposed in this application are set forth in the Examples section below.

Polymerases

[036] Provided herein are polymerases, compositions comprising a polymerase, and methods of using a polymerase. Disclosed in this application, the polymerase is a DNA polymerase. In one embodiment, the implementation of the polymerase according to the present disclosure, also referred to in this application "altered polymerase", is based on the amino acid sequence of the reference polymerase. The altered polymerase includes substitution mutations at one or more residues compared to the reference polymerase. The substitution mutation may be in the same position or in a functionally equivalent position as compared to the reference polymerase. Reference polymerases and functionally equivalent positions are described in detail in this application. A person skilled in the art will easily understand that the modified polymerase described in this application does not occur in nature.

[037] Disclosed in this application, the reference polymerase has an error rate suitable for SBS reactions; however, the use of a reference polymerase in SBS reactions with a shorter turn-on time increases the error rate. The modified polymerase described in this application maintains error rates in excess of those observed for reference polymerases, even when the modified polymerase is used in SBS reactions with shorter turn-on times. In one embodiment, reduced error rates occur when the modified polymerase is tested using a fast turn-on time. Inclusion refers to the amount of time the DNA polymerase is in contact with the template. In this application, the slow turn-on time is the turn-on time used in a standard run using the MiniSeq™ benchtop sequencing system. The slow turn-on time is 40 to 50 seconds. In the present application, the fast cycle time refers to the turn-on phase, which is from 10 seconds to 40 seconds. In one embodiment, the fast cycle time is an on time of 40 seconds or less, 30 seconds or less, 20 seconds or less, 18 seconds or less, 16 seconds or less, 14 seconds or less, or 12 seconds or less. In one embodiment, the fast cycle time is an on time of at least 10 seconds, at least 12 seconds, at least 14 seconds, at least 16 seconds, at least 18 seconds, at least 20 seconds, or at least 30 seconds. In one embodiment, the fast cycle time is an on time of less than 40 seconds, less than 30 seconds, less than 20 seconds, less than 18 seconds, less than 16 seconds, less than 14 seconds, less than 12 seconds, or less than 10 seconds.

[038] The modified polymerase described in this application can be used in SBS reactions for runs of various lengths. "Run" refers to the number of nucleotides identified on the template. A run typically includes a run based on a first primer (eg primer read1) which reads one strand of the template and a run based on a second primer (eg primer read2) which reads the complementary strand of the template. In one embodiment, the number of nucleotides identified using the first or second primer may be from 10 to 150 nucleotides. In one embodiment, the number of nucleotides identified using the first primer or the second primer may be no more than 150 nucleotides, no more than 130 nucleotides, no more than 110 nucleotides, no more than 90 nucleotides, no more than 70 nucleotides, no more than 50 nucleotides, no more than 30 nucleotides or no more than 20 nucleotides. In one embodiment, the number of nucleotides identified using the first primer or the second primer may be at least 10, at least 20, at least 30, at least 50, at least 70, at least 90, at least at least 110 or at least 130 nucleotides.

[039] In some embodiments, the modified polymerase is based on a type B family DNA polymerase. The modified polymerase can be based on, for example, archaeal B family DNA polymerase, human DNA polymerase-a, or phage polymerase.

[040] Family B archaeal DNA polymerases are well known in the art, as illustrated in US Pat. No. 8,283,149. In some embodiments, the archaeal DNA polymerase is a DNA polymerase from hyperthermophilic archaea and is thermostable.

[041] In some embodiments, the family B archaeal DNA polymerase is from a genus such as, for example, Thermococcus, Pyrococcus, or Methanococcus. Members of the genus Thermococcus are well known in the art and include, but are not limited to, T. 4557, T. barophilus, T. gammatolerans, T. onnurineus, T. sibiricus, T. kodakarensis, T. gorgonarius, and T. waiotapuensis. Members of the genus Pyrococcus are well known in the art and include, but are not limited to, P. NA2, P. abyssi, P. furiosus, P. horikoshii, P. yayanosii, P. endeavori, P. glycovorans, and P. woesei. Members of the genus Methanococcus are well known in the art and include, but are not limited to, M aeolicus, M. maripaludis, M. vannielii, M voltae, M. thermolithotrophicus, and M. jannaschii.

[042] In one embodiment, the modified polymerase is based on Vent®, Deep Vent®, 9°N, Pfu, KOD, or Pab polymerase. Vent® and Deep Vent® are the commercial names used for family B DNA polymerases isolated from hyperthermophilic archaea Thermococcus litoraiis. Polymerase 9°N is a family B polymerase isolated from Thermococcus sp. Pfu polymerase is a family B polymerase isolated from Pyrococcus furiosus. The KOD polymerase is a family B polymerase isolated from Thermococcus kodakaraenis. The Pab polymerase is a family B polymerase isolated from Pyrococcus abyssi. Twa is a family B polymerase isolated from T. waiotapuensis. Examples of Vent®, Deep Vent®, 9°N, Pfu, KOD, Pab and Twa polymerases are disclosed in FIG. one.

[043] In some embodiments, the archaeal B family DNA polymerase is derived from a phage, such as, for example, T4 phage, RB69, or phi29.

[044] FIG. 1 shows a sequence alignment of proteins having the amino acid sequences shown in SEQ ID NOs: 1-7. The alignment indicates amino acids that are conserved across the various family B polymerases. One of skill in the art will appreciate that conserved amino acids and conserved regions are likely to be conserved because they are important for the functioning of polymerases and therefore exhibit a correlation between structure and functioning of polymerases. The alignment also shows regions of variability in the various family B polymerases. From such data, regions of the polymerase in which substitutions, in particular conservative substitutions, can be resolved without undue impact on the biological activity of the altered polymerase can be determined by one skilled in the art.

[045] The modified polymerase described herein is a polymerase based on the amino acid sequence of a known polymerase (also referred to herein as a reference polymerase) and further includes substitution type mutations at one or more residues. In one embodiment, the substitution mutation is at a position that is functionally equivalent to the amino acid of the reference polymerase. By "functionally equivalent" is meant that the altered polymerase has an amino acid substitution at an amino acid position in the reference polymerase that plays the same functional role in both the reference polymerase and the altered polymerase.

[046] In general, functionally equivalent substitution mutations in two or more different polymerases occur at homologous amino acid positions in the amino acid sequences of the polymerases. Therefore, the use in this application of the term "functionally equivalent" also covers mutations that are "positionally equivalent" or "homologous" to a given mutation, regardless of whether the specific function of the mutated amino acid is known. The locations of functionally equivalent and positionally equivalent amino acid residues in the amino acid sequences of two or more different polymerases can be identified based on sequence alignment and/or molecular modeling. An example of sequence alignment to identify positionally equivalent and/or functionally equivalent residues is shown in FIG. 1. For example, residues in Twa, KOD, Pab, Pfu, Deep Vent, and Vent polymerases in FIG. 1 that are vertically aligned are considered to be positionally equivalent as well as functionally equivalent to the corresponding residue in the 9°N polymerase amino acid sequence. Thus, for example, residue 349 in polymerases 9°N, Twa, KOD, Pfu, Deep Vent, and Pab and residue 351 in Vent polymerase are functionally equivalent and positionally equivalent. Similarly, for example, residue 633 in 9°N, Twa, KOD and Pab polymerases, residue 634 in Pfu and Deep Vent polymerases, and residue 636 in Vent polymerase are functionally equivalent and positionally equivalent. One skilled in the art can readily identify functionally equivalent residues in DNA polymerases.

[047] In some embodiments, a substitution mutation includes mutation to a residue having a non-polar side chain. Amino acids having non-polar side chains are well known in the art and include, for example: alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine.

[048] In some embodiments, a substitution mutation includes mutation to a residue having a polar side chain. Amino acids having polar side chains are well known in the art and include, for example: arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, cysteine, tyrosine, and threonine.

