# Patent

## Title
DNA polymerases

## Abstract

The technology provided herein relates to novel variants of DNA-Polymerases exhibiting high termo-stability as well as a strong strand displacement activity; to nucleic acid molecules encoding said DNA-Polymerases, vectors, host cells containing the nucleic acids and methods for preparation and producing such enzymes; compositions comprising at least one of the DNA-Polymerases; and methods for using such enzymes in DNA sequencing and/or DNA amplification processes.

## Background
FIELD OF THE DISCLOSURE
The technology provided herein relates to novel variants of DNA-Polymerases exhibiting high termo-stability as well as a strong strand displacement activity; to nucleic acid molecules encoding said DNA-Polymerases, vectors, host cells containing the nucleic acids and methods for preparation and producing such enzymes; compositions comprising at least one of the DNA-Polymerases; and methods for using such enzymes in DNA sequencing and/or DNA amplification processes.
BACKGROUND
Sequence-specific DNA amplification has many applications in molecular biology research and medical diagnostics. At the present time, there are two main strategies for amplifying a defined sequence of nucleic acid: polymerase chain reaction (PCR) and isothermal amplification. The polymerase chain reaction relies upon instrument-based thermal cycling to denature template DNA, followed by the annealing of primers at specific sites in the denatured template and extension of the primers by a thermostable DNA polymerase (such as Taq polymerase) in order to exponentially increase the amount of DNA [U.S. Pat. Nos. 4,683,202 and 4,683,195].
Isothermal amplification of DNA may require the same three steps and an initial high temperature incubation to denature the template DNA for initiation of the process, but the amplification of the DNA to take place at one defined temperature.
A variety of isothermal amplification methods have been developed, for example: strand displacement amplification (SDA) [Walker, G. T. et al. Nucleic Acids Res 20, 1691-6 (1992); and Walker, G. T., Little, M. C., Nadeau, J. G. & Shank, D. D. Proc Natl Acad Sci USA 89, 392-6 (1992)], rolling circle amplification [Fire, A. & Xu, S. Q. Proc Natl Acad Sci USA 92, 4641-5 (1995)], cross priming amplification (CPA) [Xu, G. et al. Sci. Rep. 2, 246; D01:10.1038/srep00246 (2012)] and loop mediated amplification [Notomi, T. et al. Nucleic Acids Res 28, e63 (2000)].
These methods, like many other isothermal amplification methods, require the use of a DNA polymerase with a strong strand displacement activity. At the same time, sequence-specific DNA synthesis or amplification needs a high temperature of the reaction (as a rule 60° C. or over) for the specific annealing of primers. Thus, a DNA polymerase suitable for these methods must be a thermostable DNA polymerase with a strong strand displacement activity.
At present time, only the large fragment of Bst DNA polymerase is suitable for methods of sequence-specific isothermal DNA amplification, such as LAMP or CPA. A relative thermostability and strong strand displacement activity of Bst DNA polymerase make it helpful for other applications. For example, usage of Bst in Illumina's Next Generation Sequencing technology for carrying out cluster amplification of DNA molecules can be mentioned.
The Bst DNA polymerase or Bacillus stearothermophilus DNA Polymerase I is a typical member of polymerase family A and its structure looks like structure of Taq DNA polymerase or other members of the family [Kiefer, J. R. et al. (1997) Structure, 5, 95-108] but a mechanism of strand displacement of Bst and other A-family polymerases is unclear. Bst polymerase is one of the most popular enzymes with strand displacement activity because its optimum is at about 63° C. and it is suitable for sequence-specific amplification like LAMP. Unfortunately, Bst polymerase can be used at temperatures only up to 68-70° C. At temperature 68° C. or higher it is inactivated [Xu, G. et al. Sci. Rep. 2, 246; D01:10.1038/srep00246 (2012)]. So, Bst cannot be used in methods requiring heat denaturation of DNA, such as PCR or polymerase chain displacement reaction (PCDR) [Harris, C. L. et al. BioTechniques 54:93-97 (February 2013) doi 10.2144/000113951].
Additionally, initial heat denaturation of DNA can many times increase the sensitivity of isothermal DNA amplification like LAMP [Aryan, E., et al. (2010) Microbiol. Res., 165, 211-220; Geojith G., et al. (2010) J Microbiol Methods, 84, 71-73; Neonakis, I. K., et al. (2011) Eur. J. Clin. Microbiol. Infect. Dis., 30, 937-942].
Therefore the availability of novel DNA polymerases with high thermo stability and a strong strand displacement activity would be highly advantageous.
SUMMARY OF THE DISCLOSURE
In a first aspect, embodiments of the disclosure provide novel modified DNA-polymerases within the Type-A family of polymerases, wherein the polymerase comprises substitutions of at least two amino acid residues in the amino acid sequence of the naturally occurring DNA-polymerase at positions corresponding to 738 and 743 relative to the numbering of the amino acid sequence of wild-type Thermus aquaticus (Taq) DNA polymerase (SEQ ID NO: 1), wherein the substitutions are 738F and 743F, and wherein said modified DNA polymerase, or homologs thereof, have an increased strand display activity relative to the corresponding naturally occurring unmodified DNA-polymerase.
In other aspects, the modified thermostable DNA-polymerases as described herein may be useful for, or used in, performing DNA sequencing and DNA amplification, preferably LAMP or PCDR DNA amplification.
In further aspects, embodiments of this disclosure provide nucleic acids encoding modified thermostable DNA-polymerases variants with strand display activity as disclosed herein, as well as vectors and host cells comprising such nucleic acids.
In other aspects, this disclosure relates to compositions comprising modified thermostable DNA-polymerases as described herein, wherein the compositions may be useful for, or used in, performing DNA sequencing and DNA amplification, preferably LAMP or PCDR DNA amplification.
In a further aspect, embodiments of this disclosure relate to methods for producing the DNA-polymerase variants in a host cell by transforming the host cell with a DNA construct, advantageously including a promoter having transcriptional activity in the host cell, cultivating the transformed host cell in a suitable culture medium to allow expression of said DNA-polymerases and producing the DNA-polymerases. The method may also include recovering the produced DNA-polymerases.
In an advantageous embodiment of this disclosure, the DNA-polymerase has the nucleic acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or variants, homologs, derivative or functional equivalents or functional fragments thereof.
In a further aspect, embodiments relates to kits for amplification of a target nucleic acid, said kit comprises the modified DNA polymerase or a composition according to the present disclosure.
Before the disclosure is described in detail, it is to be understood that this disclosure is not limited to the particular component parts of the devices described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.
To provide a comprehensive disclosure without unduly lengthening the specification, the applicant hereby incorporates by reference each of the patents and patent applications referenced above, in particular the disclosure of WO 01/80880 A2 and US 2009/0081185 A1.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an agarose gel showing the results of LAMP DNA amplification with a DNA polymerase of the present disclosure and Bst large fragment DNA polymerase.
FIG. 2 shows an agarose gel comparing the results of PCR amplification of a 8 kb fragment of λ DNA with a DNA-Polymerase of the present disclosure and Taq DNA polymerases from different suppliers.
FIG. 3 shows an agarose gel comparing the results of PCR and PCDR DNA amplification with a DNA polymerase of the present disclosure and KlenTaq DNA polymerase.
FIG. 4 shows an amino acid sequence of wild-type Thermus aquaticus (Taq) DNA-polymerase (SEQ ID NO: 1).
FIG. 5 shows an amino acid sequence (SEQ ID NO: 2) of the SD DNA-Polymerase as an embodiment of the modified DNA-Polymerase according to the present disclosure, derived from a parent Taq DNA-Polymerase.
FIG. 6 shows a nucleic acid sequence (SEQ ID NO: 9) of the SD DNA-Polymerase as an embodiment of the modified DNA-Polymerase according to the present disclosure, derived from a parent Taq DNA-Polymerase.
FIG. 7 shows an amino acid sequence of wild-type Thermus thermophiles (Tth) DNA polymerase (SEQ ID NO: 3).
FIG. 8 shows an amino acid sequence (SEQ ID NO: 4) of a DNA-Polymerase variant according to the present disclosure, derived from a parent Tth DNA-Polymerase.
FIG. 9 shows a nucleic acid sequence (SEQ ID NO: 10) of a DNA-Polymerase variant according to the present disclosure, derived from a parent Tth DNA-Polymerase.
FIG. 10 shows an amino acid sequence of wild-type Thermus flavus (Tfl) DNA polymerase (SEQ ID NO: 5).
FIG. 11 shows an amino acid sequence (SEQ ID NO: 6) of a DNA-Polymerase variant according to the present disclosure, derived from a parent Tfl DNA-Polymerase.
FIG. 12 shows a nucleic acid sequence (SEQ ID NO: 11) of a DNA-Polymerase variant according to the present disclosure, derived from a parent Tfl DNA-Polymerase.
FIG. 13 is a nucleic acid sequence alignment showing a consensus sequence of wild-type DNA-Polymerases within the Type-A family.
FIG. 14 shows an amino acid sequence motifs (SEQ ID NOs: 12, 13, 14) comprised in embodiments of DNA-Polymerase variants according to the present disclosure.
FIG. 15 shows an amino acid sequence of wild-type Bacillus stearothermophilus (Bst) DNA Polymerase I (SEQ ID NO: 15).
FIG. 16 shows nucleic acid sequence of murine G3PDH cDNA (SEQ ID NO: 16)
FIG. 17 shows nucleic acid sequences of primers for the amplification of murine G3PDH cDNA.
FIG. 18 shows the sequence of amplified DNA fragment of murine G3PDH cDNA.
FIG. 19 is an agarose gel showing a long-distance PCR with the SD DNA polymerase.
FIG. 20 (A-C) are diagrams showing the comparison of SD and Taq DNA polymerases in real-time PCR and PCDR amplifications.



