Phi29 DNA polymerase mutant with improved primer recognition

Abstract

To provide mutants of Phi29 DNA polymerase with improved primer recognition, compared to the wild-type enzyme.SOLUTION: Provided is a Phi29-type DNA polymerase comprising one or both of the mutations K64R or M97K. The provided mutants are capable of using shorter and longer random synthetic DNA primers more efficiently than wild-type Phi29 DNA polymerase and generating more amplification products in Multiple Displacement Amplification reactions.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of the priority date of U.S. Provisional Application No. 62 / 849,252, filed on May 17, 2019, the content of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing in computer - readable form, which is hereby incorporated by reference in this specification.

Background Art

[0003] Background Phi29 DNA polymerase (Phi29 DNApol) is a monomeric enzyme (66 kDa) responsible for replicating bacteriophage genomes (19285 bp) by catalyzing both protein-primed initiation at both ends of linear dsDNA molecules and the complete elongation of each DNA strand (Blanco and Salas, 1984; 1985). Phi29 DNApol belongs to DNA polymerase family B (Bernad et al, 1987) and exhibits a typical right-hand fold, containing not only palm, thumb, and finger subdomains, but also two additional domains called TPR1 and TPR2 (Rodriguez et al, 2005; Kamtekar et al, 2006; Berman et al, 2007). Phi29 DNApol exhibits unique properties that enable its application in numerous DNA amplification and sequencing technologies and platforms: highly processive DNA synthesis, enabling the enzyme to incorporate over 70,000 nucleotides per DNA binding event in the absence of processivity factors (Blanco et al, 1989); extremely good strand displacement, enabling polymerization linked to the unwinding of double-stranded DNA in the absence of helicase-type enzymes (Blanco et al, 1989); and a very low error insertion rate (10 -4 ~10 -6 ) and efficient calibration of inserted errors, along with the built-in 10 6 ~10 8 High synthetic fidelity, where the accuracy is reduced to a maximum of one error per nucleotide (Esteban et al, 1993 and 1994).

[0004] These properties make Phi29 DNApol an optimal choice for isothermal multiple displacement amplification (MDA) (Dean et al, 2002) and rolling circle amplification (RCA) (Lizardi et al, 1998). These DNA amplification techniques are based on a combination of Phi29 DNApol and either randomly synthesized primers (RPs), which are mainly hexanucleotides or hexamers, or DNA primases that can synthesize DNA primers in situ during the amplification reaction (Picher et al, 2016).

[0005] Current sequencing techniques often require DNA amplification because the amount of DNA obtained from a particular sample (e.g., a single cell) is insufficient for the sequencing process. Unfortunately, DNA amplification carries the risk of introducing errors, creating asymmetry (bias), and even facilitating the simultaneous amplification of trace amounts of contaminated DNA. Therefore, key parameters determining amplification quality are the absence of contaminants and artifacts in the reaction product, broad and uniform coverage, a low nucleotide error rate, and the ability to correct single nucleotide variants (SNVs), copy number variants (CNVs), and structural variants.

[0006] The potential amplification bias in current MDA methods based on random hexamers stems from priming imbalances resulting from differing sequence-dependent hybridization rates of the oligonucleotides. More importantly, self-pairing hexamers tend to generate primer-derived, input-independent DNA amplification artifacts due to exponential amplification.

[0007] It has been shown that using longer primers instead of hexamers and raising the reaction temperature to 40°C significantly reduces DNA amplification artifacts (Alsmadi et al, 2009). The most likely reason behind this behavior is that higher temperatures reduce the likelihood of stable self-pairing of primers, thus reducing their subsequent amplification. However, to carry out amplification reactions at such a high temperature as 40°C (10°C higher than the optimal temperature for Phi29 DNApol), a thermally stable or thermally resistant Phi29 DNApol variant is required. In this regard, several mutant Phi29 DNApol have been described as exhibiting improved thermal stability (Povilaitis et al, 2016). [Brief explanation of the drawing]

[0008] The accompanying drawings incorporated herein and forming part of this specification illustrate exemplary embodiments and, together with the description, will be of further use to enable those skilled in the art to practice and use these embodiments and other embodiments that are apparent to those skilled in the art. The present invention will be described more specifically in conjunction with the following drawings. [Figure 1] 3D structure of Phi29 DNApol complexed with DNA and dNTPs (PDB id:2PYL). Most of the protein is shown in white, except for the subdomains of Sam (dark green), TPR2 (cyan), and TPR1 (yellow). The N-terminal 3'-5' exonuclease domain is not fully depicted (only two segments are shown (yellow), one containing Arg96 and the other containing Lys64). For the primer strand (cyan), numbers corresponding to each nucleotide position are indicated. The template strand (light green), incoming nucleotides (magenta), and two activated metal ions (beige) are also shown. [Figure 2]A) Schematic diagram of wild-type (WT) Phi29 DNApol amino acid residues involved in interaction with the first 10 nucleotides of the primer chain, derived from the crystal structure (PDB id: 2PYL). The nucleotide numbered 1 is at the very 3' end and is often called the "primer end," being closest to the enzyme active site. B) This diagram shows the acquisition of new interactions with the primer chain resulting from various mutations (shown in magenta). The colored arrows indicate whether a phosphodiester (red), sugar (orange), or base (green) is involved in the interaction. Mutants T499K and T499R are predicted to interact with the same positions on the complementary / template chain (shown by dashed arrows). [Figure 3-1] Amplification efficiency of WT Phi29 DNApol or designed variants when using 1 ng of human genomic DNA as input to the amplification reaction, combined with TthPrimPol or random primers of different lengths (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)). [Figure 3-2] This is a continuation of Figure 3-1. [Figure 3-3] This is a continuation of Figure 3-2. [Figure 4] The balance of exonuclease activity and polymerase activity of WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K as a function of provided deoxyribonucleotide (dNTP) concentration. [Figure 5]Amplification efficiency of WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K when using 1 ng of human genomic DNA as input to the amplification reaction, combined with random primers of different lengths (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)). For each primer length N, the vertical bars are shown from left to right in the following order: WT Phi29 DNApol, mutant K64R, mutant M97K, double mutant K64R / M97K. [Figure 6] Amplification yield observed in the absence of input DNA, using either WT Phi29 DNApol, mutant K64R, mutant M97K, or double mutant K64R / M97K, combined with randomly synthesized primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)) under low ionic strength conditions. For each primer length N, the vertical bars are shown from left to right in the following order: WT Phi29 DNApol, mutant K64R, mutant M97K, double mutant K64R / M97K. [Figure 7] Amplification yield observed in the absence of input DNA, using either WT Phi29 DNApol, mutant K64R, mutant M97K, or double mutant K64R / M97K, combined with randomly synthesized primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)) under high ionic strength conditions. For each primer length N, the vertical bars are shown from left to right in the following order: WT Phi29 DNApol, mutant K64R, mutant M97K, double mutant K64R / M97K. [Figure 8]Amplification efficiency of WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K, combined with randomly synthesized primers of different lengths (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)), when using 1 ng of human genomic DNA as input to the amplification reaction under high ionic strength conditions. For each primer length N, the vertical bars are shown from left to right in the following order: WT Phi29 DNApol, mutant K64R, mutant M97K, double mutant K64R / M97K. [Figure 9-1] Amplification efficiency of WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K, combined with randomly synthesized primers of different lengths (tetramer (4N), pentamer (5N), hexamer (6N)), when using different amounts of human genomic DNA input (1, 10, 100 pg, and 1 ng) in the amplification reaction under low and high ionic intensity conditions. For each primer length N, the vertical bars are shown from left to right in the following order: WT Phi29 DNApol, mutant K64R, mutant M97K, double mutant K64R / M97K. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-2. [Figure 10] Amplification of 1, 10, 100 pg, and 1 ng of human genomic DNA by multiple substitution amplification (MDA) combining the Phi29 DNApol variant and TthPrimPol. For each DNA quantity, the vertical bars are shown from left to right in the following order: WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K. [Figure 11-1] Estimated coverage values ​​obtained from CovCheck analysis of amplification reactions using hexamers (6N), pentamers (5N), and tetramers (4N) were used in each case, combined with Phi29 DNApol variants and different amounts of human genomic DNA input, to reach conditions where differences in coverage could be observed. [Figure 11-2] It is a continuation of FIG. 11-1. [Figure 12] Estimated coverage values obtained from CovCheck analysis of an amplification reaction performed by combining TthPrimPol and a Phi29 DNApol variant. SUMMARY OF THE INVENTION

[0009] Summary Modified DNA polymerases can be useful for various applications such as DNA sequencing, DNA amplification, library preparation, DNA genotyping, etc. The present invention provides a recombinant Phi29 DNA polymerase containing mutations that confer improved properties particularly desirable for these or other applications. Such changes in the amino acid sequence can improve the performance of multiple displacement DNA amplification (MDA) by using shorter random synthetic primers, resulting in a reduction of amplification artifacts, an increase in the rate of sequence-dependent hybridization, and thus an improved coverage spread and uniformity. "Phi29" may also be written as "φ29".