[049] In some embodiments, a substitution type mutation comprises a mutation to a residue having a hydrophobic side chain. Amino acids having hydrophobic side chains are well known in the art and include, for example: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan.

[050] In some embodiments, a substitution mutation includes mutation to a residue having an uncharged side chain. Amino acids with uncharged side chains are well known in the art and include, for example: glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine, among others.

[051] In one embodiment, the modified polymerase has an amino acid sequence that is similar in structure to the reference polymerase disclosed herein. In one embodiment, the implementation of the reference polymerase is a polymerase that includes the amino acid sequence 9°N (SEQ ID NO: 1). Optionally, the reference polymerase is SEQ ID NO: 1 with the following substitution mutations: Met129Ala, Asp141 Ala, Glu143Ala, Cys223Ser, Leu408Ala, Tyr409Ala, Pro410lle and Ala485Val. A polymerase having the amino acid sequence 9°N (SEQ ID NO: 1) with substitution mutations Met129Ala, Asp141Ala, Glu143Ala, Cys223Ser, Leu408Ala, Tyr409Ala, Pro41Olle and Ala485Val is disclosed in SEQ ID NO: 8 and is also referred to in this application as polymerase Po1812. Other reference sequences include SEQ ID NO: 2, 3, 4, 5, 6, or 7. Optionally, the reference polymerase is SEQ ID NO: 2, 3, 4, 5, 6, or 7 with substitution type, functional, and positional mutations. equivalent to the following substitution mutations in SEQ ID NO: 1: Met129Ala, Asp141Ala, Glu143Ala, Cys223Ser, Leu408Ala, Tyr409Ala, Pro410lle and Ala485Val.

[052] As used herein, an altered polymerase may be "structurally similar" to a reference polymerase if the amino acid sequence of the altered polymerase has a certain degree of sequence similarity and/or sequence identity compared to the reference polymerase.

[053] Structural similarity between two amino acid sequences can be determined by aligning the residues of two sequences (eg, a candidate polymerase and a reference polymerase described herein) to optimize the number of identical amino acids along their sequence length; gaps are allowed in the alignment in one or both sequences to optimize the number of identical amino acids, although the amino acids in each sequence must still remain in their correct order. A candidate polymerase is a polymerase that is compared to a reference polymerase. A candidate polymerase that has structural similarity to the reference polymerase and polymerase activity is an altered polymerase.

[054] Unless otherwise indicated herein, a pairwise comparison analysis of amino acid sequences or nucleotide sequences can be performed, for example, using the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), Pearson & Lipman, Proc. Nat'l. Acad. sci. USA 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) or by visual evaluation (See generally, Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., updated 2004).

[055] One example of an algorithm that is suitable for determining structural similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). BLAST analysis software is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying sequence pairs with a high score of similarity (HSP) by identifying short "words" of length W in the requested sequence that either match or satisfy some positive threshold score T when aligned with a "word" of the same length in the sequence. from the database. T is referred to as the neighborhood word similarity score threshold (Altschul et al., J. Mol. Biol. 215:403-410 (1990)). These initial matches of neighboring "words" serve as seeds to initiate searches to find longer HSPs containing them. Word matches are then extended in both directions along each sequence as long as the total alignment score can increase. Total scores are calculated using the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatched residues; always less than 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the total score. Word hits stop extending in each direction if: the total alignment score decreases by X from the maximum value it has reached; the total score drops to zero or below, due to the accumulation of one or more negative-scoring residual alignments, or the end of any sequence is reached. The W, T and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses word length (W) equal to 11, expectation (E) equal to 10, cutoff equal to 100, M equal to 5, N equal to -4 and comparison of both strands as default parameters. For amino acid sequences in BLASTP, the default parameters are word length (W) of 3, expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci USA 89:10915).

[056] In addition to computing percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)) . One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which is an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the lowest sum probability when comparing the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[057] When comparing two amino acid sequences, structural similarity may be indicated by a percentage of "identity", or may be indicated by a percentage of "similarity". "Identity" refers to the presence of identical amino acids. "Similarity" refers to the presence of not only identical amino acids, but also the presence of conservative substitutions. A conservative amino acid substitution in a protein may be selected from other members of the class to which the amino acid belongs. For example, it is well known in the field of protein biochemistry that an amino acid belonging to a group of amino acids having a certain size or characteristics (such as charge, hydrophobicity, or hydrophilicity) can be replaced by another amino acid without changing the activity of the protein, especially in regions of the protein that are not directly associated with biological activity. For example, non-polar amino acids include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Hydrophobic amino acids include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. Polar amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, cysteine, tyrosine, and threonine. Uncharged amino acids include, among others, glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine.

[058] Thus, in the present application, a reference to a polymerase described in this application, such as a reference to an amino acid sequence representing one or more of the SEQ ID NOs described in this application, may include a protein with amino acid sequence similarity to a reference polymerase, constituting at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[059] Alternatively, in the present application, a reference to a polymerase described in this application, such as a reference to an amino acid sequence representing one or more SEQ ID NOs described in this application, may include a protein with amino acid sequence identity with reference polymerase comprising at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[060] This disclosure describes a set of mutations that lead to the production of a polymerase having one or more of the activities described in this application. The polymerase described in this application may include any number of mutations, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least at least 17 or at least 18 mutations compared to a reference polymerase such as SEQ ID NO: 1 or SEQ ID NO: 8. Similarly, the polymerase described herein may include mutations in any combination. For example, Table 1 provides examples of specific modified polymerases that include various combinations of the mutations described herein. A checkmark (√) indicates the presence of the specified mutation. These mutations, e.g., Y497G, F152G, V278L, etc., are mutations at positions in SEQ ID NO: 1.

[062] The altered polymerase of the present disclosure includes a substitution mutation at a position functionally equivalent to Tyr497 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Tyr497 is a mutation to a non-polar, hydrophobic, or uncharged amino acid, eg, Gly.

[063] In one embodiment, the altered polymerase comprises a substitution type mutation at a position functionally equivalent to Pne152 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution mutation at a position functionally equivalent to Phe152 is a mutation to a non-polar, hydrophobic, or uncharged amino acid, eg, Gly.

[064] In one embodiment, the altered polymerase includes a substitution type mutation at a position functionally equivalent to Val278 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution mutation at a position functionally equivalent to Val278 is a mutation to a non-polar or hydrophobic amino acid, eg, Leu.

[065] In one embodiment, the altered polymerase includes a substitution mutation at a position functionally equivalent to Met329 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution type mutation at a position functionally equivalent to Met329 is a mutation to a polar amino acid, eg, His.

[066] In one embodiment, the altered polymerase includes a substitution mutation at a position functionally equivalent to Val471 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution mutation at a position functionally equivalent to Val471 is a mutation to a polar or uncharged amino acid, eg, Ser.

[067] In one embodiment, the altered polymerase includes a substitution mutation at a position functionally equivalent to Thr514 in polymerase 9°N (SEQ ID NO: 1), as is known in the art and illustrated in US Patent Application No. 2016/0032377 . In one embodiment, the substitution type mutation at a position functionally equivalent to Thr514 is a mutation to a non-polar or hydrophobic amino acid, eg, Ala. In some embodiments, other substitution mutations that can be used in combination with a non-polar or hydrophobic amino acid at a position functionally equivalent to Thr514 include Phe152, Val278, M329, Val471, Lue631, Glu734, or a combination thereof. In one embodiment, the substitution mutation at a position functionally equivalent to Thr514 is a mutation to a polar or uncharged amino acid, eg, Ser. In some embodiments, other substitution mutations that can be used in combination with a polar or uncharged amino acid at a position functionally equivalent to Thr514 include Lys476, Lys477, lle521, Thr590, or a combination thereof.

[068] In one embodiment, the altered polymerase includes a substitution mutation at a position functionally equivalent to Leu631 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution mutation at a position functionally equivalent to Leu631 is a mutation to a non-polar or hydrophobic amino acid, eg, Met.