DETAILED DESCRIPTION OF THIS DISCLOSURE
Disclosed herein are variants of DNA-polymerases within the Type-A family of polymerases having high thermo stability and a strong strand displacement activity.
In particular, DNA-polymerase variants according to the present disclosure have the properties of a strong strand displacement activity, suitable for performing LAMP and PCDR DNA amplification, high thermostability suitable for performing PCDR and PCR DNA amplification, and a high efficiency of long (over 2 kb) DNA sequences amplification in PCR. Said properties being obtained by at least two substitutions of Phe for amino acid residues in a thermostable DNA polymerase of A-family at positions corresponding to Lys738 and Ala743 of the wild-type Taq DNA polymerase.
The present disclosure reveals enzymes with an amino-acid sequence derived from the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 or variants, modified forms, homologs, fusion proteins, functional equivalents or functional fragments thereof, having one or more modifications at one or more positions selected from the group of positions that correspond structurally or by amino acid sequence homology to the positions 738 and 743 of SEQ ID NO:1.
As used herein, “cell”, “cell line”, and “cell culture” can be used interchangeably and all such designations include progeny. Thus, the words “transformants” or “transformed cells” includes the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included.
As used herein, a “chimeric” protein refers to a mutant protein whose amino acid sequence represents a fusion product of subsequences of the amino acid sequences from at least two distinct proteins. A chimeric protein preferably is not produced by direct manipulation of amino acid sequences, but, rather, is expressed from a “chimeric” gene that encodes the chimeric amino acid sequence.
The term “complementary” as used herein refers to a relationship between two nucleic acid sequences. One nucleic acid sequence is complementary to a second nucleic acid sequence if it is capable of forming a duplex with the second nucleic acid, wherein each residue of the duplex forms a guanosine-cytidine (G-C) or adenosine-thymidine (A-T) base pair or an equivalent base pair. Equivalent base pairs can include nucleoside or nucleotide analogues other than guanosine, cytidine, adenosine, or thymidine.
The terms “DNA template”, or “template” as used herein, refer to a nucleic acid that is used by a polymerase to synthesize a new complementary nucleic acid.
The term “DNA-Polymerase variants” or “modified DNA-Polymerase” means any DNA-Polymerase within the the Type-A family of polymerases obtained by site-directed or random mutagenesis, insertion, deletion, recombination and/or any other protein engineering method, which leads to a DNA-Polymerase that differ in their amino acid sequence from the corresponding wildtype DNA-Polymerase. The terms “wildtype DNA-Polymerase”, “wildtype enzyme”, or “wildtype” in accordance with the disclosure describe a DNA-Polymerase enzyme with an amino acid sequence found in nature or a fragment thereof.
The term “derivative” as used herein, refers to a nucleic acid molecule that has similar binding characteristics to a target nucleic acid sequence as a nucleic acid molecule according to one of the claimed sequences.
The term “expression clone” refers to DNA sequences containing a desired coding sequence and control sequences in operable linkage, so that hosts transformed with these sequences are capable of producing the encoded proteins. The term “expression system” refers to a host transformed with an expression clone. To effect transformation, the expression clone may be included on a vector; however, the relevant DNA may also be integrated into the host chromosome.
The term “fusion proteins” comprises all proteins derived from a modified DNA-Polymerase according to the present disclosure by covalently fusing additional amino-acid sequences at the C- and/or N-terminus. The source and composition of the additional amino-acid sequence is either natural from any living organisms or virus or unnatural. In particular, the fusion protein may be a “recombinant” polypeptide, which is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process involved uses of recombinant nucleic acid techniques. In reference to structure, recombinant polynucleotides or polypeptides contain sequences from different sources. In particular, it encompasses polypeptides made by generating a sequence comprising two or more fragments, which are not naturally contiguous or operably linked to each other. Thus, for example, products made by transforming cells with any unnaturally occurring vector are encompassed.
The term “functional fragment” or “effective fragment” means a fragment or portion of a DNA-Polymerase variant according to the present disclosure that retains about the same enzymatic function or effect.
The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of a recoverable bioactive polypeptide or precursor.
The term “homologous polypeptide” or “homolog” according to the present disclosure comprises any enzyme with a sequence identity of at least 70% or preferably at least 80%, 85%, 90%, 95%, 97% or 99% to a DNA-Polymerase variant according to the present disclosure including functional fragments or effective fragments thereof.
The term “homologue of the nucleic acid molecule” refers to a nucleic acid molecule the sequence of which has one or more nucleotides added, deleted, substituted or otherwise chemically modified in comparison to a nucleic acid molecule according to one of the claimed sequences, provided always that the homologue retains substantially the same binding properties as the latter.
The term “host cell” in relation to the present disclosure includes any cell that comprises either the nucleic acid molecule or an expression vector as described above and which is used in the recombinant production of an enzyme having the specific properties as defined herein or in the methods of the present disclosure.
The term “isolated” describes any molecule separated from its natural source.
The term “modified form” or “variant” means that the enzyme has been modified from its original form (parent/wildtype, wt) but retains at least the same enzymatic functional characteristics as that of the wild-type enzyme in addition to an increased strand display activity relative to the corresponding naturally occurring unmodified wild-type DNA-Polymerase.
The term “modification” as used herein, refers for example to substitutions, insertions or deletions of amino acid residues at specific positions in an amino acid sequence as well as the phosphorylation, acetylation like palmitoylation, methylation, sulphation, glycosylation, lipidation like isoprenylation, farnesylation, attachment of a fatty acid moiety, glypiation and/or ubiquitinylation of specific positions on the polypeptide, or combinations thereof.
The term “mutation” refers to the substitution or replacement of single or multiple nucleotide triplets, insertions or deletions of one or more codons, homologous or heterologous recombination between different genes, fusion of additional coding sequences at either end of the encoding sequence, or insertion of additional encoding sequences or any combination of these methods, which result in a polynucleic acid sequence encoding the desired protein. Thus, the term “mutations” also refers to all of the changes in the polypeptide sequence encoded by the polynucleic acid sequence modified by one or more of the above described changes. Amino acid residues are abbreviated according to the following Table 1 either in one- or in three-letter code.
The term “nucleic acid molecule” or “nucleic acid” is intended to indicate any single- or double stranded nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA, Peptide nucleic acid (PNA) or LNA origin.
The term “oligonucleotide” as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. Oligonucleotides can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphotriester method, the diethylphosphoramidite method, and the solid support method. A review of synthesis methods is provided in [Goodchild J., Bioconjug. Chem. V. 1 (1990), P. 165-187].
The term “plasmid”, “vector system” or “expression vector” means a construct capable of in vivo or in vitro expression. In the context of the present disclosure, these constructs may be used to introduce genes encoding enzymes into host cells.
“Percent sequence identity”, with respect to two amino acid or polynucleotide sequences, refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in two optimally aligned polypeptide sequences are identical. Percent identity can be determined, for example, by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in “Atlas of Protein Sequence and Structure”, M. O. Dayhoff et., Suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., which adapts the local homology algorithm of Smith and Waterman (1981) Advances in Appl. Math. 2:482-489 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters 5 recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Likewise, computer programs for determining percent homology are also readily available.
The term “polynucleotide” corresponds to any genetic material of any length and any sequence, comprising single-stranded and double-stranded DNA and RNA molecules, including regulatory elements, structural genes, groups of genes, plasmids, whole genomes and fragments thereof.
The term “position” in a polynucleotide or polypeptide refers to specific single bases or amino acid residues in the sequence of the polynucleotide or polypeptide, respectively.
The term “primer” as used herein refers to an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated. Synthesis of a primer extension product, which is complementary to a nucleic acid strand, is initiated in the presence of the requisite four different nucleoside triphosphates and a thermostable DNA polymerase in an appropriate buffer at a suitable temperature. A “buffer” includes cofactors (such as divalent metal ions) and salt (to provide the appropriate ionic strength), adjusted to the desired pH.
“Strand displacement activity”, as used herein, refers to the phenomenon by which an enzyme, such as a DNA polymerase, causes the dissociation of a paired nucleic acid from its complementary strand in a direction from 5′ towards 3′, in conjunction with, and close to, the template-dependent nucleic acid synthesis. The strand displacement starts at the 5′ end of a paired nucleic acid sequence and the enzyme therefore carries out the nucleic acid synthesis. The neosynthesized nucleic acid and the displaced nucleic acid generally have the same nucleotide sequence, which is complementary to the template nucleic acid strand. The strand displacement activity may be situated on the same molecule as that conferring the activity of nucleic acid synthesis, and particularly the DNA synthesis.
“Strong strand displacement activity” of DNA polymerase, as used herein, allows to use the enzyme for carrying out LAMP reactions like the large fragment of Bst polymerase.
The term “stringent conditions” relates to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium). Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60° C. for longer probes. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide and the like.
The term “thermostable enzyme” or “thermostable polymerase”, as used herein, refers to an enzyme which is stable to heat and has an elevated temperature reaction optimum. The thermostable enzyme of the present invention catalyzes primer extension optimally at a temperature between 60 and 90° C., and is usable under the temperature cycling conditions typically used in cycle sequence reactions and polymerase chain reaction amplifications (described in U.S. Pat. No. 4,965,188). The modified DNA polymerase according to the present disclosure are preferably thermostable at a temperature of at least 80° C.
The term “variant of the nucleic acid molecule” refers herein to a nucleic acid molecule which is substantially similar in structure and biological activity to a nucleic acid molecule according to one of the claimed sequences.