[0010] The recombinant Phi29 DNA polymerase contains one or two mutations selected from the group consisting of K64R and M97K. DETAILED DESCRIPTION OF THE INVENTION 〈0000082〉

[0011] Detailed Description I. Definitions "Isolated" means that a molecule is the major species present in a composition, i.e., it is more abundant on a molar basis than other individual macromolecular species in the composition. Generally, an isolated molecule can account for more than 80%, more than 90%, more than 95%, more than 98%, or more than 99% of the macromolecular species present in the composition and is the purified species of interest. Solvent species, small molecules (<500 daltons), stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for the purposes of this definition.

[0012] As used herein, the term "recombinant nucleic acid" refers to a nucleic acid molecule containing two or more linked nucleotide sequences that are not normally linked to each other in nature.

[0013] As used herein, the term "recombinant cell" refers to a cell containing a recombinant nucleic acid, such as a cell of an animal, plant, fungus or microorganism (e.g., bacterium).

[0014] Terms used to describe the sequence relationship between two or more nucleotide or amino acid sequences include "reference sequence", "selected from", "comparison window", "identical", "percentage of sequence identity", "substantially identical", "complementary" and "substantially complementary".

[0015] A "reference sequence" is a defined sequence used as a basis for sequence comparison and can be a subset of a larger sequence, such as a complete cDNA, protein, or gene sequence.

[0016] Because two nucleic acids or polypeptides can each contain (1) sequences that are similar between the two nucleic acids (i.e., only a portion of the complete nucleic acid or polypeptide sequence), or (2) sequences that diverge between the two nucleic acids, sequence comparison between two (or more) nucleic acids or polypeptides is typically performed by comparing the sequences of the two nucleic acids over a "comparison window" that identifies and compares local regions of sequence similarity.

[0017] A "comparison window" refers to a conceptual segment typically consisting of at least 12 consecutive nucleotides or 4 consecutive amino acid residues that is compared to a reference sequence. The comparison window often has a length of at least 15 or at least 25 nucleotides, or at least 5 or at least 8 amino acids. For optimal alignment of the two sequences, the comparison window may contain approximately 20% or less of additions or deletions (i.e., gaps) compared to the reference sequence (without additions or deletions). Optimal sequence alignment for aligning the comparison window can be performed by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI)) or by inspection, and the best alignment obtained by any of these methods (i.e., the one that yields the highest homology percentage across the comparison window) is selected.

[0018] A nucleotide sequence or amino acid sequence is considered "identical" to a reference sequence if, when aligned to maximize correspondence (match) over the length of the nucleotide or amino acid sequence, the two sequences are identical.

[0019] To calculate the "percentage of sequence identity" between two sequences, compare two sequences that are optimally aligned across a comparison window, count the positions where the same nucleotide or amino acid appears in both sequences, divide the number of matching positions by the total number of positions within the comparison window (i.e., the window size), and multiply the result by 100 to obtain the percentage of sequence identity.

[0020] Unless otherwise specified, the comparison window used when comparing two arrays is the length of the shorter array.

[0021] These methods are further described in Natl. Acad. Sci. USA 85:2444; Higgins & Sharp (1988) Gene 73:237-244; Higgins & Sharp, CABIOS 5:151-153 (1989); Corpet et al. (1988) Nucleic Acids Research 16:10881-90; Huang et al. (1992) Computer Applications in the Biosciences 8:155-65; and Pearson et al. (1994) Methods in Molecular Biology 24:307-31. Alignment is often performed by scrutiny and manual alignment.

[0022] A nucleotide or amino acid sequence is considered "substantially identical" to a reference sequence if it exhibits at least 80% sequence identity across the comparison window. Therefore, sequences exhibiting at least 85%, 90%, 95%, 98%, or 99% sequence identity with a reference sequence are also considered "substantially identical." Naturally, two sequences that are identical to each other are also considered "substantially identical."

[0023] As used herein, the term "transcriptional regulatory sequence" refers to a first nucleotide sequence that regulates the transcription of a second nucleotide sequence to which it is functionally linked.

[0024] As used herein, a nucleotide sequence is "functionally linked" to a transcriptional regulatory sequence if the transcriptional regulatory sequence functions within the cell to regulate the transcription of the nucleotide sequence. This includes promoting the transcription of the nucleotide sequence through interactions between polymerases and promoters.

[0025] A "promoter" is a transcriptional regulatory sequence that facilitates the transcription of a DNA nucleotide sequence into an RNA transcript, at least sufficiently so. The transcript transcribed from a promoter typically includes the sequence from the promoter downstream of the transcription start site, and, in the case of mRNA, the downstream sequence encoding the amino acid sequence. Due to its predictable location just upstream of the transcription start site, the promoter is the most well-characterized transcriptional regulatory sequence. Promoters contain sequences that regulate the recognition, binding, and transcription initiation activity of RNA polymerase. These sequences can be cis-acting or trans-acting. Promoters can be constitutive or regulated, depending on their regulatory nature. They are often described as having two distinct segments: a core promoter region and an extended promoter region.

[0026] The core promoter contains a sequence sufficient for RNA polymerase recognition, binding, and transcription initiation. The core promoter includes the transcription initiation site, the RNA polymerase binding site, and other common transcription binding sites, where the pre-initiation complex is formed and the general transcription mechanism is assembled. The pre-initiation complex is generally located within 50 nucleotides (nt) of the transcription initiation site (TSS).

[0027] The core promoter also contains the sequence of the ribosome binding site necessary for translation from mRNA to polypeptide.

[0028] The extended promoter region includes the so-called proximal promoter, which extends approximately 250 nucleotides (i.e., -250 nt) upstream of the transcription start site. This contains primary regulatory elements, such as binding sites for specific transcription factors. Many genes are known to have transcriptional regulatory elements located even further upstream. In particular, fragments containing the majority of a gene's transcriptional regulatory elements can extend more than 700 nt upstream from the transcription start site. In some genes, transcriptional regulatory sequences have been found several thousand nucleotides upstream of the transcription start site.

[0029] As used herein, a first nucleotide sequence is "heterogeneous" with respect to a second nucleotide sequence if the first nucleotide sequence is not naturally bound to the second nucleotide sequence, for example, if they are not functionally linked. Conversely, a polypeptide is "heterogeneous" with respect to a transcriptional regulatory sequence if the polypeptide is encoded by a nucleotide sequence heterogeneous with respect to that transcriptional regulatory sequence.

[0030] As used in this specification, the term "allele variant" refers to a naturally occurring variation of a gene.

[0031] As used herein, the term "artificial variant" refers to a gene or protein that has undergone one or more genetic modifications to a gene or protein that exists in nature.