[069] In one embodiment, the altered polymerase comprises a substitution type mutation at a position functionally equivalent to Glu734 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution mutation at a position functionally equivalent to Glu734 is a mutation to a polar or uncharged amino acid, eg, Arg.

[070] In one embodiment, the altered polymerase comprises a substitution mutation at a position functionally equivalent to Lys476 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution mutation at a position functionally equivalent to Lys476 is a mutation to a non-polar or hydrophobic amino acid, eg, Trp.

[071] In one embodiment, the altered polymerase comprises a substitution mutation at a position functionally equivalent to Lys477 in polymerase 9°N (SEQ ID NO: 1), as is known in the art and illustrated in US Patent disclosure No. 9,765,309. In one embodiment, the substitution mutation at a position functionally equivalent to Lys477 is a mutation to a non-polar or hydrophobic amino acid, eg, Met.

[072] In one embodiment, the altered polymerase includes a substitution mutation at a position functionally equivalent to Ie521 in polymerase 9°N (SEQ ID NO: 1), as is known in the art and illustrated in US Patent Application No. 2016/0032377 . In one embodiment, the substitution type mutation at a position functionally equivalent to Pe521 is a mutation to a non-polar amino acid, eg, Leu.

[073] In one embodiment, the altered polymerase includes a substitution mutation at a position functionally equivalent to Thr590 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution mutation at a position functionally equivalent to Thr590 is a mutation to a non-polar or hydrophobic amino acid, eg, lle.

[074] In one embodiment, the altered polymerase comprises a substitution mutation at a position functionally equivalent to Arg247 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution type mutation at a position functionally equivalent to Arg247 is a mutation to a non-polar or uncharged amino acid, eg, Tyr.

[075] In one embodiment, the altered polymerase comprises a substitution mutation at a position functionally equivalent to Glu599 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution type mutation at a position functionally equivalent to Glu599 is a mutation to a polar or uncharged amino acid, eg, Asp.

[076] In one embodiment, the altered polymerase includes a substitution mutation at a position functionally equivalent to Lys620 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution type mutation at a position functionally equivalent to Lys620 is a mutation to a polar or uncharged amino acid, eg, Arg.

[077] In one embodiment, the altered polymerase includes a substitution mutation at a position functionally equivalent to His633 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution mutation at a position functionally equivalent to His633 is a mutation to a non-polar, hydrophobic, or uncharged amino acid, eg, Gly.

[078] In one embodiment, the altered polymerase comprises a substitution mutation at a position functionally equivalent to Val661 in polymerase 9°N (SEQ ID NO: 1). In one embodiment, the substitution type mutation at a position functionally equivalent to Val661 is a mutation to a polar or uncharged amino acid, eg, Asp.

[079] In one embodiment, the altered polymerase comprises at least one substitution type mutation at a position functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Lys408, Tyr409, Pro410, Ala485, or a combination thereof. In one embodiment, the substitution mutation at a position functionally equivalent to Met129, Asp141, Glu143, Lys408, or Tyr409 is a mutation to a non-polar or hydrophobic amino acid, eg, Ala. In one embodiment, the substitution type mutation at a position functionally equivalent to Cys223 is a mutation to a polar or uncharged amino acid, eg, Ser. In one embodiment, the substitution mutation at a position functionally equivalent to Pro410 is a mutation to a non-polar or hydrophobic amino acid, eg, lle. In one embodiment, the substitution mutation at a position functionally equivalent to A1a485 is a mutation to a non-polar or hydrophobic amino acid, eg, Val.

[080] In one embodiment, the altered polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least one, at least two, at least three, at least four, at least five, at least at least six or seven amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Phe152, Val278, Met329, Val471, Thr514, Leu631 and Glu734 in the amino acid sequence of DNA polymerase 9°N. In one embodiment, the altered polymerase also includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[081] In one embodiment, the altered polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least one, at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Lys476, Lys477, Thr514, lle521 and Thr590 in the amino acid sequence of DNA polymerase 9°N. In one embodiment, the altered polymerase also includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[082] In one embodiment, the altered polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to the amino acid selected from Arg247, Glu599, Lys620, His633 and Val661 in the amino acid sequence of DNA polymerase 9°N. In one embodiment, the altered polymerase also includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[083] In one embodiment, the altered polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr497, at least one, at least two, at least three, at least four, at least five, at least six or seven amino acid substitution mutations at a position functionally equivalent to Phe152, Val278, Met329, Val471, Thr514, Leu631, or Glu734 in the amino acid sequence of DNA polymerase 9°N, and (iii) at least one, at least two, at least three, at least four or five amino acid substitution mutations at a position functionally equivalent to Lys476, Lys477, Thr514, Ile521, or Thr590 in the amino acid sequence of DNA polymerase 9°N. In one embodiment, the altered polymerase also includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[084] In one embodiment, the altered polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr497, at least one, at least two, at least three, at least four, at least five, at least six or seven amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Phe152, Val278, Met329, Val471, Thr514, Leu631, and Glu734 in the amino acid sequence of DNA polymerase 9°N, and at least one, at least two , at least three, at least four or five amino acid substitution type mutations at positions functionally equivalent to an amino acid selected from Arg247, Glu599, Lys620, His633, or Val661 in the amino acid sequence of DNA polymerase 9°N. In one embodiment, the altered polymerase also includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[085] In one embodiment, the altered polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr497, at least one, at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Lys476, Lys477, Thr514, Ile521, or Thr590 in the amino acid sequence of DNA polymerase 9°N, and at least one, at least two, at least three, at least four, or five mutations in a type of amino acid substitution at positions functionally equivalent to an amino acid selected from Arg247, Glu599, Lys620, His633, or Val661 in the amino acid sequence of DNA polymerase 9°N. In one embodiment, the altered polymerase also includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[086] In one embodiment, the altered polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr497, at least one, at least two, at least three, at least four, at least five, or six mutations in type of amino acid substitution at positions functionally equivalent to an amino acid selected from Phe152, Val278, Met329, Val471, Leu631 and Glu734 in the amino acid sequence of DNA polymerase 9°N, at least one, at least two, at least three, at least at least four or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Lys476, Lys477, Thr514, Ile521, or Thr590 in the amino acid sequence of DNA polymerase 9°N, and at least one, at least two, at least at least three, at least four or five amino acid substitution type mutations at positions functionally equivalent to an amino acid selected from Arg247, Glu599, Lys6 20, His633 or Val661 in the amino acid sequence of DNA polymerase 9°N. In one embodiment, the altered polymerase also includes amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N.

[087] Specific examples of modified polymerases include Pol 1550 (SEQ ID NO: 10), Pol 1558 (SEQ ID NO: 11), Pol 1563 (SEQ ID NO: 12), Pol 1565 (SEQ ID NO: 13), Pol 1630 (SEQ ID NO: 14), Pol 1634 (SEQ ID NO: 15), Pol 1641 (SEQ ID NO: 16), Pol 1573 (SEQ ID NO: 17), Pol 1576 (SEQ ID NO: 18), Pol 1584 (SEQ ID NO: 19), Pol 1586 (SEQ ID NO: 20), Pol 1601 (SEQ ID NO: 21), Pol 1611 (SEQ ID NO: 22), Pol 1671 (SEQ ID NO: 23), Pol 1677 (SEQ ID NO: 24), Pol 1682 (SEQ ID NO: 25), Pol 1680 (SEQ ID NO: 27), Pol 1745 (SEQ ID NO: 28), Pol 1758 (SEQ ID NO: 29), Pol 1761 (SEQ ID NO: 30), Pol 1762 (SEQ ID NO: 31), Pol 1765 (SEQ ID NO: 32), Pol 1769 (SEQ ID NO: 33) and Pol 1770 (SEQ ID NO: 34).