TABLE 1







Amino acid abbreviations










Abbreviations
Amino acid















A
Ala
Alanine



C
Cys
Cysteine



D
Asp
Aspartic acid



E
Glu
Glutamic acid



F
Phe
Phenylalanine



G
Gly
Glycine



H
His
Histidine



I
Ile
Isoleucine



K
Lys
Lysine



L
Leu
Leucine



M
Met
Methionine



N
Asn
Asparagine



P
Pro
Proline



Q
Gln
Glutamine



R
Arg
Arginine



S
Ser
Serine



T
Thr
Threonine



V
Val
Valine



W
Trp
Tryptophan



Y
Tyr
Tyrosine










Mutations or variations are described by use of the following nomenclature: position; substituted amino acid residue(s). According to this nomenclature, the substitution of, for instance, an alanine residue for a glycine residue at position 20 is indicated as 20G. When an amino acid residue at a given position is substituted with two or more alternative amino acid residues these residues are separated by a comma or a slash. For example, substitution of alanine at position 20 with either glycine or glutamic acid is indicated as 20G/E, or 20G, 20E.
Furthermore, the following nomenclature could also be used: amino acid residue in the protein scaffold; position; substituted amino acid residue(s). According to this nomenclature, the substitution of, for instance, an alanine residue for a glycine residue at position 20 is indicated as Ala20Gly or A20G, or 20G. The deletion of alanine in the same position is shown as Ala20* or A20*. The insertion of an additional amino acid residue (e.g. a glycine) is indicated as Ala20AlaGly or A20AG. The deletion of a consecutive stretch of amino acid residues (e.g. between alanine at position 20 and glycine at position 21) is indicated as A(Ala20-Gly21) or A(A20-G21). When a sequence contains a deletion in comparison to the parent protein used for numbering, an insertion in such a position (e.g. an alanine in the deleted position 20) is indicated as *20Ala or *20A. Multiple mutations are separated by a plus sign or a slash. For example, two mutations in positions 20 and 21 substituting alanine and glutamic acid for glycine and serine, respectively, are indicated as A20G+E21S or A20G/E21S. When an amino acid residue at a given position is substituted with two or more alternative amino acid residues these residues are separated by a comma or a slash. For example, substitution of alanine at position 30 with either glycine or glutamic acid is indicated as A20G,E or A20G/E, or A20G, A20E. When a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an alanine in position 20 is mentioned but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid residue (i.e. any one of R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V).
The terms “conservative mutation”, or “conservative substitution”, respectively, refer to an amino acid mutation that a person skilled in the art would consider a conservative to a first mutation. “Conservative” in this context means a similar amino acid in terms of the amino acid characteristics. If, for example, a mutation leads at a specific position to a substitution of a non-aliphatic amino acid residue (e.g. Ser) with an aliphatic amino acid residue (e.g. Leu) then a substitution at the same position with a different aliphatic amino acid (e.g. Ile or Val) is referred to as a conservative mutation. Further amino acid characteristics include size of the residue, hydrophobicity, polarity, charge, pK-value, and other amino acid characteristics known in the art. Accordingly, a conservative mutation may include substitution such as basic for basic, acidic for acidic, polar for polar etc. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone C. D. and Barton G. J. (1993) “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput. Appl Biosci. 9: 745-756; Taylor W. R. (1986) “The classification of amino acid conservation” J. Theor. Biol. 119; 205-218). Conservative substitutions may be made, for example, according to the table below which describes a generally accepted Venn diagram grouping of amino acids.