[0032] As used herein, the term “variation” generally refers to an alteration, variant, or polymorphism of a nucleotide sequence compared to the wild type. Such alterations, variants, or polymorphisms may be, for example, relative to a reference genome in a genome database. Variations include, but are not limited to, single nucleotide variations (SNVs), substitutions, insertions or deletions (collectively called “indels”), and repeats.

[0033] II. Introduction Novel strategies to reduce amplification artifacts and amplification bias arising from sequence-dependent hybridization rates may benefit from the use of shorter DNA primers than the current gold standard, hexamers. These strategies require obtaining Phi29 DNApol variants that recognize, stably bind to, and efficiently utilize these shorter DNA primers, potentially significantly improving current DNA amplification techniques.

[0034] The availability of the 3D structure of Phi29 DNApol, complexed with DNA and incoming nucleotides (Berman et al, 2007), allowed for a detailed examination of the amino acid residues directly involved in the interaction with the primer chain (Figure 1). The ligands for these primer chains (see scheme in Figure 2A) are as follows: • R96 (interacts with the phosphodiester between nucleotides 7 and 8 of the primer). • R306 (interacts with the phosphodiester between nucleotides 8 and 9 of the primer). • R308 (interacts with the phosphodiester between nucleotides 9 and 10 of the primer). • K498 (interacts with the sugar of the first 3' nucleotide of the primer). • Y500 (interacts with the phosphodiester between nucleotides 1 and 2 of the primer). • K529 (interacts with the phosphodiester between nucleotides 1 and 2 of the primer).

[0035] Based on these few contacts, Phi29 DNApol establishes a direct interaction that extends across the first 10 bases of the primer strand, suggesting that such a size gives the primer maximum binding stability. The absence of contact between nucleotides 3 and 6 is quite surprising. Surprisingly, current MDA techniques using Phi29 DNApol are based on the provision of random hexamers, which are only slightly stabilized by contact with a phosphodiester bond between the first two nucleotides and with a base at the 3' terminal nucleotide. For this reason, hexamers are not optimally sized to be used as initial primers for Phi29 DNApol to bind and extend. These suboptimal primers were very likely selected to have complements in any DNA sample at sufficiently short intervals to enable efficient and uniform amplification while minimizing self-hybridization artifacts known as primer dimers.

[0036] On the other hand, the alternative TruePrime DNA amplification technology (Picher et al, 2016) utilizes a DNA primase (TthPrimPol) to synthesize DNA primers on demand. However, it has not yet been established what the optimal primer size is for TthPrimPol to supply to Phi29 DNApol, nor what the fate of these primers remains shorter than the minimum size required for optimal extension by Phi29 DNApol.

[0037] Based on this information and warnings, the inventors explored the possibility of generating Phi29 DNApol variants (variants of the invention) with improved affinity for short primers, ideally within the range of 4–6 nucleotides. To this end, the inventors followed two different approaches: 1) enhancing some existing interactions, and 2) creating a novel (non-existent) enzyme:DNA ligand in the primer region.

[0038] Such improved variants are expected to be valuable in RP-based MDA methods, likely reducing primer dimer artifacts and the formation of amplified chimeras. Furthermore, in relation to TruePrime DNA amplification technology, using shorter primers that can be generated by TthPrimPol may lead to improved amplification efficiency and / or improved coverage.

[0039] In this study, detailed analysis of the 3D structure of Phi29 DNApol (Berman et al, 2007) allowed us to select five amino acid residues as candidate "gain of function" mutations. These residues are as follows: Lys64 (located in the ExoII motif), Met97 (adjacent to Arg96, the primer ligand of WT Phi29 DNApol), Thr499 (adjacent to Lys498 and Tyr500, two primer ligands of WT Phi29 DNApol), Thr534 and Lys538 (proximity to Lys529, the primer ligand of WT Phi29 DNApol). The mutations selected for these residues (summarized in Figure 2B) were as follows: • K64R leads to the acquisition of the phosphodiester interaction between residues 4 and 5 of the primer chain. • K64KG; K64KK; L63LG; L63LH are +1 insertion mutations flanking Lys64, designed to match the heterogeneity observed in different ExoII motifs of B-family DNA polymerases. These changes are also predicted to acquire interaction with primer chain residues 4 and 5. R96K is predicted to weaken the interaction with the phosphodiester bond between primer residues 7 and 8. • M97K leads to the acquisition of interaction between nucleotide 5 of the primer chain and the nitrogenous base. • M97R leads to the acquisition of interactions with amino acid residues 4 and 5 of the primer chain. • T499K leads to the acquisition of an interaction between amino acid residue 5 of the template chain and the sugar. • T499R leads to the acquisition of sugar interactions between amino acid residues 4 and 5 of the template chain. • K529R leads to the acquisition of a double interaction with the phosphodiester bond between residues 1 and 3 of the primer chain. • T534K leads to the acquisition of an interaction between amino acid residue 4 of the primer chain and the sugar. • T534R leads to the acquisition of the phosphodiester interaction between residues 3 and 4 of the primer chain. • K538R leads to the acquisition of a phosphodiester interaction between residues 2 and 3 of the primer chain.

[0040] The above mutants, designed to enhance the affinity of Phi29 DNApol to short primers, were expressed and purified according to a standard protocol for obtaining WT Phi29 DNApol. It is unpredictable whether the specific acquisition of primer-chain interaction resulting from the introduced mutations will negatively impact Phi29 DNApol function, such as translocation or processivity, or DNA amplification techniques with appropriate (TthPrimPol) and random primers.

[0041] III. Nucleic acids, expression constructs, recombinant cells, and mutant polymerase polypeptides A. Nucleic acid This specification provides nucleic acids having nucleotide sequences encoding a mutant Phi29 polymerase with improved primer recognition. The nucleotide sequence of wild-type Phi29 polymerase is shown in SEQ ID NO: 1. The nucleic acids encoding the mutant Phi29 polymerase sequence have one or both of the K64R and M97K mutations. In some embodiments, the nucleotide sequences encoding one or both of these mutations are substantially identical to the sequence in SEQ ID NO: 1.

[0042] B. Expression constructs Furthermore, this specification provides an expression construct comprising a transcriptional regulatory sequence functionally linked to a nucleotide sequence encoding the mutant Phi29 polymerase described herein. This expression construct may be in plasmid form or any other form suitable for expression in target cells.

[0043] C. Recombinant cells Furthermore, this specification provides recombinant cells containing the expression constructs described herein. In certain embodiments, such cells are bacterial cells. Such recombinant cells are useful for replicating the nucleic acid molecules of this disclosure and for producing the mutant Phi29 polymerase of this disclosure. The mutant Phi29 polymerase can be produced by culturing recombinant cells containing the expression constructs. The transcriptional regulatory sequences used may include constitutive promoters.

[0044] D. Mutant Phi29 polymerase Furthermore, this specification also provides a mutant Phi29 polymerase with improved primer recognition. The mutant Phi29 polymerase of this disclosure has an amino acid sequence substantially identical to that of SEQ ID NO: 1 (also deposited as UniProtKB - P03680) and includes one or both of the amino acid substitutions K64R and M97K.

[0045] Polymerases having substantially identical amino acid sequences can be based on naturally occurring sequences, such as allele variants, provided they contain one or both of the amino acid substitutions K64R and M97K. Such variants may have at most 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions, or deletions compared to the wild-type sequence SEQ ID NO: 1, provided that either or both of the amino acid substitutions K64R and M97K are present.

[0046] Preferably, the amino acid sequence of the DNA polymerase of the present invention has at least 80% identity with SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. More preferably, the amino acid sequence of the polymerase of the present invention has at least 90% identity with SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Even more preferably, the amino acid sequence of the polymerase of the present invention is SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0047] IV. How to use This specification provides a method for primer extension and / or nucleic acid polymerization using the mutant Phi29 polymerase described herein. The primer extension method is useful for nucleic acid replication, amplification, and sequencing.