[088] The modified polymerase described in this application may include additional mutations that are known to affect the activity of the polymerase. With this substitution, the mutation is in a position functionally equivalent to Arg713 in polymerase 9°N (SEQ ID NO: 1). Any of a variety of substitution type mutations at one or more positions that are known to result in decreased exonuclease activity can be made, as is known in the art and illustrated in US Pat. No. 8,623,628. In one embodiment, the substitution mutation at position Arg713 is a mutation to a non-polar, hydrophobic, or uncharged amino acid, eg, Gly, Met, or Ala.

[089] In one embodiment, the altered polymerase comprises a substitution type mutation at a position functionally equivalent to Arg743 or Lys705, or a combination thereof, in polymerase 9°N (SEQ ID NO: 1), as is known in the art and illustrated in the patent disclosure USA No. 8623628. In one embodiment, the substitution mutation at position Arg743 or Lys705 is a mutation to a non-polar or hydrophobic amino acid, eg, Ala.

[090] The present disclosure also provides compositions that include the modified polymerase described in this application. The composition may include components other than the modified polymerase. For example, the composition may include a buffer, a solution of nucleotides, or a combination thereof. The nucleotide solution may include nucleotides, such as labeled, synthetic, modified nucleotides, or a combination thereof. In one embodiment, the composition includes target nucleic acids, such as a library of target nucleic acids.

Mutating polymerases

[091] Various types of mutagenesis are optionally used in the present disclosure, e.g., to modify polymerases to produce variants, e.g., according to polymerase models and model-based prediction as described above, or using random or semi-random mutation approaches. In general, any available mutagenesis procedure can be used to generate polymerase mutants. Such mutagenesis procedures optionally include selection of mutant nucleic acids and polypeptides for one or more activities of interest (eg reduced pyrophospholysis, increased turnover, eg for a given nucleotide analog). Procedures that can be used include, but are not limited to: site-directed point mutagenesis, random point mutagenesis, in vitro or in vivo homologous recombination (DNA shuffling and combined PCR with overlapping primers), mutagenesis using uracil-containing templates, oligonucleotide- site-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, gap duplex DNA mutagenesis, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by whole gene synthesis, degenerate PCR , double-strand break repair, and many others known to those skilled in the art. The parent polymerase for mutation can be any of those specified herein, including available polymerase mutants such as those identified, for example, in US Pat. No. 8,460,910 and US Pat.

[092] Optionally, mutagenesis can be performed using known information about a naturally occurring polymerase molecule or a known altered or mutated polymerase (e.g., using an existing mutant polymerase), e.g., sequence, sequence comparison, physical properties, crystal structure, and/or how discussed above. However, in another class of implementations, the modification may be essentially random (eg, as in classical or "family" DNA shuffling, see, for example, Crameri et al. (1998) "DNA shuffling of a family of genes from diverse accelerates directed evolution" Nature 391:288-291).

[093] Additional information on mutation formats can be found in: Sambrook et al., Molecular Cloning-A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 ("Sambrook "); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (updated 2011) ("Ausubel")) and PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) ("Innis"). The following publications and references cited provide further details on mutation formats: Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology 4:450-455 (1993); Bass et al., Mutant Trp repressors with new DNA-binding specificities, Science 242:240-245 (1988); Bordo and Argos (1991) Suggestions for "Safe" Residue Substitutions in Site-directed Mutagenesis 217:721-729; Botstein & Shortle, Strategies and applications of in vitro mutagenesis, Science 229:1193-1201 (1985); Carter et al., Improved oligonucleotide site-directed mutagenesis using M13 vectors, Nucl. Acids Res. 13:4431-4443 (1985); Carter, Site-directed mutagenesis, Biochem. J. 237:1-7 (1986); Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors, Methods in Enzymol. 154: 382-403 (1987); Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioate method, Methods Mol. Biol. 57:369-374 (1996); Eghtedarzadeh & Henikoff, Use of oligonucleotides to generate large deletions, Nucl. Acids Res. 14:5115 (1986); Fritz et al., Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Grundstrom et al., Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Hayes (2002) Combining Computational and Experimental Screening for rapid Optimization of Protein Properties PNAS 99(25) 15926-15931; Kunkel, The efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag, Berlin)) (1987); Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc. Natl. Acad. sci. USA 82:488-492 (1985); Kunkel et al. Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol. 154, 367-382 (1987); Kramer et al. The gapped duplex DNA approach to oligonucleotide-directed mutation construction, Nucl. Acids Res. 12: 9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed construction of mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367 (1987); Kramer et al. Point Mismatch Repair, Cell 38:879-887 (1984); Kramer et al. Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations, Nucl. Acids Res. 16:7207 (1988); Ling et al. Approaches to DNA mutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997); Lorimer and Pastan Nucleic Acids Res. 23, 3067-8 (1995); Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis, Proc. Natl. Acad. sci. USA 83:7177-7181 (1986); Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis, Nucl. Acids Res. 14: 9679-9698 (1986); Nambiar et al. Total synthesis and cloning of a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakamar and Khorana, Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Sayers et al, Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802 (1988); Sayers et al. Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide, (1988) Nucl. Acids Res. 16:803-814; Sieber, et al. Nature Biotechnology, 19:456-460 (2001); Smith, In vitro mutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987); Stemmer, Nature 370, 389-91 (1994); Taylor et al. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8787 (1985); Wells et al. Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986); Wells et al. Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, Gene 34:315-323 (1985); Zoller & Smith, Oligonucleotide-directed mutagenesis using M 13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment, Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol. 154:329-350 (1987); Clackson et al. (1991) "Making antibody fragments using phage display libraries" Nature 352:624-628; Gibbs et al. (2001) "Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling" Gene 271:13-20; and Hiraga and Arnold (2003) "General method for sequence-independent site-directed chimeragenesis: J. Mol. Biol. 330:287-296. Additional information on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes suitable control options to eliminate problems using various mutagenesis methods.

Preparation and isolation of recombinant polymerases

[094] In General, nucleic acids encoding the polymerase presented in this application can be obtained by cloning, recombination, in vitro synthesis, in vitro amplification and/or other available methods. For the expression of the expression vector that encodes the polymerase presented in this application, you can use a variety of recombinant methods. Methods for producing recombinant nucleic acids, expressing and isolating expressed products are well known and described in the art. This application describes a number of examples of mutations and combinations of mutations, as well as strategies for creating the desired mutations. Methods for creating and selecting mutations in the active site of polymerases, including to modify steric properties at or near the active site to allow improved access for nucleotide analogs, can be found in this application and, for example, in WO 2007/076057 and WO 2008 /051530.

[095] Additional useful references to mutation methods, recombinant methods, and in vitro manipulation of nucleic acids (including cloning, expression, PCR, and the like) include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc. ., San Diego, Calif. (Berger); Kaufman et al. (2003) Handbook of Molecular and Cellular Methods in Biology and Medicine Second Edition Ceske (ed) CRC Press (Kaufman); The Nucleic Acid Protocols Handbook Ralph Rapley (ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley); Chen et al. (ed) PCR Cloning Protocols, Second Edition (Methods in Molecular Biology, volume 192) Humana Press; and Viljoen et al. (2005) Molecular Diagnostic PCR Handbook Springer, ISBN 1402034032.

[096] In addition, a variety of kits for purifying plasmids or other appropriate nucleic acids from cells are commercially available (see, for example, EasyPrep™ and FlexiPrep™, both from Pharmacia Biotech; StrataClean™ from Stratagene; and QIAprep™ from Qiagen). Any isolated and/or purified nucleic acid can be further manipulated to produce other nucleic acids for use in cell transfection, incorporation into related vectors to infect organisms for expression, and/or the like. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters suitable for regulating the expression of a particular target nucleic acid. The vectors optionally contain standard expression cassettes containing at least one independent terminator sequence, sequences allowing the cassette to replicate in eukaryotes or prokaryotes, or both (e.g., shuttle vectors), and selectable markers for both prokaryotic and eukaryotic systems . The vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both.