TABLE 2







Venn diagram grouping amino acids










Set

Sub-set






Hydrophobic
FWYHKMILVAGC
Aromatic
FWYH




Aliphatic
ILV


 


Polar
WYHKREDCSTNQ
Charged
HKRED




Positively
HKR




charged





Negatively
ED




charged



 


Small
VCAGSPTND
Tiny
AGS









It is also understood that the present disclosure comprises all molecules that are derived from the polynucleotides of the disclosure and all variants thereof described in this application, by posttranslational processing compared to the genetically encoded amino acid sequence. These posttranslational modifications comprise, but are not limited to, proteolytic cleavage of N-terminal sequences such as leader and/or pro-sequences, proteolytic removal of C-terminal extensions, N- and/or O-glycosylation, lipidation, acylation, deamidation, pyroglutamate formation, phosphorylation and/or others, or any combination thereof, as they occur during production/expression by the native host or any suitable expression host. These post-translational modifications may or may not have an influence on the physical or enzymatic properties of the enzymes as explored herein.
In preferred embodiments of the present disclosure, the modified DNA-Polymerases within the Type-A family of polymerases comprise substitutions of at least two amino acid residues in the amino acid sequence of the naturally occurring DNA-polymerase at positions corresponding to 738 and 743 relative to the numbering of the amino acid sequence of wild-type Thermus aquaticus (Taq) DNA-polymerase (SEQ ID NO: 1), wherein the substitutions are 738F and 743F, and wherein said modified DNA polymerase, or homologs thereof. These positions are characterized in that mutagenesis of the enzyme at these positions leads to improvement in the desired enzyme characteristics, in particular to an increased strand display activity relative to the corresponding naturally occurring unmodified DNA-polymerase while keeping their thermostability.
For increasing strand displacement activity of the enzyme, the mutant polymerase may contain: Asp at position corresponding to Ala675 of wild-type Taq DNA polymerase; Tyr or Phe at position corresponding to His676 of wild-type Taq DNA polymerase; Arg at position corresponding to Tyr686 of wild-type Taq DNA polymerase; and Thr at position corresponding to Met747 of wild-type Taq DNA polymerase.
The gene encoding Taq DNA polymerase, the nucleotide sequence of the Taq DNA polymerase gene, as well as the full amino acid sequence of the encoded protein, are described in [Lawyer, F. C. et al., J. Biol. Chem., 261, 11, 6427-6437] and U.S. Pat. No. 5,079,352.
In yet a further aspect, the disclosure relates to a nucleic acid molecule and to the use of a nucleic acid molecule selected from the group consisting of

    
    
        a) a nucleic acid molecule encoding a DNA-Polymerase according to the present disclosure;
        b) a nucleic acid molecule encoding for a modified form of the DNA-Polymerases according to the present disclosure, preferably in which one or more amino acid residues are conservatively substituted;
        c) a nucleic acid molecule that is a fraction, variant, homologue, derivative, or fragment of the nucleic acid molecule presented as SEQ ID NO:9, SEQ ID NO:10 and/or SEQ ID NO:11;
        d) a nucleic acid molecule that is capable of hybridizing to any of the nucleic acid molecules of a)-c) under stringent conditions
        e) a nucleic acid molecule that is capable of hybridizing to the complement of any of the nucleic acid molecules of a)-d) under stringent conditions
        f) a nucleic acid molecule having a sequence identity of at least 95% with any of the nucleic acid molecules of a)-e) and encoding for a DNA-Polymerase,
        g) a nucleic acid molecule having a sequence identity of at least 85% with any of the nucleic acid molecules of a)-f) and encoding for a DNA-Polymerase,
        h) or a complement of any of the nucleic acid molecules of a)-g).
    
    