[0048] Primer extension involves hybridization of the primer to a nucleic acid molecular template, followed by a polymerization reaction catalyzed by polymerase, which adds a nucleotide to the 3' end of the primer. Primers can be added exogenously to the reaction mixture or generated by a primase / polymerase. A primase is an enzyme that catalyzes the synthesis of oligonucleotides called primers, which are complementary to the nucleic acid template. One such primase is, for example, TthPrimPol.

[0049] Synthetic primers are commonly used for nucleic acid amplification. Such primers are typically about 6 to 25 nucleotides in length. When attempting to amplify a specific sequence, the primers can have sequences complementary to the target sequence. For whole-genome amplification or other nondirected amplification methodologies, random primers may be used. Random primers typically consist of a collection or set of oligonucleotides, where each position of the oligonucleotide in one or more primers within the set contains a given base. In certain situations, one or more positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) can be filled with a predetermined base or a combination of two or three bases.

[0050] A. Amplification Nucleic acid amplification, such as polymerase chain reaction (PCR) introduced by Mullis (US 5,656,493), is an essential technique used in medical and biological research. It has been successfully used in a variety of applications, including nucleic acid cloning, manipulation, or sequencing, DNA-based functional and phylogenetic analysis of genes, disease detection and diagnosis, and even forensic science and paternity testing.

[0051] B. Rolling Circle Amplification Rolling circle amplification is a method for amplifying covalently bound DNA molecules, such as single-stranded covalently bound ring-bound DNA molecules. The template DNA molecule is primed with primers, such as those provided by a primase / polymerase. The DNA polymerase performs primer extension along the ring-bound DNA molecule relative to the primers. The polymerase then replaces the hybridized copy and continues the extension of polynucleotides along the template, producing a concatenated amplification product.

[0052] C. Multiple substitution amplification (MDA) Multiple Displacement Amplification (MDA) is a non-PCR-based isothermal DNA amplification method; in this method, ssDNA strands are generated by priming and extension from a template, and these are successively reprimed and copied by strand displacement synthesis to generate a highly branched DNA structure. Because, after the initial denaturation of a double-stranded DNA sample, DNA synthesis can be successively primed and extended from many locations on the amplified molecule without requiring further denaturation rounds, MDA results in highly branched structures. As new primers are extended from a single DNA molecule template to branched regions, the branched strands are replaced by each other. MDA is also described, for example, in WO2011 / 047307A1 ("Multiple Displacement Amplification"), published on April 21, 2011. MDA can be briefly described as: "Isothermal polymerization extending primers at multiple priming sites on a self-generated ssDNA template."

[0053] In certain embodiments, the MDA utilizes random trimers, tetramers, pentamers, hexamers, heptamers, or octamers as primers to facilitate amplification at multiple sites on the initial template and its amplified copy. In certain embodiments of the disclosed method, priming is achieved using a DNA primase / polymerase such as TthPrimPol.

[0054] In certain embodiments, amplification of double-stranded linear polynucleotides involves using: 1) randomly synthesized primers and / or DNA-dependent primase / polymerase, e.g., TthPrimPol; 2) a modified DNA polymerase having strand-displacement activity, e.g., Phi29 DNApol; 3) dNTPs. In certain embodiments, the dNTP substrate is unmodified. In other embodiments, dNTPs may be modified by the attachment of labeling groups, such as fluorescent molecules. As used herein, “labeling” refers to a chemical portion attached to a molecule, such as a nucleic acid molecule. Detectable labels include, for example, fluorescent labels, luminescent labels, enzymatic labels, colorimetric labels such as colloidal gold or colored glass or plastic beads, and radioactive labels. These three types of reagents, in combination, facilitate multiple substitution amplification (MDA) of a given DNA, which is multiple times primed by either randomly synthesized primers or primase / polymerase and extended by DNA polymerase. Furthermore, combinations of randomly synthesized primers and / or primases / polymerases with DNA polymerases can perform multi-strand substitution amplification via priming of the amplified molecule with primases / polymerases and / or random oligonucleotide primers, and primer extension by DNA polymerases.

[0055] 1. DNA polymerases with strand displacement activity Amplification methods such as MDA can use DNA polymerases with strand displacement activity, such as polymerases that show strong binding to single-stranded DNA rather than double-stranded DNA. Strand displacement activity can be useful for replacing hybridized strands of DNA molecules while extending the primer position.

[0056] Phi29 DNApol is an example of a DNA polymerase with useful strand displacement activity in the methods disclosed herein. Phi29 DNApol is commercially available from, for example, New England Biolabs (Ipswich, MA, USA), ThermoFisher Scientific (Waltham, MA, USA), and Expedeon (Cambridge, UK). Phi29 DNApol possesses both inherently high processability and strand displacement ability in conjunction with DNA polymerization, enabling the generation of DNA fragments longer than 70 kb from a single enzyme:DNA binding event (Blanco et al., 1989). This potential allows Phi29 DNApol to replicate DNA templates containing secondary structures such as hairpin loops. This enzyme also possesses 3'→5' exonuclease proofreading activity (Blanco and Salas, 1985; Garmendia et al., 1992), resulting in up to 1000 times higher fidelity compared to methods based on Taq DNA polymerase.

[0057] 2. Deoxyribonucleoside triphosphate The creation and extension of primers can be achieved simply by providing deoxyribonucleotide substrates, e.g., dNTPs, through a combination of a specialized DNA primase / polymerase capable of synthesizing DNA primers, such as TthPrimPol (Picher et al, 2016), and an elongating DNA polymerase, such as Phi29 DNApol. Generally, these contain four standard bases: A, T, G, and C. However, in certain embodiments, non-natural nucleotides such as inosine may also be included. In certain embodiments, the nucleotides may have labels for detecting or capturing the polynucleotides into which they are incorporated.

[0058] D. DNA sequencing As of today, many different sequencing techniques exist, which are generally classified into "first-generation sequencing," "second-generation sequencing" (often called "next-generation sequencing" or NGS), and "third-generation sequencing" (also known as single-molecule sequencing (SMS)). First-generation sequencing mainly refers to the methods of Maxam and Gilbert (1977) or Sanger (Sanger et al., 1977; Sanger and Coulson, 1978), but today only the latter is used.

[0059] Second-generation, or next-generation, sequencing refers to techniques that use advanced technical (optical) detection methods for base positions to obtain many sequences simultaneously. An overview of existing methods is described in (Metzker, 2010).

[0060] Third-generation or single-molecule sequencing (SMS) techniques do not require prior amplification, the template is a single molecule rather than a clone or ensemble of DNA, and its sequence is often copied / read in "real time" as a result of polymerase activity and recorded online (Sam et al, 2011; Thompson and Milos, 2011).

[0061] As used herein, "high-throughput sequencing" refers to the simultaneous or near-simultaneous sequencing of thousands of nucleic acid molecules. High-throughput sequencing platforms include, but are not limited to, MPSS (massively parallel signature sequencing), Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing (Complete Genomics / BGI Shenzhen), Heliscope single-molecule sequencing, single-molecule real-time sequencing (SMRT) (PacBio), and nanopore DNA sequencing (e.g., Oxford Nanopore).

[0062] The methods described herein can be used for whole-genome sequencing, exome sequencing, and amplicon sequencing, but are not limited to these. However, the amplified molecules themselves may be amplified by specific amplicons. The amplified molecules corresponding to exomes can be isolated using sequence capture with a bait for gene sequences in the genome. The amplified transcriptome can be prepared for sequencing by reverse transcription of mRNA into double-stranded cDNA.

[0063] V. Kit Furthermore, this specification also provides kits for use when carrying out the methods described herein. As used herein, the term "kit" refers to a set of items intended to be used together.

[0064] Certain kits disclosed herein include 2, 3, 4, 5, 6, or 7 elements selected from the following: (1) PrimPol enzyme (e.g., TthPrimPol); (2) DNA polymerase (e.g., Phi29 DNApol); (3) random trimer; (4) random tetramer; (5) random pentamer; (6) random heptamer; (7) random octamer; (8) random primer; (9) dNTP; (10) reaction buffer; (11) buffer for use with any of the elements. The kit may include a container for holding the reagents. The container may be placed in a transport container. The container may be delivered by hand or by a general carrier such as the national postal system or a delivery service such as FedEx. The kit may also include a container, e.g., a box or bag, for transporting the collected blood to a central facility. The kit may also typically include instructions for use and software for data analysis and interpretation.