[097] Other useful references, for example for isolation and culture (eg, for subsequent nucleic acid isolation) include Freshney Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid S John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tiss Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Hei New York); and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Boca Raton, Fla.

[098] The present disclosure also includes nucleic acids encoding altered polymerases described in this application. A particular amino acid can be coded for by multiple codons, and certain translation systems (e.g., prokaryotic or eukaryotic cells) often show a preference for x. For example, different organisms often prefer one of several synonymous codons that code for the same amino acid. As such, the nucleic acids provided herein are optionally "codon-optimized", meaning that the nucleic acids are synthesized to include codons that are preferred for the particular translation system used to express the polymerase. For example, when it is desired to express a polymerase in a bacterial cell (or even in a particular strain of bacteria), the nucleic acid can be synthesized to include the most common codons in the genome of that bacterial cell for efficient expression of the polymerase. Such a page can be used when it is necessary to express a polymerase in a eukaryotic cell, for example, the nucleic acid may include codons that are preferred for that eukaryotic cell.

[099] There are various methods for the isolation and detection of proteins that can be used to isolate polymerases, for example, from recombinant cell cultures expressing the recombinant polymerases presented in this application. Various methods for isolating and detecting proteins are well known in the art, including, for example, those set forth in R. Scopes, Protein Purification, Springer-N.Y. (1982); Deutscher, Methods in Enzymology Vol.182: Guide to Protein Purification, Ac Press, Inc. N.Y. (1990); Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; & al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3rd Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and the links provided in them. Additional information on protein purification and detection methods can be found in Satinder Ahuja ed. Handbook of Bioseparations, Academic Press (2000).

Methods of application

[0100] The modified polymerases provided herein can be used in a sequencing procedure, such as a sequencing by synthesis (SBS) method. Briefly, SBS can be initiated by contacting target nucleic acids with one or more nucleotides (eg, labeled, synthetic, modified, or combinations thereof), DNA polymerase, and so on. Those features where the primer is extended using the target nucleic acid as a template will include a labeled nucleotide that can be detected. The turn-on time used in a sequencing run can be significantly reduced using the modified polymerases described in this application. Optionally, labeled nucleotides may further include a reversible termination property that stops further extension of the primer after the nucleotide has been added to the primer. For example, a nucleotide analogue may be added to the primer having a reversible terminator fragment such that subsequent extension cannot occur until a deblocking agent is delivered to remove the fragment. Thus, for embodiments that use reversible termination, the deblocker reagent can be delivered to the flow cell (before or after detection). Washing can be carried out between different stages of delivery. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Examples of SBS procedures, fluid systems, and detection platforms that can be easily adapted for use with a chip produced by the methods of the present disclosure are described, for example, in Bentley et al. Nature 456:53-59 (2008); WO 04/018497; W091/06678; WO 07/123744; US Pat. Nos. 7,057,026; 7,329,492; 7,211,414; 7,315,019;

[0101] Other sequencing procedures that use cyclic reactions, such as pyrosequencing, can be used. Pyrosequencing detects the release of inorganic pyrophosphate (PPi, from inorganic pyrophosphate) as certain nucleotides are incorporated into the nascent nucleic acid strand (Ronaghi, et al. Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res 11(1), 3-11 (2001); Ronaghi et al Science 281(5375), 363 (1998); US Pat. In pyrosequencing, released PPi can be detected by conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and ATP generated can be detected by photons produced by luciferase. Thus, the sequencing reaction can be monitored using a luminescence detection system. Excitation sources used for fluorescence-based detection systems are not required for pyrosequencing procedures. Suitable fluid systems, detectors, and procedures that can be used to apply pyrosequencing on a chip according to the present disclosure are described, for example, in WO 2012/058096, US patent application No. 2005/0191698 A1, US patents No. 7595883 and 7244559.

[0102] In some embodiments, methods can be used that include real-time monitoring of DNA polymerase activity. For example, incorporation of nucleotides can be detected by fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and γ-phosphate labeled nucleotides, or by null mode waveguides. Methods and reagents for FRET-based sequencing are disclosed, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33.1026-1028 (2008); Korlach et al. Proc. Natl. Acad. sci. USA 105, 1176-1181 (2008).

[0103] Some embodiments of SBS include detection of a proton released when a nucleotide is included in the extension product. For example, sequencing based on the detection of released protons can use an electrical detector and related methods that are commercially available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies) or the sequencing systems and methods disclosed in US Pat. , 8349167 and published application for US patent No. 2010/0137143 A1.

[0104] Accordingly, the present application provides methods for incorporating nucleotide analogs into DNA, including providing interaction of the following components: (i) an altered polymerase according to any of the above embodiments, (ii) a DNA template; and (iii) a solution of nucleotides. In some embodiments, the DNA template includes a clustered chip. In some embodiments, the nucleotides are modified at the 3'-hydroxyl of the sugar and include modifications at the 3'-hydroxyl of the sugar, such that the substituent is larger than the size of the naturally occurring 3'-hydroxyl group.

Nucleic acids encoding altered polymerases

[0105] The present disclosure also includes nucleic acid molecules encoding the altered polymerases described herein. For any given altered polymerase that is a mutant version of the polymerase for which the amino acid sequence and preferably also the wild-type nucleotide sequence encoding the polymerase is known, a nucleotide sequence encoding a mutant can be obtained according to the basic principles of molecular biology. For example, given that the wild-type nucleotide sequence encoding the 9°N polymerase is known, it is possible to derive a nucleotide sequence encoding any given mutant version of 9°N having one or more amino acid substitutions using a standard genetic code. Similarly, nucleotide sequences can be easily generated for mutant versions of other polymerases such as, for example, Vent® polymerase, Deep Vent® polymerase, Pfu polymerase, KOD polymerase, Pab polymerase, etc. Nucleic acid molecules having the desired nucleotide sequence can then be designed using standard molecular biology techniques known in the art.

[0106] In accordance with the embodiments presented herein, a particular nucleic acid includes not only the identical nucleic acid, but also any minor base variations, including, in particular, substitutions in cases that result in a synonymous codon (another codon defining the same amino acid residue) due to the degenerate code with conservative amino acid substitutions. The term "nucleic acid sequence" also includes a sequence that is complementary to any single strand sequence given in relation to base variations.

[0107] The nucleic acid molecules disclosed herein can also advantageously be included in a suitable expression vector for expression of the polymerase proteins they encode in a suitable host. Incorporation of the cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of transformed cells is well known to those skilled in the art, as outlined in Sambrook et al. (1989), Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory.

[0108] Such an expression vector includes a vector containing a nucleic acid according to the embodiments provided herein, operably linked to regulatory sequences, such as promoter regions, that are capable of influencing the expression of said DNA fragments. The term "operably linked" refers to a pairing in which the described components are in a relationship that allows them to function in the intended way. Such vectors can be transformed into a suitable host cell to allow protein expression in accordance with the embodiments provided herein.

[0109] The nucleic acid molecule can encode a mature protein or a protein having a prosequence, including the coding leader sequence on the precursor protein, which is then cleaved by the host cell to form the mature protein. The vectors may be, for example, plasmid, viral or phage vectors having an origin of replication and optionally a promoter for the expression of said nucleotide and optionally a promoter regulator. The vectors may contain one or more selectable markers such as, for example, an antibiotic resistance gene.

[0110] The regulatory elements required for expression include promoter sequences for RNA polymerase binding and for providing an appropriate level of transcription initiation, as well as translation initiation sequences for ribosome binding. For example, a bacterial expression vector may include a promoter, such as the lac promoter, and a Shine-Dalgarno sequence and an AUG start codon to initiate translation. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, an AUG start codon, and a termination codon for ribosome detachment. Such vectors can be obtained commercially or assembled from the sequences described by methods well known in the art.

[0111] Transcription of the DNA encoding the polymerase by higher eukaryotes can be optimized by including an enhancer sequence in the vector. Enhancers are cis-acting DNA elements that act on a promoter to increase the level of transcription. Vectors also typically include origins of replication in addition to selectable markers.