A nucleotide or nucleic acid is considered to hybridize to one of the above nucleotides if it is capable of hybridizing under conditions of medium stringency, more preferably high stringency, even more preferably under very high stringency conditions.
The nucleic acid molecule of the present disclosure may comprise nucleotide sequences that encode for SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or an effective fragment thereof or a variant, modified form, homologue or derivative thereof.
In particular, the disclosure provides a plasmid or vector system comprising a nucleic acid sequence encoding a modified DNA-Polymerase as described herein or a homologue or functional fragment thereof.
When compared with wild-type DNA-Polymerases within the Type-A family of polymerases, modified DNA-Polymerases of the disclosure are characterized inter alia by a high termo-stability as well as a strong strand displacement activity. In particular, the modified DNA-Polymerase variants according to the present disclosure are suitable for performing LAMP and PCDR DNA amplification, showing high thermostability suitable for performing PCDR and PCR DNA amplification, and have a high efficiency of long (over 2 kb) DNA sequences amplification in PCR.
In other words, the mutant thermostable enzymes of the present disclosure represent a significant improvement over thermostable DNA polymerases described in the literature. In particular, the DNA polymerase of the invention combines high thermostability and strong strand displacement, and provides the following properties: 1) high efficiency in PCR amplification and especially in long PCR; 2) high efficiency in LAMP; and 3) high efficiency in PCDR DNA amplification.
In an advantageous embodiment, the modified DNA-polymerases within the Type-A family of polymerases comprises substitutions of at least two amino acid residues in the amino acid sequence of the naturally occurring DNA-polymerase at positions corresponding to 738 and 743 relative to the numbering of the amino acid sequence of wild-type Thermus aquaticus (Taq) DNA-polymerase (SEQ ID NO: 1), wherein the substitutions are 738F and 743F, and wherein said modified DNA polymerase, or homologs thereof, have an increased strand display activity relative to the corresponding naturally occurring unmodified DNA-polymerase.
In advantageous embodiments, the naturally occurring Type-A DNA polymerase is a thermostable Thermus species DNA polymerase, in particular to Type-A DNA polymerases derived from a thermophilic bacterium that belongs to the Deinococcus-Thermus group.
Preferably, the naturally occurring Type-A DNA polymerase is derived from a thermophilic bacterium selected from the group consisting of Thermus aquaticus (Taq), Thermus thermophiles (Tth) and Thermus Flavus (Tfl), or derivative thereof. As shown in FIG. 13 these wild-type DNA-Polymerases show a consensus sequence of 21 amino acids beginning at the respective positions shown in FIG. 13, wherein position 738 of the Taq Polymerase corresponds to position 737 of the Tfl-Polymerase and to position 740 of the Tth Polymerase. Furthermore, position 743 of the Taq Polymerase corresponds to position 742 of the Tfl-Polymerase and to position 745 of the Tth Polymerase. Eleven of the 21 amino acids also line up with the 21-amino acid sequence of Eco- beginning at position 830 and of the 21-amino acid sequence of Mtu- beginning at position 805.
Embodiments of the present disclosure pertains to Type-A DNA polymerases comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.
In an advantageous embodiment, the modified DNA Polymerases comprise further a substitution at one or more of the positions corresponding to a position relative to the numbering of the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1), selected from the group of:
a) A675
b) H676
c) Y686
d) M747
In another advantageous embodiment, the modified DNA polymerases comprise further a substitution at one or more of the positions corresponding to a position relative to the numbering of the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1), selected from the group of:
a) A675D
b) H676Y/F
c) Y686R
d) M747T
In one embodiment, the described modified DNA Polymerase comprises further the substitutions A675D, H676Y/F, Y686R and M747T relative to the numbering of the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1).
In another embodiment, the modified DNA polymerases, or homologs thereof, have a reduced 5′-3′ exonuclease activity. In a preferred embodiment, the mutant DNA polymerase is a mutated Taq DNA polymerase with reduced 5′-3′ exonuclease activity, wherein said mutated Taq DNA polymerase comprises substitutions of mentioned above amino acid sequences from Bst polymerase for homologue sequences from Taq polymerase.
A derivative of DNA polymerase with reduced 5′-3′ exonuclease activity may be a truncated form of the enzyme with up to 50 amino acids deleted from the N-terminus of the enzyme. The DNA polymerase with reduced 5′-3′ exonuclease activity may be a truncated form of the enzyme with up to 100 amino acids deleted from the N-terminal of the enzyme. Preferably, it is a truncated form of the polymerase with up to 235 amino acids deleted from the N-terminus of the native enzyme. An example of one such mutant enzyme may be the one described in U.S. Pat. No. 5,616,494.
A derivative of DNA polymerase with reduced 5′-3′ exonuclease activity may also be a truncated form of the enzyme with up to 280 amino acids deleted from the N-terminus of the native enzyme. An example of one such mutant enzyme may be the one described in U.S. Pat. No. 5,436,149.
In an embodiment, the modified DNA polymerases according to the comprise a substitution at a position corresponding to position G46 relative to the numbering of the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1), in particular the substitution G46D.
In a further embodiment, the modified DNA polymerase comprises an N-terminal deletion.
In one example, a mutant DNA polymerase of the present disclosure with reduced 5′-3′ exonuclease activity may comprise a 5′-3′ exonuclease reducing mutation, such as Asp residue at position 46 of wild-type Taq DNA polymerase. Additionally, the mutant DNA polymerase of the present disclosure may include Asn at position 543 of wild-type Taq DNA polymerase for improving processivity of the polymerase [Ignatov, K. B., et al., (1998) FEBS letters, 425, 249-50; Ignatov, K. B., et al., (1999) FEBS letters, 448, 145-8], and may also include Tyr at position 667 for sequencing capabilities [Tabor, S., et al., (1995) Proc Natl Acad Sci USA, 92, 6339-43].
In a further embodiment, the modified DNA polymerases comprise a substitution at a position corresponding to position F667 relative to the numbering of the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1), in particular the substitution F667Y.
In an advantageous embodiment, the modified DNA polymerase according to the disclosure is a chimeric DNA-polymerase, wherein in an example the chimeric DNA-polymerase comprises an amino acid sequences of a modified DNA polymerase according to the present disclosure and an amino acid sequences of a wild-type Bacillus stearothermophilus DNA Polymerase I, in particular of a wild-type Bacillus stearothermophilus DNA Polymerase I having the amino acid sequence of SEQ ID NO 15. In particular, said chimeric DNA polymerase comprises the amino acid sequences of the wild-type Bacillus stearothermophilus DNA Polymerase I from Asp653 to Arg738 and from Arg779 to Pro795.
An advantageous embodiment of the chimeric DNA polymerase contains substitutions of Bst DNA polymerase fragments from Asp653 to Glu731 and from Arg779 to Pro795 for Taq DNA polymerase fragments from Asp610 to Glu688 and from Arg736 to Pro752, correspondingly. The obtained DNA polymerase had strong strand displacement activity, high thermostability and was able to perform PCR like Taq polymerase and LAMP reaction like Bst polymerase (“SD DNA polymerase”). The amino acid sequence of SD DNA polymerase is given in SEQ ID No: 2.
In one embodiment the modified DNA Polymerase comprises the amino acid sequence NFNVRSFAERTAMNT (SEQ ID NO. 12) derived from the Bst DNA Polymerase. Preferably, this amino acid sequence is at a position between amino acid residues corresponding to Arg736 and Pro752 relative to the numbering of the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1).
In another embodiment, the modified DNA polymerase comprises the amino acid sequence DYGXXXXXXITRK (SEQ ID NO: 13), in particular this amino acid sequence is at a position between amino acid residues corresponding to Ser674 and Glu688 relative to the numbering of the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1). In an advantageous embodiment, this amino acid sequence is DYGLAQNLNITRK (SEQ ID NO. 14).
In an advantageous embodiment, the modified DNA polymerases according to the present disclosure is derived from the wild-type Thermus aquaticus (Taq) DNA polymerase shown in SEQ ID NO. 1 by at least two substitutions L738F and A743F, or homologs thereof, wherein said modified DNA polymerase, or said homologs, are thermostable at a temperature of at least 80° C. and have an increased strand display activity relative to the wild-type Taq DNA polymerase and wherein the homologs have an sequence identity of at least 80% to the wild-type Taq DNA polymerase of SEQ ID NO. 1.
In another advantageous embodiment, the modified DNA polymerases according to the present disclosure is derived from the wild-type Thermus thermophiles (Tth) DNA polymerase shown in SEQ ID NO. 3 by at least two substitutions L740F and A745F, or homologs thereof, wherein said modified DNA polymerase, or said homologs, are thermostable at a temperature of at least 80° C. and have an increased strand display activity relative to the wild-type Tth DNA polymerase and wherein the homologs have an sequence identity of at least 80% to the wild-type Tth DNA polymerase of SEQ ID NO. 3.
In another advantageous embodiment, the modified DNA polymerases according to the present disclosure is derived from the wild-type Thermus Flavus (Tfl) DNA polymerase shown in SEQ ID NO. 5 by at least two substitutions L737F and A742F, or homologs thereof, wherein said modified DNA polymerase, or said homologs, are thermostable at a temperature of at least 80° C. and have an increased strand display activity relative to the wild-type Tfl DNA polymerase and wherein the homologs have an sequence identity of at least 80% to the wild-type Tfl DNA polymerase of SEQ ID NO. 5.
As mentioned above, said homologs have a sequence identity of at least 80%, in particular of at least 85%, in particular of at least 90% to the corresponding modified DNA-polymerase. There is no requirement that the polypeptides of the present disclosure comprise a full-length native polypeptide sequence of a DNA polymerase, in particular of Taq DNA-Polymerase. Rather, the polypeptide can also have a sequence that is modified from a native polypeptide sequence using the techniques known to those of skill in the art and/or taught in this specification. In some particular embodiments, the polypeptide is an enzyme variant that comprises a sequence that has DNA polymerase function, which is thermostable and has a strong strand display activity. Those of ordinary skill in the art will understand that it is be possible to reduce, increase or decrease the number of amino acids in polypeptide variants according to the present disclosure, so long as the active site and activity of the polypeptide having DNA polymerase activity are maintained. For example, there are a wide variety of variants that can be prepared to meet the needs according to the present disclosure and the teachings of this paragraph and the remainder of the specification can be used to prepare variants based on a large number of polypeptides that have DNA polymerase activity together with the before mentioned advantages.
Therefore, the disclosure pertains also to homologs and functional fragments of the DNA polymerases according to the present disclosure which has at least a minimum percentage sequence identity and/or percent homology to the polypeptides according to the present disclosure, wherein the minimum percent identity and/or homology is at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%.
In an advantageous embodiment of the disclosure, the described DNA polymerase variants have an increased strand display activity when incubated at temperatures>60° C., in particular at temperatures>90° C. during a standard DNA amplification procedure like PCR or LAMP.
It is also understood that the amino acid sequences of the modified DNA-Polymerases according to the present disclosure and homologs thereof may be produced as a N- and/or C-terminal fusion protein, for example to aid in extraction, detection and/or purification and/or to add functional properties to the DNA-Polymerase molecule. The fusion protein partner may be any protein or peptide including any polypeptide sequence derived from the native host, any other naturally occurring amino-acid sequence as well as synthetic sequences. Examples of fusion protein partners include, but are not limited to, glutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/or transcriptional activation domains), FLAG-, MYC-tags or other tags well known to anyone skilled in the art. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably, the fusion protein will not hinder the activity of the protein sequence of interest.
The modified DNA-Polymerases described herein may also be used in enzyme compositions conjunction with one or more additional proteins for the use in DNA sequencing and/or DNA amplification.
In another aspect of the present disclosure, method of producing a modified DNA polymerase are disclosed comprising the steps of: (a) culturing the host cell according to claim 33 in a suitable culture medium under suitable conditions to produce said DNA polymerase; (b) obtaining said produced DNA polymerase.
In order to produce modified DNA-Polymerase enzymes, the DNA encoding the enzyme can be chemically synthesized from published sequences or obtained directly from host cells harboring the gene (e.g., by cDNA library screening or PCR amplification). The modified DNA-Polymerase gene can be included in an expression cassette and/or cloned into a suitable expression vector by standard molecular cloning techniques. Such expression cassettes or vectors often contain sequences that assist initiation and termination of transcription (e.g., promoters and terminators), and may contain selectable markers. Cassettes can also be comprised of plus or minus strand mRNA, and their expression may or may not include an amplification step before translation of the mRNA. The expression cassette or vector can be introduced in a suitable expression host cell which will then express the corresponding DNA-Polymerase gene. Particularly suitable expression hosts are bacterial expression host genera including Escherichia (e.g., Escherichia coli), Pseudomonas (e.g., P. fluorescens or P. stutzerei), Proteus (e.g., Proteus mirabilis), Ralstonia (e.g., Ralstonia eutropha), Streptomyces, Staphylococcus (e.g., S. carnosus), Lactococcus (e.g., L. lactis), lactic acid bacteria or Bacillus (subtilis, megaterium, licheniformis, etc.).