[0065] Exemplary aspects 1. Phi29 type DNA polymerase containing either or both of the K64R or M97K mutations. 2. A Phi29 type DNA polymerase having an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 3. A method for replicating, amplifying, or sequencing template DNA, wherein the DNA is at least (a) DNA polymerase according to embodiment 1 or 2, (b) buffer solution; (c) Magnesium chloride, (d) Primer, and (e) Nucleoside triphosphate The method comprising the step of contacting a reaction mixture containing the following. 4. A kit for carrying out the method described in Embodiment 3, comprising (a) the DNA polymerase described in Embodiment 1 or 2, (b) a buffer, and (c) magnesium chloride. 5. A kit for carrying out the method of embodiment 3, comprising the DNA polymerase described in embodiment 1 or 2, (a) PrimPol enzyme (e.g., TthPrimPol), (b) Random trimmer, (c) Random Tetramer, (d) Random pentamers, (e) random heptamers, (f) Random octamer, (g)dNTP, (h) Reaction buffer, (i) Buffer for use with any of the above elements The kit includes one or more of the following. 6. A Phi29 type DNA polymerase having an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1, and containing one or both of the amino acid substitutions K64R and M97K. 7. The Phi29 type DNA polymerase according to embodiment 6, having the sequence SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 8. Phi29 type DNA polymerase according to embodiment 6, having 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or one or fewer amino acid substitutions, additions, or deletions in addition to one amino acid substitutions K64R and M97K. 9. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a Phi29 type DNA polymerase, wherein the Phi29 type DNA polymerase has an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1, and the Phi29 type DNA polymerase comprises one or both of the amino acid substitutions K64R and M97K. 10. The isolated nucleic acid molecule according to embodiment 9, wherein the Phi29 type DNA polymerase has the sequence SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 11. The isolated nucleic acid molecule according to embodiment 9, wherein the Phi29 type DNA polymerase has 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or one or fewer amino acid substitutions, additions, or deletions in addition to one amino acid substitutions K64R and M97K. 12. Recombinant nucleic acid comprising a transcriptional regulatory sequence functionally linked to a Phi29-type DNA polymerase as described in any of embodiments 9 to 11. 13. The recombinant nucleic acid according to embodiment 12, wherein the transcriptional regulatory sequence includes a bacterial or mammalian promoter. 14. Recombinant nucleic acids according to embodiment 12, contained in a vector selected from plasmid vectors, viral vectors, cosmids, and transposons. 15. Recombinant nucleic acid according to embodiment 14, comprising a cloning site positioned relative to a nucleotide sequence encoding Phi29 DNA polymerase such that a transcriptional regulatory sequence inserted into the cloning site is functionally linked to the nucleotide sequence encoding Phi29 DNA polymerase. 16. Recombinant cells comprising recombinant nucleic acid as described in any of embodiments 12 to 15. 17. (a) A step of contacting a nucleic acid template molecule with a Phi29 type DNA polymerase and a reagent sufficient for primer extension, as described in any of embodiments 1, 2 and 6-8; and (b) The step of extending the primer using the nucleic acid template with the polymerase. Methods that include... 18. The method according to embodiment 17, wherein the reagent sufficient for primer extension includes an oligonucleotide primer. 19. The method according to embodiment 18, wherein the oligonucleotide primer comprises one or more of trimers, tetramers, pentamers, hexamers, octamers, nonamers, or 10mers. 20. The method according to embodiment 19, wherein the primer is a random primer. 21. The method according to embodiment 18, wherein the oligonucleotide primer has a link of 5 to 25 nucleotides. 22. The method according to embodiment 17, wherein the reagent sufficient for primer extension includes a primase / polymerase (e.g., TthPrimPol). 23. The method according to embodiment 17, wherein the primer extension is performed at any of the following temperatures: approximately 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C, or at a temperature above 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C. 24. The method according to embodiment 17, wherein the template nucleic acid molecule is present in an amount of 1 ng, 100 pg, 10 pg, or 1 pg or less. 25. The method according to embodiment 17, wherein the primer extension includes (1) multiple substitution amplification ("MDA") or (2) rolling circle amplification. 26. The method according to embodiment 17, wherein the primer extension includes a multiple annealing and looping-based amplification cycle (MALBAC). 27. The method according to any one of embodiments 17 to 26, wherein the Phi29 type DNA polymerase contains both the substitution K64R and M97K. [Examples]

[0066] Example 1: Screening to detect which mutants can use shorter randomly synthesized primers compared to WT Phi29 DNApol in multiple substitution amplification reactions. Figure 3 shows the amplification of 1 ng of human genomic DNA by multiple substitution amplification (MDA) using the Phi29 DNApol variant combined with TthPrimPol or randomly synthesized primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)).

[0067] As shown in Figure 3, WT Phi29 DNApol efficiently utilized pentamers, hexamers, heptamers, and octamers, as well as TthPrimPol, to amplify human genomic DNA. Trimers and tetramers were not suitable for amplification.

[0068] Of the group of Phi29 DNApol variants created, six (K538R, T534K, T534R, L63LH, K64KG, and K64KK) were completely inactive in MDA, regardless of primer size or the use of alternative TthPrimPol. Another set of mutants (K529R, M97R, R96K, L63LG, and T499K) exhibited inferior amplification performance compared to WT Phi29 DNApol, demonstrating lower amplification yields and / or limitations in using specific primer sizes. For example, mutant M97R was able to efficiently use pentamers and hexamers, but heptamers and octamers did not induce amplification. Similarly, mutant R96K was only able to use hexamers from a randomly synthesized set of primers. Surprisingly, the insertion mutant L63LG was able to amplify DNA with pentamers, hexamers, heptamers, and octamers, but the combination with TthPrimPol did not result in any amplification product. Conversely, the mutant T499K was able to slightly amplify DNA in the presence of TthPrimPol, but none of the randomly synthesized primers promoted MDA.

[0069] The mutant T499R exhibited behavior very similar to that of WT Phi29 DNApol.

[0070] Finally, the mutants K64R and M97K showed significant improvement compared to WT Phi29 DNApol. Both mutants were the only ones that could produce a tetramer, while WT Phi29 DNApol and the remaining mutants did not show amplification yield.

[0071] The two "gain-of-function" mutations described above were introduced into the same polypeptide to create the dual mutant K64R / M97K; this was thoroughly characterized in comparison to the WT Phi29 DNApol and the single mutants K64R and M97K, as shown in the following examples.

[0072] Example 2: In the Phi29 DNApol mutant M97K and the double mutant K64R / M97K, polymerase activity is dominant over exonuclease activity. Figure 4 shows the dynamic equilibrium analysis of the 3'-5' exonuclease activity and 5'-3' polymerization activity of the optimal mutants of the present invention (K64R, M97K, and the double mutant K64R / M97K) against WT Phi29 DNApol. 5'-labeled primers TIFF0007864893000001.tif4128 is the template Using DNA double helix formed by hybridizing to TIFF0007864893000002.tif4128, DNA synthesis and degradation coupling were analyzed as a function of dNTP concentrations (0, 10, 25, 50, 100, and 500 nM). In the absence of dNTPs, exonuclease degradation of the primer ends was observed. This degradation pattern reflects the level of exonuclease activity of the variants of the invention against WT Phi29 DNApol. As the dNTP concentration increases, 5'-3' polymerization gradually surpasses the exonuclease activity; net dNMP incorporation is observed as an increase in the size of the labeled primer, specifying the dNTP concentration required to obtain efficient primer extension for each variant. As seen in Figure 4, the variant K64R exhibited a Pol / Exo equilibrium nearly identical to that shown by the WT enzyme, reaching the 28-mer position at 25 nM dNTPs. On the other hand, the mutant M97K and the double mutant K64R / M97K reached the same position (28mer) at the lowest dNTP concentration tested (10nM), indicating that the polymerase activity of these mutants is dominant over that of the exonuclease.