[0112] The present disclosure also provides a kit for conducting a nucleotide incorporation reaction. The kit includes at least one modified polymerase described in this application, and a solution of nucleotides in a suitable packaging material in an amount sufficient for at least one nucleotide incorporation reaction. Optionally, other reagents are also included, such as buffers and solutions necessary to apply the modified polymerase and nucleotide solution. Instructions for use of the packaged components are also usually included.

[0113] In some embodiments, the nucleotide solution includes labeled nucleotides. In some embodiments, the nucleotides are synthetic nucleotides. In some embodiments, the nucleotides are modified nucleotides. In some embodiments, the modified nucleotide has been modified at the 3'-hydroxyl of the sugar such that the substituent is larger than the naturally occurring 3'-hydroxyl group. In some embodiments, the modified nucleotides include a modified nucleotide or nucleoside molecule that includes a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3'-OH blocking group covalently attached to it, such that a group is attached to the 3' carbon atom structures

,

,

where Z is any of -C(R') ₂ -O-R'', -C(R') ₂ -N(R'') ₂ , -C(R') ₂ -N(H)R'',-C(R')2-S-R'' and -C(R') ₂ -F,

where each R'' represents or is part of a removable protective group;

each R' is independently hydrogen, alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amido, or a detectable label attached via a linking group; or (R') ₂ is an alkylidene group of the formula ═C(R''') ₂ , where each R''' may be the same or different and is selected from the group consisting of hydrogen and halogen atoms and alkyl groups; and

where the molecule can react to form an intermediate in which each R'' is replaced by H or, where Z is -C(R') ₂ -F, F is replaced by OH, SH or NH ₂ , preferably OH, and the intermediate dissociates under aqueous conditions to form a molecule with free 3'OH;

provided that where Z is -C(R') ₂ -S-R'', both R' groups are not H.

In some embodiments, R' of the modified nucleotide or nucleoside is alkyl or substituted alkyl. In some embodiments, the modified nucleotide or nucleoside -Z has the formula -C(R') ₂ -N ₃ . In some embodiments, Z is an azidomethyl group.

[0114] In some embodiments, the modified nucleotides are labeled with a fluorescent label to enable their detection. In some embodiments, the modified nucleotides include a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker. In some embodiments, the detectable label includes a fluorescent label.

[0115] Used in this application, the expression "packaging material" refers to one or more physical structures used to contain the contents of the set. The packaging material is made by known methods, preferably to provide a sterile environment free of contaminants. The packaging material has a label indicating that the components can be used to carry out the nucleotide incorporation reaction. In addition, the packaging material contains instructions indicating how the materials in the kit are used to carry out the nucleotide incorporation reaction. In this application, the term "packaging" refers to a solid matrix or material, such as glass, plastic, paper, foil, and the like, capable of holding the polypeptides within the specified boundaries. "Instructions for use" typically includes a specific expression describing the concentration of a reagent or at least one parameter of the assay method, such as the relative amounts of reagent and sample to be mixed, the exposure times for the reagent/sample mixture, temperature, buffer conditions, and so on. similar.

[0116] The full disclosures of patents, patent documents, and publications cited in the Background of the Invention, the Detailed Description of the Invention, and elsewhere in this application are incorporated by reference in their entirety as if each were included separately.

[0117] Exemplary embodiments of the present invention are discussed and reference is made to possible variations within the scope of the present invention. These and other variations, combinations and modifications to the present invention will be apparent to those skilled in the art without departing from the scope of the invention, and it should be understood that the present invention is not limited to the exemplary embodiments set forth in this application. Accordingly, the present invention is to be limited only by the following claims and their equivalents.

Examples

[0118] The present invention is illustrated by the following examples. It should be understood that specific examples, materials, quantities and procedures should be interpreted in a broad sense in accordance with the scope and essence of the invention set forth in this application.

Example 1

General methods of analysis and conditions

[0119] Unless otherwise indicated, this application describes the general conditions of analysis used in the examples described in this application.

A. Cloning and expression of polymerases

[0120] Methods for producing recombinant nucleic acids, expressing and isolating the expressed products are known and described in the art. Mutagenesis was performed in the coding region that encodes polymerase 9°N (SEQ ID NO: 1) using standard site-directed mutagenesis methodology. PCR based approaches have been used to amplify mutated coding regions and add a His tag. For each mutation performed, the correct sequence of the altered coding region was confirmed by determining the sequence of the cloned DNA.

[0121] His-tagged mutant polymerase coding regions were subcloned into the pET11a vector and transformed into BL21 Star (DE3) expressing cells (Invitrogen). Overnight cultures from selected individual colonies were used to inoculate expression cultures in 2.8 liter flasks. Cultures were grown at 37° C. to an OD600 of about 0.8, then protein expression was induced with 0.2 mM IPTG followed by 4 hours of additional growth. Cultures were centrifuged at 7000 rpm for 20 minutes. Cell pellets were stored at -20° C. until purification.

[0122] The pellets were thawed and lysed with 5-fold w/v. lysis buffer (50 mM Tris-HCI pH 7.5, 1 mM EDTA, 0.1% BME, and 5% glycerol) in the presence of Ready-Lyse and Omnicleave reagents (Epicentre) according to the manufacturer's recommendations. The final NaCl concentration was raised to 500 mM and the lysate was incubated on ice for 5 minutes. After centrifugation, the supernatant was incubated at 80°C for about 70 minutes. All further purification was carried out at 4°C. The supernatant was cooled for 30 minutes before centrifugation and purified using 5 ml Ni Sepharose HP (GE) columns. The columns were pre-equilibrated with buffer A (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 5% glycerol, 500 mM NaCl and 20 mM imidazole). The column was eluted with a 75 ml gradient of 20 to 500 mM imidazole. Peak fractions were pooled and diluted with 10% glycerol to match the conductivity of SP buffer A (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 5% glycerol) and loaded onto a 5 ml SP Sepharose (GE) column. The column was eluted with a 100 ml gradient of 150 to 1000 mM NaCl. Peak fractions were pooled, dialyzed in storage buffer (10 mM Tris-HCl pH 7.5, 300 mM KCL, 0.1 mM EDTA and 50% glycerol) and stored at -20°C.

B. Error rate and phasing analysis

[0123] Sequencing experiments were used to compare error rates and phasing values. Unless otherwise noted, experiments were performed on the MiniSeq™ system (Illumina, Inc., San Diego, CA) according to the manufacturer's instructions. For example, a separate incorporation mix (IMX) was prepared for each polymerase and used in a short run (35 cycles at reading 1) or a long run (227 cycles, of which 151 at reading 1 and 76 at reading 2) . Medium yield MiniSeq standard reagent cartridge formulations were used, replacing the standard polymerase with the test polymerase at 90 μg/mL. The incubation time of the IMX on the flow cell was varied as indicated in the examples given in this application. The DNA library used was prepared according to the standard TruSeq™ Nano protocol (Illumina, Inc.) with a target insert size of 350 bp. using E. coli genomic DNA; PhiX DNA (Illumina, Inc) was added to the resulting library in a molar ratio of approximately 1:10. Illumina RTA software was used to estimate error rates for both genomes and phasing levels.

Example 2

Altered polymerase sequencing efficiency

[0124] A number of altered polymerases were identified that had error rates and phasing levels in the short run at short on-time that were not significantly higher than the control polymerase used in the short run at standard on-time. Table 2 below compares the sequencing efficiency of the altered polymerases listed in Table 1 at an on-time of 14 seconds relative to the control polymerase shown by Pol 812 (SEQ ID NO: 8). Quality metrics used to evaluate altered polymerases were phasing-to-read 1 (“R1 phasing”) and cumulative error rates for E. coli sequencing controls (“E. coli error”) and bacteriophage PhiX (“PhiX error”) . The readings were normalized to the corresponding Pol 812 phasing and error rate at standard (46 seconds) turn-on time ("812 STD"). For example, the R1 Pol 812 phasing rate at short turn-on time (“812 Fast”) is 6 times higher than the R1 phasing at standard turn-on time, while the accumulated error rate of E. coli and PhiX is 12.5 and 6.4 times higher. , respectively. Similarly, the phasing rate of R1 Pol 963 (SEQ ID NO: 9) at short turn-on time is 5.9 times higher than the phasing rate of R1 Pol 812 at standard turn-on time, while the accumulated error rate of E. coli and PhiX for Pol 963 is higher 4.6 and 3.6 times, respectively.