The above methods of cloning and expression of the modified DNA-Polymerase gene are suitable both for industrial scale expression and for use in high throughput screens for the evaluation of mutated variants.
The modified DNA-Polymerases according to the present disclosure or chimeric variants thereof may be constructed in a number of ways, most easily via the construction of a recombinant DNA molecule, followed by expression of the protein product. The protein may then be expressed from the DNA, using expression vectors maintained within host cells. DNA cloning, manipulation and protein expression are all standard techniques in the art, and details of suitable techniques may be found in [Sambrook et al, ‘Molecular cloning—A Laboratory Manual’].
The present invention, therefore, also provides DNA encoding the mutant thermostable DNA polymerase, along with vector containing this DNA, host cells containing this vector, and cultures of such cells, as well as methods for making the enzyme.
As described above, the DNA-Polymerase proteins can be expressed in a variety of expression systems and accordingly the appropriate down-stream processing and purification procedures have to be selected. The protein of interest can be secreted into the extracellular or periplasmic space or expressed intracellular. In an advantageous embodiment of the disclosure the DNA-Polymerase variant is expressed in a microbial host and the protein is secreted into the periplasmic or extracellular space. Cells expressing the DNA-Polymerase variants are preserved by methods well known to anyone skilled in the art, such as, but not limited, to cryo stocks. Cultures of the expressing organism are prepared at an appropriate volume with standard methods of fermentation. In a preferred embodiment, cultures for protein expression are inoculated from a cryo stock and the volume of the culture increased successively in the appropriate containers. In a preferred embodiment the cells are grown in a fermenter and optionally growth conditions such as pH, temperature, oxygen and/or nutrient supply are controlled. A first step of purification comprises the separation of cells from supernatant using one or more of several techniques, such as sedimentation, microfiltration, centrifugation, flocculation or other. In a preferred embodiment the method applied is microfiltration. In case of intracellular expression the cells are subjected to treatments that result in a release of the protein from the intracellular space. These treatments may comprise for example pressure, enzymatic, osmotic shock, freezing, ultrasonic or other treatment to produce a cellular extract which may or may not be subjected to further purification.
In an advantageous embodiment of the disclosure the DNA-Polymerase is secreted into the supernatant and an optional step of purification comprises the concentration of the supernatant by ultrafiltration. Further protein purification from the supernatant or concentrated supernatant may be performed with one or more of several methods comprising extraction or fractionation methods such as ammonium sulfate or ethanol or acid precipitation, or chromatographic methods including but not limited to ion-exchange, hydrophobic interaction, hydroxylapatite, size fractionation by gel-filtration, phosphocellulose or lectin chromatography and affinity chromatography or any combination thereof. In a more preferred method the affinity-tagged protein is purified by metal-chelate affinity chromatography to obtain a high purity protein.
The preferred purification method yields a purity of the protein of >30%, in a more preferred method the purity is >50%, >60%, >70%, or >80%. In an even more preferred method the purity is >90%, in a yet more preferred method the purity is >95% and in a most preferred method the purity is >98%.
In another advantageous embodiment of the disclosure the supernatant or the supernatant partially purified by ultra filtration or the concentrated and/or diafiltrated supernatant is dried by any one of several technical methods such as, but not limited to, spray-drying, lyophilisation, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyer drying or any combination thereof. In a further advantageous embodiment of the disclosure the fermented cell-suspension including the expressed DNA-Polymerase variants is dried as a whole using processes such as, but not limited to, fluidized bed drying, conveyer drying, spray drying or drum drying or any combination thereof.
In a further embodiment of the invention there is provided an isolated nucleic acid sequence comprising sequence encoding a polypeptide as described supra. Thus, a nucleic acid sequence encoding any of the polypeptides or polypeptide fusion proteins described herein are also included as part of the instant disclosure. The skilled artisan will understand that a variety of nucleic acid sequence may be used to encode identical polypeptides in view of the degeneracy of genetic code. In certain cases for example the codon encoding any particular amino acid may be altered to improve cellular expression.
Additionally, the present invention includes kits containing the enzymes of the disclosure in combination with other reagents, suitable for use in laboratory experiments.
The following methods and examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.
Methods and Examples
In the following examples, materials and methods of the present disclosure are provided including the determination of catalytic properties of enzymes obtained by the method. It should be understood that these examples are for illustrative purpose only and are not to be construed as limiting this disclosure in any manner. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
EXAMPLES
The Examples relate to testing of mutant thermostable enzyme of the invention, such as SD DNA polymerase.
SD DNA polymerase is a mutant thermostable DNA polymerase with a strong strand displacement activity, which is suitable for both LAMP and PCR methods of DNA amplification. It was designed from Taq DNA polymerase. SD DNA polymerase has the 5′-3′ polymerase and 5′-3′ strand displacement activities, but it does not have any exonuclease activity. The 5′-3′ exonuclease activity was reduced by G46D mutation, and the 5′-3′ strand displacement activity was achieved by substitutions of Bst DNA polymerase fragments from Asp653 to Glu731 and from Arg779 to Pro795 for Taq DNA polymerase fragments from Asp610 to Glu688 and from Arg736 to Pro752, respectively. The amino acid sequence of SD DNA polymerase is given in SEQ ID No: 2.
The nucleic acid encoding SD DNA polymerase (SEQ ID No: 9) was synthesized by Evrogen JSC (Russia) and cloned into the plasmid pTTQ18 under control of Tac promoter. The resulting plasmid vector pTTQ-SD was purified, and the nucleotide sequence encoding the mutant enzyme was verified by sequencing.
Bst DNA polymerase (Large fragment) was obtained from New England Biolabs, Inc. USA. Commercially available Taq DNA polymerases were obtained from: Promega Corporation, USA (GoTaq polymerase); Bioline Limited, GB (MyTaq polymerase); and Evrogen JSC, Rus.
dNTPs were obtained from Bioline Limited (London, GB). Lambda DNA and HeLa gDNA were obtained from New England Biolabs, Inc. USA. Murine cDNA library was obtained from Evrogen JSC, Russia.
LAMP reactions were performed with control template DNA and primers for Group B Streptococcus (GBS) obtained from Meridian Bioscience Inc., USA. (http://www.meridianbioscience.com)
As mentioned above, the Examples are illustrative of, but not binding on, the present invention. Any methods, preparations, solutions and such like, which are not specifically defined, may be found in Sambrook et al. All solutions are aqueous and made up in sterile, deionised water, unless otherwise specified.
Example 1
Preparation of Chimeric Thermostable Enzyme Using an Expression Vector (Plasmid pTTQ-SD)
E. coli JM 109 cells were transformed with the plasmid pTTQ-SD according to the method of Dower et al. [1988, Nucl. Acid. Res., V. 16, P. 6127]. The transformed cells were grown to an optical density of A.600=0.3 in 7 L of LB medium containing ampicillin (150 mkg/ml) at 37° C. Expression of the chimeric gene encoding the chimeric polymerase was induced by IPTG (1 mM). The cells were further incubated for 10-12 h at 37° C. Cells were harvested by centrifugation.
The cells (35 g) were suspended in 70 ml of buffer A (20 mM K-phosphate pH 7.0, 2 mM DTT, 0.5 mM EDTA) containing 0.2M NaCl and 0.1 mM PMSF. The cellular walls were disrupted with an ultrasonic disintegrator (MSE, 150 wt) at maximum amplitude for 15 minutes (30 pulses, each for 30 sec) and with cooling on ice. The suspension was then centrifuged at 40,000 g, the pellet discarded, and 5% polyethylenimine was added to the supernatant to a final concentration of 0.1%. The precipitate was separated by centrifugation, and the remaining proteins were precipitated with ammonium sulfate at 45% saturation. The resulting polymerase-containing precipitate was collected by centrifugation at 20,000 g and dissolved in buffer A (30 ml) containing 0.05 M NaCl and 0.2% Tween-20, heated for 15 minutes at 70° C. in the presence of 5 mM MgCl2, and centrifuged for 10 minutes at 40,000 g.
The supernatant was loaded on to a (2.5×20 cm) phosphocellulose P-11 column (Whatman) equilibrated in buffer A containing 0.05 M NaCl, and washed out with the same buffer. The proteins were eluted with a linear gradient of NaCl concentrations ranging from 50 to 400 mM in buffer A. The gradient volume was 800 ml, and the flow rate was 60 ml/h. The polymerase was eluted at NaCl concentrations ranging from 100 to 200 mM.
The fractions were tested for polymerase activity, and assayed via incorporation of radioactive-labelled nucleotide 32P(dATP) into the acid-insoluble pellet [Myers T. W., Gelfand D. H., (1991) Biochemistry, v 30, N 31, p 7661-7666].
Specifically, the amount of the enzyme that incorporated 10 nmol of deoxynucleotide triphosphates into the acid-insoluble fraction within 30 minutes under conditions described below was taken as one unit of activity. The reaction mixture (50 mkL) contained 25 mM N-Tris [Hydroxymethyl]methyl-3-aminopropanesulphonic acid (TAPS), pH 9.3, 50 mM KCl, 2 mM MgCl2; 1 mM β-mercaptoethanol; 0.2 mM of each dNTP's, 1 mkCi 32P(dATP), and 12.5 mkg of activated salmon sperm DNA. The polymerase activity was determined at 73° C. (Salmon sperm DNA (12.5 mg/ml) was activated in 10 mM Tris-HCl (pH 7.2) containing 5 mM MgCl2 with pancreatic DNase I (0.03 U/ml) at 4° C. for 1 h and then heated at 95° C. for 5 minutes.)
Fractions containing the polymerase activity were combined, dialyzed against buffer A containing 50 mM NaCl and loaded on to a column (0.6×6 cm) of DEAE-cellulose (Whatman) equilibrated with same buffer. The proteins were eluted with a linear gradient of NaCl concentrations ranging from 50 to 250 mM in buffer A. The gradient volume was 150 ml, and the flow rate was 15 ml/h. The polymerase was eluted at 150-200 mM NaCl. Polymerase activity was assayed as described above. Yield of polymerase activity was 1,475,000 units.
The purified enzymes were stored at −20° C. in the following buffer: 100 mM NaCl; 10 mM Tris HCl pH 7.5; 1 mM DTT; 0.2% Tween 20 and 50% (v/v) glycerol.
Homogeneity of the polymerase preparations was not less than 95% according to SDS electrophoresis data on a 10% polyacrylamide gel.
Example 2
Comparison of Strand Displacement Activity and Thermostability of SD DNA Polymerase with Bst Large Fragment DNA Polymerase in LAMP Reaction
Displacement activity and thermostability of SD and Bst DNA polymerase were compared in LAMP reaction.
LAMP amplification was performed with control template DNA and primers for Group B Streptococcus (GBS) from Meridian Bioscience Inc. (http://www.meridianbioscience.com)
SD and Bst DNA polymerase (NEB) were compared in two reactions buffers: GoTaq buffer (Promega) and Encyclo (Evrogen, www.evrogen.com). Reaction mixtures (50 μl) contained: 50 units of DNA polymerase (SD or Bst), 3.5 mM MgCl2, 0.5 mM dNTP (each), 2 μl GBS control template DNA, and GBS primers: F3T3—0.2 μM, B3—0.2 μM, FIP—0.8 μM, BIP—0.8 μM, FL—0.8 μM, BL—0.8 μM.
LAMP reactions were performed at 63° C. for 45 min, with or without initial preheating at 92° C. for 2 min.
The results are shown in FIG. 1, and indicate that the mutant enzyme of the invention (SD polymerase) demonstrates in the LAMP reaction a similar with Bst strand displacement activity and the efficiency of DNA, but SD polymerase has much higher thermostability than Bst polymerase.
Example 3
Comparison of SD DNA Polymerase with Taq DNA Polymerase in Long PCR
Example 3 illustrates a high efficiency of SD polymerase in PCR amplification. SD polymerase was compared with Taq polymerases from different suppliers like GoTaq polymerase and GoTaq buffer from Promega; MyTaq polymerase and MyTaq buffer from Bioline; and Taq polymerase and Encyclo buffer from Evrogen
An 8 kb fragment of λ DNA was amplified with 2.5, 5, 10 and 15 units of SD or Taq DNA polymerase. Reaction mixture (50 μl) contained: 5 ng λ DNA as template, 0.25 mM dNTP (each), 10 pmol (0.2 μM) of each primer, 1×PCR buffer, and 3 mM MgCl2.
PCR was carried out for 25 cycles: preheating for 2 min at 92° C.; cycling for 30 sec at 92° C., 30 sec at 60° C. and 2 min 40 sec (20 sec/kb) at 68° C.
FIG. 2 demonstrates that SD polymerase provides much higher efficiency of PCR then Taq. This result can be explained by displacement activity of SD polymerase, which solves problems with secondary structures of DNA templates.
Example 4
The Usage of SD DNA Polymerase for Performing PCDR Amplification
PCDR is a novel method of DNA sequence specific amplification. The idea of this method was described in [Harris, C. L. et al. BioTechniques 54:93-97 (February 2013) doi 10.2144/000113951] and U.S. Pat. No. 8,252,558 B2.
PCDR requires a heat denaturation of DNA like PCR and strand displacement activity of DNA polymerase like LAMP. So, we attempted at using the SD polymerase for performing PCDR.
In PCDR, four primers are employed in the reaction—amplification is initiated from both the outer primers and the inner primers and by using a polymerase with strand displacement activity, PCDR enables increased template amplification per cycle compared to the standard two primer reaction and thus enhanced sensitivity in PCR applications. In our variants of PCDR we used tetra- and hex-primer systems.
PCDR with four primers generates four fragments (amplicons): one long or common fragment (I); two middle fragments (II, III); and one short fragment (IV).
Theoretical kinetics of amplification for fragments:

2n;   (I)

n×2(n-1) or (n×2n):2;   (II), (III)

(n2+3n)×2(n-2) or (n2+3n)×2n:4   (IV)

(wherein n is a number of cycle).

Thus PCDR amplification of the short fragment (IV) outperforms PCR amplification (n2+3n):4 times (wherein n is the cycle number).
PCDR with six primers generates 9 fragments. Amplification of the shortest amplicon of this system has the following theoretical kinetic:

[(n2+3n)×2(n-2)]2+(n×2n)

(wherein n is the cycle number). It outperforms PCR amplification (n2+3n)2×2(n-4)+n or (n2+3n)2×2n:16+n times.

Reaction mixtures (50 mkl) contained: 1× GoTaq buffer; 3 mM MgCl2; 0.375 mM dNTPs (each); primers 20 pmol (each); about 0.05 ng of Murine cDNA library as a template. PCR and PCDR were performed for 20 cycles: 92° C.—30 sec, 65° C.—1 min; preheating: 92° C.—1 min 30 sec. Reactions were carried out with 25 units of SD polymerase or with 25 units of KlenTaq polymerase (KlenTaq was used for control reactions, as an enzyme without strand displacement activity).
The results are shown in FIG. 3, and indicate that PCDR provides much higher efficiency and sensitivity than PCR, and that SD polymerase can be an enzyme of choice for performing PCDR.
In our experiments we used primers for Murine G3PDH cDNA (SEQ ID NO: 16). The sequence of amplified DNA fragment is shown in FIG. 18. Sequences of forward primers (F1, F2, F3) and reverse primers (R1, R2, R3) are highlighted in grey.
Example 5
A Long-Distance PCR of a 17.5 kb Human gDNA Fragment by SD DNA Polymerase
The strand-displacement activity of SD polymerase solves problems with secondary structures of templates and improves an efficiency of a long-distance amplification. A fitness of this polymerase for performing a long PCR was estimated by PCR amplification of a 17.5 kb DNA fragment from Human genomic DNA. The reaction was carried out with 2.5 U of SD polymerase for 35 cycles: 92° C.—25 sec, 66.5° C.—1 min, 69° C.—9 min; initial preheating 45 sec at 92° C. Reaction mixture (25 μl) contained: 100 ng of Human gDNA (NEB) as template, 0.2 mM dNTPs (each), 5 pmol (0.2 μM) of each primer (HG1 and HG2, see table 3), 1× reaction buffer, and 2.75 mM MgCl2. The elongation time was 9 minutes or 30 sec/kb.
FIG. 19 shows the result of the long-distance amplification. SD polymerase provided fast and efficient long PCR from Human gDNA. It should be emphasized that the result was achieved by using SD polymerase alone, without any “proofreading” additives, such as Vent or Pfu polymerases.
An amplification of a 17.5 kb fragment of Human β-globin gene was carried out for 35 cycles with 2.5 U of SD polymerase and 100 ng of Human gDNA as a template (lanes 1, 2). The elongation time was 9 minutes or 30 sec/kb. M1—λ/Hind III DNA marker; M2—1 kb DNA ladder.