[0073] Example 3: The mutants of the present invention (K64R, M97K, and K64R / M97K) can use shorter randomly synthesized primers in multiple substitution amplification reactions compared to WT Phi29 DNApol. Figure 5 shows the amplification of 1 ng of human genomic DNA by multiple substitution amplification (MDA) using selected Phi29 DNApol variants (K64R, M97K, and the double mutant K64R / M97K) combined with randomly synthesized primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)). The amplification yields shown are the mean values ​​from two independent experiments, each containing three replicates. The standard deviations from the two experiments are shown.

[0074] As shown in Figure 5, none of the enzymes tested were able to efficiently amplify genomic DNA using random synthesis trimmers. Only the double mutant K64R / M97K showed a yield close to 1 μg.

[0075] The three variants of the present invention were able to induce amplification using tetramers, but no amplification was observed in WT Phi29 DNApol. Variant K64R showed the lowest amplification yield (2.7 μg), mutant M97K showed a slightly higher yield (3.8 μg), and the double mutant K64R / M97K showed a much higher yield (12.9 μg). The highest yield observed in the double mutant represents a synergistic effect of both mutations present in the same polypeptide.

[0076] WT Phi29 DNApol and the three variants of the present invention were able to efficiently utilize random pentamers to initiate amplification. In this case as well, the double mutant K64R / M97K yielded the highest yield (over 20 μg of amplified DNA), clearly outperforming the performance of single variants and the WT enzyme.

[0077] A similar comparison pattern was observed with random hexamers, but in both cases, the amplification yield was higher.

[0078] Using random heptamers, WT Phi29 DNApol maintained the same yield as obtained with hexamers, but the three variants of the present invention tended to reduce amplification efficiency, resulting in DNA levels similar to those obtained with random pentamers.

[0079] In the case of octamers, both single mutants K64R and M97K showed lower amplification yields than WT Phi29 DNApol. On the other hand, the double mutant K64R / M97K clearly outperformed WT Phi29 DNApol, as occurred under all conditions tested, and demonstrated robust and efficient amplification values ​​regardless of the length of the randomly synthesized primers used to initiate amplification.

[0080] Example 4: Effect of ionic intensity on background amplification observed in the absence of input DNA in the non-template control (NTC). Figure 6 shows the amplification yields observed in the absence of input DNA when WT Phi29 DNApol or selected variants of the present invention (K64R, M97K, and K64R / M97K) are combined with randomly synthesized primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)).

[0081] Under the tested low ionic strength conditions (20 mM KCl; 57 mM NaCl), both the M97K single mutant and the K64R / M97K double mutant showed significant amplification yields in the absence of input DNA when using pentamers and hexamers, and in the case of the double mutant, also significant amplification yields when using tetramers (see Figure 6). However, the amplification yields were significantly lower than those obtained when using DNA (1 ng) as input under the same conditions (see Figure 5), suggesting the involvement of different amplification mechanisms. In this field, the primer dimer amplification ability of Phi29 DNApol in the absence of input DNA is well known (Alsmadi et al, 2009), and it is possible that the stability of primer dimers is enhanced under the tested conditions in the M97K single mutant and the K64R / M97K double mutant.

[0082] Figure 7 shows the amplification yield observed when ionic strength conditions were increased by adding ammonium sulfate [(NH4)2SO4] in the absence of input DNA. In the presence of ammonium sulfate (45 mM), the amplification levels observed in the absence of input DNA completely disappear for all variants and primer sizes.

[0083] Example 5: High ionic intensity conditions enhance the robustness and efficiency of the double mutant K64R / M97K for DNA amplification using random primers of different lengths. Figure 8 shows the amplification of 1 ng of human genomic DNA by multiple substitution amplification (MDA) under high ionic strength conditions (20 mM KCl; 57 mM NaCl; 45 mM (NH4)2SO4) using WT Phi29 DNApol or selected variants of the present invention (K64R, M97K, and K64R / M97K) combined with randomly synthesized primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)). The amplification yields shown are the mean values ​​from two independent experiments, each containing three repeats. The standard deviations from the two experiments are shown.

[0084] As shown in Figure 8, none of the enzymes tested were able to efficiently amplify genomic DNA using random synthesis trimmers. Only the double mutant K64R / M97K showed a yield close to 600 ng.

[0085] In contrast to those observed under previous conditions (see Figure 5), the tetramer was efficiently utilized only by the M97K mono-mutant and the K64R / M97K double mutant, while the mono-mutant K64R yielded only a small yield of nearly 1 μg. Notably, the amplified yields observed in the M97K mono-mutant and the K64R / M97K double mutant were increased compared to those obtained in the absence of ammonium sulfate (from 4 μg to 12 μg and from 13 μg to 16 μg, respectively).

[0086] For pentamers and hexamers, the M97K single mutant and the K64R / M97K double mutant showed similar results, clearly exceeding the amplification yields obtained with the WT enzyme or the K64R variant. As demonstrated in the absence of ammonium sulfate, the WT Phi29 DNApol showed a higher yield than the K64R variant.

[0087] In the case of heptamers, only the double mutant K64R / M97K maintained the amplification yield obtained with shorter randomly synthesized primers and / or in the absence of ammonium sulfate. Both WT Phi29 DNApol and the K64R variant showed a significant decrease in yield, exhibiting the same values ​​under these conditions. The yield obtained with the M97K mutant was also decreased compared to the previous conditions.

[0088] Finally, the octamer was efficiently developed only by the double mutant K64R / M97K, while the other three enzymes showed very low amplification yields.

[0089] The Phi29 DNApol double mutant K64R / M97K retains amplification performance under both low and high ionic intensity conditions, which may be a result of gain-of-function obtained through additional contact between the enzyme and the nitrogen base from nucleotide 5 of the primer and the phosphodiester bond between nucleotides 4 and 5 (see Figure 2). These additional contacts enable the enzyme to successfully stabilize primers of different sizes under different ionic intensity conditions.

[0090] Example 6: The double mutant K64R / M97K provides highly sensitive amplification of minute amounts of DNA, regardless of the primer size tested. Figure 9 shows the amplification of different amounts of human genomic DNA (1, 10, 100 pg, and 1 ng) by multiple substitution amplification (MDA) combining WT Phi29 DNApol or selected variants of the present invention (K64R, M97K, and K64R / M97K) with randomly synthesized primers of different sizes (tetramer (4N), pentamer (5N), or hexamer (6N)) under low ionic intensity (20 mM KCl; 57 mM NaCl) or high ionic intensity (20 mM KCl; 57 mM NaCl; 45 mM (NH4)2SO4).

[0091] Under low ionic intensity conditions (Figure 9, top panel), the dual mutant K64R / M97K yielded the most consistent and highest amplification yield across all conditions tested.

[0092] In the case of randomly synthesized tetramers, as previously shown (see Figure 5), WT Phi29 DNApol was unable to amplify any of the DNA inputs tested. The K64R variant yielded a detectable yield only with 1 ng of DNA input and lacked sensitivity to amplify smaller amounts of DNA. On the other hand, both the M97K and M97K / K64R mutants efficiently amplified the DNA inputs tested, and the dual mutants yielded higher yields in all cases.

[0093] In the case of randomly synthesized pentamers, all enzymes were able to utilize them to initiate amplification, but exhibited different levels of sensitivity and efficiency. WT Phi29 DNApol showed a significant decrease in amplification yield as the amount of DNA input decreased, while the three variants of the present invention maintained moderate efficiency under all tested conditions. The double mutant M97K / K64R exhibited the highest amplification efficiency among the three variants of the present invention, regardless of the amount of DNA input, and therefore showed the best sensitivity.