[0125] Table 2: The performance of the modified polymerases are listed in Table 1.

[0126] Altered polymerases having relative phasing rates of no more than 3.0 and cumulative error rates of no more than 2.0 with short turn-on times are particularly attractive candidates for use in fast SBS. Examples of such polymerases are indicated in bold and asterisk in Table 2. Additional results are shown in FIG. 2 and 3.

[0127] FIG. 2 shows the reduction in phasing and cumulative errors at short turn-on times demonstrated for one of the altered polymerases identified in Example 2, Pol 1558 (SEQ ID NO: 11), compared to the Pol 812 control (left panels). The two enzymes show comparable phasing rates and error rates at standard turn-on times (right panels).

[0128] FIG. 3A shows the reduction in R1 phasing error rates and E. coli cumulative errors with short on-time demonstrated for selected altered polymerases identified in Example 2, Pol 1558, Pol 1671 (SEQ ID NO: 23), Pol 1682 (SEQ ID NO: 25). ) and Pol 1745 (SEQ ID NO: 28), compared to controls Pol 812 and Pol 963. The dotted lines in the top and bottom panels show the accumulated E. coli error and R1 phasing frequency demonstrated by Pol 812 at standard turn-on time.

[0129] FIG. 3B compares the phasing and prephasing rates of the same altered polymerases with respect to Pol 812 and Pol 963 controls, demonstrating a reduction in phasing rates with Pols 1558, 1671, 1682, and 1745.

Example 3

Activity of Altered Polymerases under Long Run Conditions

[0130] Selected altered polymerases identified in Example 2 were evaluated using different run lengths. PhiX cumulative error rates for each of the modified polymerases at standard on time (46 seconds) were compared to phasing error rates and cumulative error rates at short on time (22 seconds) during long sequencing reads (250 cycles at read 1 followed by 250 cycles at reading 2, total 500 cycles). Longer run conditions result in more sequence information (i.e. more nucleotide identity) per run and are similar to conditions often used in sequencing platforms such as whole genome sequencing. The results are shown in FIG. four.

[0131] FIG. Figure 4 compares PhiX cumulative error rates for Pol 1550 (SEQ ID NO: 10) and Pol 1558 (SEQ ID NO: 11) with that for control Pol 812 (SEQ ID NO: 8) at standard and short turn-on times during long reads. sequencing (2×250 cycles). Both mutants show a marked reduction in error rates after performing paired-end sequencing. In addition, Pol 1550 shows a significant reduction in error rate compared to Pol 812 during the first sequencing read.

Example 4

Evaluation of sequencing parameters on NovaSeq™

[0132] One of the altered polymerases identified in Example 2, Pol 1671, was evaluated on the Illumina NovaSeq™ platform using the S1 flow cell and NovaSeq™ sequencing chemistry. Error rates, phasing levels, and other sequencing quality parameters were determined for reads 1 and 2 at short and standard turn-on times. The results for Pol 1671 and Pol 812 are summarized in FIG. 5.

[0133] FIG. 5A shows a comparison of NovaSeq™ sequencing performance for Pol 1671 (SEQ ID NO: 23) demonstrated at short on-times (10 sec) with those for control Pol 812 (SEQ ID NO: 8) demonstrated at standard (40 sec) and short (10 sec) turn-on time. The upper panels show the percentage of clusters that passed the filter ("Clusters PF"); the lower panels show the PhiX cumulative error rate. Light open circles indicate Pol 812 performance at standard on time, while dark open circles indicate Pol 812 performance at short on time. All Pol 1671 values indicated by solid circles have a short turn-on time. The results show that Pol 1671 shows comparable performance in both readings at short turn-on times with that of Pol 812 at standard turn-on times.

[0134] FIG. 5B summarizes the PhiX cumulative error rates, Q30 values, and phasing rates exhibited by Pol 1671 compared to Pol 812 control for NovaSeq™ reads 1 and 2 at standard (40 sec) and short (22 sec) turn-on times. A significant improvement in sequencing quality for both reads was observed with Pol 1671.

[0135] Full disclosure of all patents, patent applications and publications, and materials available electronically (including, for example, providing nucleotide sequences, for example, in GenBank and RefSeq, and providing amino acid sequences, for example, in SwissProt, PIR, PRF , PDB, and translations from the annotated coding regions in GenBank and RefSeq) cited in this application are incorporated herein by reference in their entirety. Additional materials referenced in publications (such as additional tables, additional figures, additional materials and methods, and/or additional experimental data) are also incorporated herein by reference in their entirety. In the event that there is any inconsistency between the disclosure of this application and the disclosure(s) of any document incorporated herein by reference, the disclosure of this application shall prevail. The above detailed disclosure and examples are provided for clarity of understanding only. No unnecessary restrictions should be understood from this. The present invention is not limited to the exact details shown and described, so long as variations obvious to a person skilled in the art will be included in the present invention defined by the claims.

[0136] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so on, used in the description and claims, should be understood as modified in all cases by the term "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in the description and claims are approximations that may vary depending on the desired properties that seek to obtain using the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in the light of the number of significant digits given and using normal rounding techniques.

[0137] While the numerical ranges and parameters defining the broad scope of the invention are approximate, the numerical values set forth in the specific examples are as accurate as possible. However, all numerical values inherently contain a range necessarily resulting from the standard deviation found in the respective test measurements.

[0138] All headings are for the convenience of the reader and should not be used to limit the meaning of the text following the heading unless otherwise noted.

Claims (61)