TABLE 3







Primer HG1
5′-ACATGATTAGCAAAAGGGCCTAGCTTGGACTCAGA


 


Primer HG2
5′-TGCACCTGCTCTGTGATTATGACTATCCCACAGTC









Example 6
Real-Time PCR and PCDR Amplifications with SD and Taq DNA Polymerases
Real-time amplifications of Murine G3PDH cDNA sequence were carried with AmpliFluor primer AF3 (see table 4). This AmpliFluor primer is similar with inner primer F3 but includes a hairpin structure with a quencher (BHQ2) and a fluorescent reporter (HEX) at 5′ end.
Reaction mixtures (25 μl) contained: 5 units of SD or Taq DNA polymerase; 1× GoTaq buffer (Promega, USA); 2.75 mM MgCl2; 0.25 mM dNTPs (each); 0.2 μM inner primers AF3 and R3 (each), 0.1 μM outer primers F2 and R2 (each), and 0.05 μM outer primers F1 and R1 (each); 10, 1, 0.1, 0.01 or 0.001 pg of Murine cDNA library as a template. PCR mixtures contained only two inner primers AF3 and R3. PCDR mixtures additionally contained two (F2, R2) or four (F2, R2 and F1, R1) outer primers.
Amplifications were carried out by using BioRad CFX96 PCR machine and the following protocol: initial preheating—92° C. for 2 min, cycling—92° C. for 15 sec, 66° C. for 40 sec.
SD and Taq DNA polymerases were compared in real-time amplifications of a Murine G3PDH cDNA sequence. SD polymerase does not possess 5′-3′ exonuclease activity, so the usage of TaqMan probes for performing real-time reactions is unable. For carrying out amplifications in a real-time manner, an AmpliFluor direct primer was used. This AmpliFluor primer AF3 was similar with inner primer F3 but included a hairpin structure with a quencher (BHQ2) and a fluorescent reporter (HEX) at 5′ end. PCR real-time mixtures contained two primers: R3 and AmpliFluor AF3. PCDR real-time mixtures contained four (F2, R2, R3 and AF3) or six (F1, R1, F2, R2, R3 and AF3) primers including the AmpliFluor. The relative sensitivity of the polymerases was determined in PCR, tetra- and hex-primer PCDR, using 10-fold dilutions of Murine cDNA library (from 10 to 0.001 pg per reaction). The results of real-time amplifications are shown in Table 5. Amplification curves for 1, 1/102 and 1/104 dilutions are shown in FIG. 20: A (for PCR), B (for tetra-primer PCDR) and C (for hex-primer PCDR).








TABLE 4







AmpliFluor AF3
5′-(HEX)CAGGATGCgcatcctg(BHQ2)cacc



accaactg









These results show that using SD polymerase, instead of Taq, improved Cq values. The Cq values were reduced by one cycle in PCR, four cycles in tetra-primer PCDR and six cycles in hex-primer PCDR (Table 5, FIG. 20).
FIG. 20 shows the comparison of SD and Taq DNA polymerases in real-time PCR and PCDR amplifications. Amplifications of a Murine G3PDH cDNA sequence were carried out with 5 units of SD (blue curves) or Taq (red curves) DNA polymerase. AmpliFluor direct primer (AF3) was used in all reactions for performing the reactions in a real-time manner. Reaction mixtures contained the following amounts of cDNA library as a template: 10 ng (triangles), 0.1 ng (circles), 0.001 ng (squires) and no template control reactions.
FIG. 20 (A): PCR amplification was carried out with two primers: R3 and AmpliFluor AF3.
FIG. 20 (B): Tetra-primer PCDR was carried out with four primers: F2, R2, R3 and AF3.
FIG. 20 (C): Hex-primer PCDR was carried out with six primers: F1, R1, F2, R2, R3 and AF3.
The usage of SD polymerase reduced Cq values by one cycle in PCR, four cycles in tetra-primer PCDR and six cycles in hex-primer PCDR.
The sensitivity of the hex-primer PCDR with SD polymerase was 100 times higher than PCR with Taq polymerase (Table 5). Real-time PCDR with Taq polymerase did not significantly improve the sensitivity in comparison to PCR.









TABLE 5









Cq number















Taq

Taq
SD
Taq


Template
SD
Two
SD
Four
Six
Six


dilution
Two primers
primers
Four primers
primers
primers
primers





1
22.07 ± 0.148
23.24 ± 0.214
19.61 ± 0.085
23.15 ± 0.060
17.58 ± 0.093
22.58 ± 0.168


1/101
25.80 ± 0.021
27.00 ± 0.009
22.90 ± 0.008
26.63 ± 0.241
20.86 ± 0.058
26.30 ± 0.201


1/102
29.14 ± 0.049
30.43 ± 0.022
26.08 ± 0.125
30.06 ± 0.187
23.88 ± 0.032
29.83 ± 0.299


1/103
32.07 ± 0.030
33.80 ± 0.093
28.94 ± 0.331
33.26 ± 0.068
26.45 ± 0.153
33.02 ± 0.189


1/104
35.92 ± 0.125
37.10 ± 0.303
32.51 ± 0.001
35.72 ± 0.618
30.08 ± 1.048
36.21 ± 0.378


Efficiency (%)
96.9
94.9
106.1
106.5
112.3
97.0


R2
0.998
0.999
0.998
0.993
0.992
0.997









Table 5 shows the results of the comparison of quantitative PCR and PCDR with Taq and SD DNA polymerase. Amplifications of a Murine G3PDH cDNA sequence were carried out with SD or Taq DNA polymerase and with using 10-fold dilutions of Murine cDNA library (from 10 to 0.001 pg per reaction). Reactions contained two (in PCR), four or six (in PCDR) primers including AmpliFluor direct primer AF3.

## Claims

1. An enzyme composition comprising at least one modified DNA polymerase, the modified DNA polymerase being within the Type-A family of polymerases, wherein the modified DNA polymerase comprises substitutions of at least two amino acid residues in the amino acid sequence of a corresponding naturally occurring unmodified DNA polymerase, at positions corresponding to 738 and 743 relative to the numbering of the amino acid sequence of wild-type Thermus aquaticus (Taq) DNA polymerase (SEQ ID NO: 1), wherein the substitutions are 738F and 743F, said modified DNA polymerase has an increased strand displacement activity relative to the corresponding naturally occurring unmodified DNA polymerase, and the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO: 2.

2. The enzyme composition according to claim 1, wherein said modified DNA polymerase is thermostable at a temperature of at least 80° C.

3. The enzyme composition according to claim 1, wherein said modified DNA polymerase has a reduced 5′-3′ exonuclease activity.

4. A kit for amplification of a target nucleic acid, said kit comprising a modified DNA polymerase, the modified DNA polymerase being within the Type-A family of polymerases, wherein the modified DNA polymerase comprises substitutions of at least two amino acid residues in the amino acid sequence of a corresponding naturally occurring unmodified DNA polymerase, at positions corresponding to 738 and 743 relative to the numbering of the amino acid sequence of wild-type Thermus aquaticus (Taq) DNA polymerase (SEQ ID NO: 1), wherein the substitutions are 738F and 743F, said modified DNA polymerase has an increased strand displacement activity relative to the corresponding naturally occurring unmodified DNA polymerase, and the modified DNA polymerase comprises the amino acid sequence of SEQ ID NO: 2.