[0094] In the case of randomly synthesized hexamers, all enzymes were able to efficiently utilize them to initiate amplification of each DNA input tested, showing significant amplification yields in all cases. The three variants of the present invention outperformed WT Phi29 DNApol, showing higher amplification yields when analyzing small amounts of DNA input. Similar to the pentamer, the double mutant M97K / K64R showed the highest amplification efficiency among the three variants of the present invention, regardless of the amount of DNA input.

[0095] As previously shown (see Figure 6), both the M97K variant and the M97K / K64R variant showed significant amplification yields in the absence of input DNA (template-free control: NTC) when using pentamers and hexamers. For this reason, the same sensitivity analysis was performed under high ionic strength conditions (20 mM KCl; 57 mM NaCl; 45 mM (NH4)2SO4) to prevent the influence of this artifact caused by primer dimer amplification.

[0096] Under high ionic strength conditions (see Figure 9, bottom panel), randomly synthesized tetramers showed similar usage patterns among the tested variants compared to low ionic strength conditions, but with increased amplification yields in most cases. The exception to this rule was variant M97K, which showed lower yields with 1 pg and 10 pg DNA inputs.

[0097] Under high ionic strength conditions for randomly synthesized pentamers, the M97K variant and the M97K / K64R variant showed the best performance in terms of sensitivity and efficiency, and demonstrated higher amplification yields compared to low ionic strength conditions with the same amount of DNA input. Increasing the ionic strength of the reaction reduced the amplification efficiency of the K64R variant when testing limited amounts of DNA (1 pg and 10 pg), but the efficiency was similar (100 pg) or higher (1 ng) for the other two input amounts. Surprisingly, under all of these conditions, WT Phi29 DNApol outperformed the K64R variant.

[0098] Under high ionic strength conditions for randomly synthesized hexamers, the dual mutant K64R / M97K was the only variant that increased the observed yield at all DNA inputs compared to the results obtained under low ionic strength conditions. The single mutant M97K showed lower yields at the smallest inputs (1 pg and 10 pg), but increased yields at 100 pg and 1 ng of DNA input; this indicates a decrease in sensitivity. The variants K64R and WT Phi29 DNApol exhibited similar behavior. As with pentamers, under these conditions, WT Phi29 DNApol yielded higher amplification yields than the K64R variant in all cases.

[0099] In summary, the double mutant K64R / M97K demonstrated the best performance in terms of amplification efficiency and sensitivity under both low and high ionic intensity conditions in amplification reactions using all tested DNA primers.

[0100] Example 7: Amplification efficiency and sensitivity are not altered by using the variant of the present invention when the primers are generated by TthPrimPol. Figure 10 shows the amplification of 1, 10, 100 pg, and 1 ng of human genomic DNA by multiple substitution amplification (MDA) combining WT Phi29 DNApol or selected variants of the present invention (K64R, M97K, and K64R / M97K) with the DNA primase TthPrimPol (Picher et al, 2016), which can synthesize primers for Phi29 DNApol during the reaction.

[0101] As shown in Figure 10, no significant difference in yield was observed between WT Phi29 DNA pol and the variant of the present invention tested, resulting in similar levels of sensitivity and efficiency under this setup.

[0102] Example 8: Selected variants of the present invention (K64R, M97K, and K64R / M97K) improve amplification coverage measured by CovCheck technology. The CovCheck technology enables coverage analysis of whole-genome amplification using a PCR panel containing 24 different primer pairs that amplify small portions from each human chromosome. The CovCheck technology has been validated by comparing CovCheck coverage values ​​with actual coverage obtained from low-pass whole-genome sequencing, and excellent correlation values ​​have been obtained. (https: / / www.expedeon.com / products / genomics / dna-rna-products / covcheck-pcr-kits / ).

[0103] To analyze the amplification coverage obtained for each variant, a limited amount of input material was selected: 30 pg of human genomic DNA. This amount of DNA is equivalent to five times the amount of a human diploid genome and may be the minimum amount to ensure that a sufficient number of copies of each chromosome are available for amplification. Below this level, due to the random distribution of molecules in the purified DNA sample, specific regions or complete chromosomes may not be present in the amplification input, resulting in uncovered regions in the amplification product not due to amplification failure, but due to the absence of the template.

[0104] Figure 11 shows the estimated coverage values ​​obtained from CovCheck analysis of amplification reactions using hexamers, pentamers, and tetramers in combination with WT Phi29 DNApol or selected variants of the present invention and 30 pg of human genomic DNA input. The coverage values ​​are the average of 6 independent reactions per condition.

[0105] In the case of randomly synthesized hexamers, amplification coverage is improved when using the three variants of the present invention compared to the coverage values ​​obtained with WT Phi29 DNApol.

[0106] In the case of randomly synthesized pentamers, all enzymes showed coverage values ​​of over 90% under these conditions. Therefore, no significant difference was observed. However, the M97K variant stood out, showing perfect coverage in the six replicates tested.

[0107] In the case of randomly synthesized tetramers, only the M97K variant and the M97K / K64R variant yielded amplified DNA; this is consistent with the amplification sensitivity shown by the WT Phi29 DNApol and K64R variant when combined with tetramers (Figure 9). CovCheck analysis revealed excellent amplification coverage (99%) in both cases; this suggests the advantage of using the variants of the present invention in combination with the shortest possible primers to maximize amplification coverage and uniformity and prevent amplification bias and sequence loss.

[0108] When using the enzyme (TthPrimPol) to create DNA primers for Phi29 DNApol, the estimated coverage values ​​obtained from CovCheck analysis of amplification reactions performed using 30 pg (equivalent to 5 genomes) of human genomic DNA as input to the amplification reaction, with TthPrimPol combined with WT Phi29 DNApol or the variant of the present invention, are shown in Figure 12. The coverage values ​​are the average of 12 independent reactions per condition. The CovCheck analysis also reveals improved amplification coverage when using the variant of the present invention, supporting their advantages in increasing the uniformity of the amplified product relative to the original DNA input.

[0109] References (incorporated herein by reference) TIFF0007864893000003.tif134158TIFF0007864893000004.tif239159TIFF0007864893000005.tif95159

[0110] As used herein, unless otherwise specified, the following meanings apply: The term “may” is used in a permissive sense (i.e., having the potential to) rather than a compulsory sense (i.e., having the potential to). Terms such as “include,” “including,” and “includes” mean “including, but not limited to.” The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, a reference to “an element” includes combinations of two or more elements, despite the use of other terms and phrases for one or more elements, such as “one or more.” The term “or” is non-exclusive unless otherwise specified, i.e., encompasses both “and” and “or.” The term “any of” between a modifier and a sequence means that the modifier modifies each member of the sequence. Thus, for example, the phrase "at least any of 1, 2, or 3" means "at least one, at least two, or at least three." In certain embodiments, an invention that "comprises" various elements may also "consist essentially of" these elements. The term "consisting essentially of" means that it includes the enumerated elements and other elements that do not substantially affect the basic and novel characteristics of the claimed combination.

[0111] The description and drawings herein are not intended to limit the invention to any particular form disclosed; rather, it should be understood that the invention should cover all modifications, equivalents, and substitutes within the spirit and scope of the invention as defined in the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in consideration of this description. Accordingly, this description and drawings should be interpreted as illustrative only and are intended to teach those skilled in the art a general way of carrying out the invention. It should be understood that the forms of the invention shown and described herein should be considered examples of various embodiments. Substitutions of elements and materials exemplified and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be used independently, all of which will be apparent to those skilled in the art after benefiting from the description of the invention. Modifications to the elements described herein may be made without departing from the spirit and scope of the invention as set forth in the appended claims. The headings used herein are for organizational purposes only and are not intended to limit the scope of the description.