1. Recombinant DNA polymerase containing an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 of DNA polymerase 9°N, while the recombinant DNA polymerase contains a mutation in the amino acid substitution type in a position functionally equivalent to Tyr497, and at least two amino acid substitution mutations at positions functionally equivalent to Phe152, Met329, or Val471 in the amino acid sequence of DNA polymerase 9°N. 2. The polymerase of claim 1, wherein the substitution mutation at a position functionally equivalent to Tyr497 contains a mutation to a non-polar, hydrophobic, or uncharged amino acid. 3. The polymerase of claim 1, wherein the substitution type mutation at a position functionally equivalent to Tyr497 contains a mutation to Gly. 4. The polymerase of claim 1, wherein the substitution mutation at a position functionally equivalent to Phe152 contains a mutation to a non-polar, hydrophobic, or uncharged amino acid. 5. The polymerase of claim 1, wherein the substitution type mutation at a position functionally equivalent to Phe152 contains a mutation to Gly. 6. The polymerase of claim 1, wherein the polymerase further comprises a substitution mutation at a position functionally equivalent to Val278, wherein said substitution mutation contains a mutation to a non-polar or hydrophobic amino acid. 7. The polymerase of claim 1, wherein the polymerase further comprises a substitution mutation at a position functionally equivalent to Val278, wherein said substitution mutation contains a pre-Leu mutation. 8. The polymerase of claim 1, wherein the substitution type mutation at a position functionally equivalent to Met329 contains a mutation to a polar amino acid. 9. The polymerase of claim 1, wherein the substitution type mutation at a position functionally equivalent to Met329 contains a mutation prior to His. 10. The polymerase of claim 1, wherein the substitution mutation at a position functionally equivalent to Val471 contains a mutation to a polar or uncharged amino acid. 11. The polymerase according to claim 1, wherein the substitution type mutation at a position functionally equivalent to Val471 contains a mutation to Ser. 12. The polymerase of claim 1, wherein the polymerase further comprises a substitution type mutation at a position functionally equivalent to Thr514, wherein said substitution type mutation contains a mutation to a non-polar or hydrophobic amino acid. 13. The polymerase of claim 1, wherein the polymerase further comprises a substitution type mutation at a position functionally equivalent to Thr514, wherein said substitution type mutation contains a mutation to Ala. 14. The polymerase of claim 1, wherein the polymerase further comprises a substitution type mutation at a position functionally equivalent to Leu631, wherein said substitution type mutation contains a mutation to a non-polar or hydrophobic amino acid. 15. The polymerase of claim 1, wherein the polymerase further comprises a substitution type mutation at a position functionally equivalent to Leu631, wherein said substitution type mutation contains a mutation to Met. 16. The polymerase of claim 1, wherein the polymerase further comprises a substitution type mutation at a position functionally equivalent to Glu734, wherein said substitution type mutation contains a mutation to a polar amino acid. 17. The polymerase of claim 1, wherein the polymerase further comprises a substitution type mutation at a position functionally equivalent to Glu734, wherein said substitution type mutation contains a mutation to Arg. 18. The polymerase according to claim 1, where the specified polymerase contains at least two, at least three, at least four, at least five, at least six or seven mutations in the type of amino acid substitution in positions functionally equivalent to the amino acid, selected from Phe152, Val278, Met329, Val471, Thr514, Leu631 and Glu734 in the amino acid sequence of DNA polymerase 9°N. 19. Polymerase according to any one of paragraphs. 1-18, wherein the polymerase additionally contains amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N. 20. Recombinant DNA polymerase containing an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 DNA polymerase 9°N, while the recombinant DNA polymerase contains a mutation in the amino acid substitution type in a position functionally equivalent to Tyr497, and at least one amino acid substitution mutation at a position functionally equivalent to Arg247, Glu599, Lys620, His633, or Val661 in the amino acid sequence of DNA polymerase 9°N. 21. The polymerase of claim 20, wherein the substitution type mutation at a position functionally equivalent to Arg247 contains a mutation to a non-polar or uncharged amino acid. 22. The polymerase of claim 20, wherein the substitution type mutation at a position functionally equivalent to Arg247 contains a mutation to Tyr. 23. The polymerase of claim 20, wherein the substitution type mutation at a position functionally equivalent to Glu599 contains a mutation to a polar amino acid. 24. The polymerase of claim 20, wherein the substitution type mutation at a position functionally equivalent to Glu599 contains a mutation to Asp. 25. The polymerase of claim 20, wherein the substitution type mutation at a position functionally equivalent to Lys620 contains a mutation to a polar amino acid. 26. The polymerase of claim 20, wherein the substitution type mutation at a position functionally equivalent to Lys620 contains a mutation to Arg. 27. The polymerase of claim 20, wherein the substitution mutation at a position functionally equivalent to His633 contains a mutation to a non-polar, hydrophobic, or uncharged amino acid. 28. The polymerase of claim 20, wherein the substitution type mutation at a position functionally equivalent to His633 contains a mutation to Gly. 29. The polymerase of claim 20, wherein the substitution mutation at a position functionally equivalent to Val661 contains a mutation to a polar amino acid. 30. The polymerase of claim 20, wherein the substitution type mutation at a position functionally equivalent to Val661 contains a mutation to Asp. 31. The polymerase of claim 20, wherein the polymerase contains at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Arg247, Glu599, Lys620, His633, and Val661 in the amino acid sequence of DNA polymerase 9°N. 32. Polymerase according to any one of paragraphs. 20-31, where the polymerase additionally contains amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N. 33. A recombinant DNA polymerase containing an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 DNA polymerase 9°N, while the recombinant DNA polymerase contains (i) a mutation in the type of amino acid substitution at position functionally equivalent to Tyr497, (ii) at least two amino acid substitution mutations at positions functionally equivalent to Phe152, Met329, or Val471 in the amino acid sequence of DNA polymerase 9°N, and (iii) at least one amino acid substitution mutation at a position functionally equivalent to Arg247, Glu599, Lys620, His633, or Val661 in the amino acid sequence of DNA polymerase 9°N. 34. The polymerase of claim 33, wherein said polymerase contains at least three, at least four, at least five, at least six or seven amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Phe152, Val278 , Met329, Val471, Thr514, Leu631 and Glu734 in the amino acid sequence of DNA polymerase 9°N. 35. The polymerase of claim 33, wherein the polymerase contains at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Arg247, Glu599, Lys620, His633, or Val661 in the amino acid sequence of DNA polymerase 9°N. 36. Polymerase according to any one of paragraphs. 33-35, wherein the polymerase additionally contains amino acid substitution mutations at positions functionally equivalent to the amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the amino acid sequence of DNA polymerase 9°N. 37. Polymerase according to any one of paragraphs. 1-36, wherein said polymerase is a DNA polymerase of the type B family. 38. Recombinant DNA polymerase containing the amino acid sequence according to any of SEQ ID NO: 10-34. 39. A nucleic acid molecule encoding a polymerase, as defined in any one of paragraphs. 1-36 and 38. 40. Expression vector containing the nucleic acid molecule according to claim 39. 41. A host cell containing the vector of claim 40, wherein said host cell expresses a protein encoded by the expression vector of claim 40. 42. The method of incorporating modified nucleotides into a growing DNA chain, including ensuring the interaction of the following components: (i) a polymerase according to any one of paragraphs. 1-36 and 38, (ii) DNA template; and (iii) nucleotide solution. 43. The method of claim 42, wherein the DNA template contains a clustered chip. 44. Kit for carrying out the reaction of the inclusion of nucleotides, containing: polymerase, as defined in any of paragraphs. 1-36 and 38, and a solution of nucleotides. 45. The kit of claim 44, wherein the nucleotide solution contains labeled nucleotides. 46. The kit of claim 44 wherein the nucleotides comprise synthetic nucleotides. 47. The kit of claim 44, wherein the nucleotides contain modified nucleotides. 48. The kit of claim 44 wherein the modified nucleotides are modified at the 3'-hydroxyl of the sugar such that the substituent is larger than the naturally occurring 3'-hydroxyl group. 49. The kit according to claim 47, wherein the modified nucleotides comprise a modified nucleotide or nucleoside molecule that contains a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3'-OH blocking group covalently attached to it, thus to 3' -carbon atom is attached to the structure group

, wherein Z is any of -C(R') ₂ -O-R'', -C(R') ₂ -N(R'') ₂ , -C(R') ₂ -N(H)R '', -C(R') ₂ -S-R'' and -C(R') ₂ -F, with each R'' representing or being part of a removable protective group; each R' is independently hydrogen, alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amido, or a detectable label attached via a linking group; or (R')2 is an alkylidene group of the formula =C(R''') ₂ , wherein each R''' may be the same or different and is selected from the group consisting of hydrogen and halogen atoms and alkyl groups; and wherein said molecule can react to form an intermediate in which each R'' is replaced by H, or where Z is -C(R') ₂ -F, F is replaced by OH, SH or NH ₂ , preferably OH, moreover, the intermediate product dissociates in aqueous conditions with the formation of a molecule with a free 3'OH; provided that where Z is -C(R') ₂ -S-R'', both R' groups are not H. 50. The kit of claim 49 wherein R' of the modified nucleotide or nucleoside is alkyl or substituted alkyl. 51. The kit according to claim 49, wherein -Z of the modified nucleotide or nucleoside has the formula -C(R') ₂ -N ₃ . 52. The kit according to claim 51, where Z is an azidomethyl group. 53. The kit of claim 47 wherein the modified nucleotides are labeled with a fluorescent label to enable their detection. 54. The kit of claim 47 wherein the modified nucleotides comprise a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker. 55. The kit of claim 54, wherein the detectable label contains a fluorescent label. 56. The kit according to claim 44, additionally containing one or more template DNA molecules and/or primers.

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