[0112] All publications, patents, and patent applications described herein are incorporated herein by reference to the same extent as any individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0113] Sequence List Italicized amino acids are not expressed in some forms. SEQ ID NO 1: Wild-type Phi29 DNA polymerase (UniProtKB - P03680) TIFF0007864893000006.tif215138TIFF0007864893000007.tif240140TIFF0007864893000008.tif47138SEQ ID NO 2: K64R Phi29 DNApol variant TIFF0007864893000009.tif175138TIFF0007864893000010.tif240138TIFF0007864893000011.tif87140SEQ ID NO 3: M97K Phi29 DNApol variant TIFF0007864893000012.tif136138TIFF0007864893000013.tif240138TIFF0007864893000014.tif126140SEQ ID NO 4: K64R / M97K Phi29 DNApol double mutant TIFF0007864893000015.tif87138TIFF0007864893000016.tif245138TIFF0007864893000017.tif166140

[0114] Sequence information SEQUENCE LISTING <110> 4BASEBIO SL <120> PHI29 DNA POLYMERASE MUTANTS WITH IMPROVED PRIMER RECOGNITION <150> US 62 / 849,252 <151> 2019-05-17 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 575 <212> PRT <213> Bacillus subtilis <220> <223> Phi29DNApol wild type <400> 1 Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr 1 5 10 15 Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25 30 Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met 35 40 45 Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asn Leu Lys 50 55 60 Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys 65 70 75 80 Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95 Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110 Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125 Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140 Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro 145 150 155 160 Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170 175 Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser 180 185 190 Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205 Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220 Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys 225 230 235 240 Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255 Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270 Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285 Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295 300 Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly 305 310 315 320 Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335 Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350 Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365 Tyr Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380 Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr 385 390 395 400 Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410 415 Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe 420 425 430 Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445 Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460 Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu 465 470 475 480 Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495 Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510 Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525 Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535 540 Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln 545 550 555 560 Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 565 570 575 <210> 2 <211> 575 <212> PRT <213> Artificial Sequence <220> <223> K64R Phi29DNApol mutant <400> 2 Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr 1 5 10 15 Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25 30 Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met 35 40 45 Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asn Leu Arg 50 55 60 Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys 65 70 75 80 Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95 Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110 Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125 Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140 Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro 145 150 155 160 Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170 175 Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser 180 185 190 Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205 Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220 Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys 225 230 235 240 Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255 Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270 Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285 Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295 300 Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly 305 310 315 320 Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335 Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350 Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365 Tyr Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380 Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr 385 390 395 400 Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410 415 Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe 420 425 430 Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445 Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460 Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu 465 470 475 480 Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495 Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510 Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525 Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535 540 Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln 545 550 555 560 Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 565 570 575 <210> 3 <211> 575 <212> PRT <213> Artificial Sequence <220> <223> M97K Phi29DNApol mutant <400> 3 Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr 1 5 10 15 Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25 30 Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met 35 40 45 Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asn Leu Lys 50 55 60 Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys 65 70 75 80 Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95 Lys Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110 Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125 Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140 Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro 145 150 155 160 Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170 175 Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser 180 185 190 Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205 Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220 Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys 225 230 235 240 Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255 Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270 Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285 Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295 300 Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly 305 310 315 320 Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335 Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350 Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365 Tyr Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380 Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr 385 390 395 400 Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410 415 Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe 420 425 430 Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445 Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460 Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu 465 470 475 480 Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495 Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510 Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525 Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535 540 Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln 545 550 555 560 Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 565 570 575 <210> 4 <211> 575 <212> PRT <213> Artificial Sequence <220> <223> K64R / M97K Phi29DNApol double mutant <400> 4 Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr 1 5 10 15 Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25 30 Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met 35 40 45 Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asn Leu Arg 50 55 60 Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys 65 70 75 80 Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95 Lys Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110 Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125 Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140 Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro 145 150 155 160 Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170 175 Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser 180 185 190 Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205 Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220 Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys 225 230 235 240 Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255 Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270 Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285 Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295 300 Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly 305 310 315 320 Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335 Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350 Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365 Tyr Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380 Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr 385 390 395 400 Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410 415 Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe 420 425 430 Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445 Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460 Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu 465 470 475 480 Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495 Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510 Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525 Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535 540 Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln 545 550 555 560 Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 565 570 575 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> 5'-labelled primer of DNA duplex <400> 5 gatcacagtg agtac 15 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> template of DNA duplex <400> 6 agaagtgtat ctggtactca ctgtgatc 28

Claims

1. A Phi29 type DNA polymerase having an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 1, and containing an amino acid substitution K64R numbered with respect to SEQ ID NO: 1, (a) The Phi29 type DNA polymerase has improved affinity for short primers compared to wild-type Phi29 polymerase having SEQ ID NO: 1, the short primers being 4-6 nucleotides, and the comparison is performed under the same conditions, or (b) The Phi29-type DNA polymerase is a Phi29-type DNA polymerase with improved primer recognition compared to a wild-type Phi29 polymerase having SEQ ID NO: 1, and the comparison is performed under the same conditions. The aforementioned Phi29 type DNA polymerase.

2. The Phi29 type DNA polymerase according to claim 1, comprising both amino acid substitutions K64R and M97K numbered with respect to SEQ ID NO:

1.

3. The Phi29 type DNA polymerase according to claim 1 or 2, having a sequence with SEQ ID NO: 2 or SEQ ID NO: 4, or having 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or one or fewer amino acid substitutions, additions, or deletions in addition to the amino acid substitution K64R numbered with respect to SEQ ID NO:

1.

4. The Phi29 type DNA polymerase according to claim 3, comprising both amino acid substitutions K64R and M97K numbered with respect to SEQ ID NO:

1.

5. The Phi29-type DNA polymerase according to any one of claims 1 to 4, wherein the Phi29-type DNA polymerase has improved affinity for a short primer compared to a wild-type Phi29 polymerase having SEQ ID NO: 1, the short primer being 4 to 6 nucleotides, and the comparison is performed under the same conditions.

6. Use of a Phi29-type DNA polymerase according to any one of claims 1 to 5 to improve affinity for a short primer compared to a wild-type Phi29 polymerase having SEQ ID NO: 1, wherein the short primer is 4 to 6 nucleotides, and the comparison is performed under the same conditions.

7. A method for replicating, amplifying, or sequencing template DNA, The DNA, at least (a) Phi29 type DNA polymerase according to any one of claims 1 to 5, (b) buffer solution; (c) Magnesium chloride, (d) Primer, and (e) Nucleoside triphosphate Step of contacting with a reaction mixture containing The method, including the method described above.

8. A kit for carrying out the method of claim 7, (a) Phi29 type DNA polymerase according to any one of claims 1 to 5, (b) Buffer, and (c) Magnesium chloride The kit includes the above.

9. A kit for carrying out the method of claim 7, A Phi29 type DNA polymerase according to any one of claims 1 to 5, (a) PrimPol enzyme, (b) Random trimmer, random primer, (c) Random tetramer, random primer, (d) Random pentamers, random primers, (e) Random heptamers, random primers, (f) Random octamer, random primer, (g) dNTP, (h) Buffer for use with any of (a) to (g) The kit includes one or more of the following.

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the Phi29 type DNA polymerase described in any one of claims 1 to 5.

11. Recombinant nucleic acid comprising a transcriptional regulatory sequence functionally linked to a nucleic acid molecule containing a nucleotide sequence encoding the Phi29 type DNA polymerase described in claim 10.

12. Recombinant cells comprising the recombinant nucleic acid described in claim 11.

13. (a) A step of contacting a DNA template molecule with a Phi29 type DNA polymerase and a reagent sufficient for primer extension according to any one of claims 1 to 5, wherein the reagent sufficient for primer extension comprises an oligonucleotide primer and a DNA primase / polymerase, and the oligonucleotide primer is a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or 10mer; and (b) The step of extending the primer using the DNA template molecule with the Phi29 type DNA polymerase. Methods that include...

14. The method according to claim 13, wherein the oligonucleotide primer is a random primer.

15. The method according to claim 13 or 14, wherein the primer extension comprises (i) multiple substitution amplification ("MDA"), (ii) rolling circle amplification, or (iii) multiple annealing and looping-based amplification cycle (MALBAC).

16. The method according to any one of claims 13 to 15, wherein the Phi29 type DNA polymerase comprises both substitutions K64R and M97K numbered with respect to SEQ ID NO: 1.

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