Phi29 DNA polymerase mutants with improved primer recognition

Abstract

Disclosed herein are mutants of bacteriophage Phi29 DNA polymerase that have improved primer recognition compared to the wild-type enzyme. Certain mutants contain one or both of the K64R or M97K mutations. The provided mutants are capable of using shorter and longer random synthetic DNA primers more efficiently than the wild-type Phi29 DNA polymerase, producing more amplification products in multiple displacement amplification (MDA) reactions. Compared to reactions performed with the wild-type Phi29 DNA polymerase, the mutants of the present invention amplify human genomic DNA polymerase with less deviation and higher coverage.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of the priority date of U.S. Provisional Application No. 62 / 849,252, filed May 17, 2019, the contents of which are incorporated herein in their entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0005] background

[0006] Phi29 DNA polymerase (Phi29 DNApol) is a monomeric enzyme (66 kDa) responsible for replicating the bacteriophage genome (19285 bp) by catalyzing both protein-primed initiation at both ends of the linear dsDNA molecule and complete elongation of each DNA strand (Blanco and Salas, 1984; 1985). Phi29 DNApol belongs to the B family of DNA polymerases (Bernad et al., 1987) and displays a common right-hand fold consisting of palm, thumb, and finger subdomains, but also has two additional domains termed 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 DNA 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); excellent strand displacement, allowing polymerization to be coupled to unwinding of double-stranded DNA in the absence of helicase-type enzymes (Blanco et al., 1989); high fidelity of synthesis, with a very low rate of misinsertion (10 -4 to 10 -6 ) and effective proofreading of inserted errors, which together enhance fidelity to 10 6 to 10 8 Only one error in each incorporated nucleotide is detected (Esteban et al., 1993 and 1994).

[0007] These properties make Phi29 DNApol an excellent choice for isothermal multiple displacement amplification (MDA) (Dean et al., 2002) and rolling circle amplification (RCA) (Lizardi et al., 1998). These DNA amplification technologies are based on combining Phi29 DNApol with either random synthetic primers (RP) (primarily hexanucleotides or hexamers) or DNA primases that can synthesize DNA primers in situ during the reaction (Picher et al., 2016).

[0008] Current sequencing technologies often require DNA amplification because the amount of DNA available from certain samples (such as single cells) is insufficient for the sequencing process. Unfortunately, DNA amplification has the risk of introducing errors, generating asymmetry (bias), and even promoting the co-amplification of trace contaminant DNA. Therefore, the key parameters that determine the quality of amplification are the absence of contamination and artifacts in the reaction products, coverage breadth and uniformity, low nucleotide error rate, and the ability to recover single nucleotide variants (SNVs), copy number variants (CNVs), and structural variants.

[0009] The potential source of amplification bias in current random hexamer-based MDA methods is priming inequality caused by different sequence-dependent hybridization kinetics of oligonucleotides. More importantly, the exponential amplification of self-pairing hexamers is prone to generating artifacts of primer-derived input-independent DNA amplification.

[0010] It has been shown that using longer primers instead of hexamers significantly reduces DNA amplification artifacts at a reaction temperature of 40°C (Alsmadi et al., 2009). The most likely reason behind this behavior is that higher temperatures reduce the likelihood of stable self-pairing of primers and, therefore, their subsequent amplification. However, in order to perform amplification reactions at temperatures as high as 40°C (10°C higher than the optimal temperature of Phi29 DNApol), thermostable or thermotolerant Phi29 DNApol variants are required. In this regard, several mutant Phi29 DNApols have been described that exhibit improved thermostability (Povilaitis et al., 2016). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated herein and form a part of this specification, illustrate exemplary embodiments and, together with this description, further serve to enable those skilled in the relevant art to make and use these embodiments and to make other embodiments apparent to those skilled in the art. The present invention will be described in more detail with reference to the following drawings, in which:

[0013] Figure 1 The three-dimensional structure of Phi29 DNApol in complex with DNA and dNTPs (PDB id: 2PYL). Most of the protein is shown in white, except for the thumb (THUMB) (dark green), TPR2 (cyan), and TPR1 (yellow) subdomains. The N-terminal 3'-5' exonuclease domain is not fully depicted (only two fragments (yellow) are shown, one containing Arg 96 , and the other contains Lys 64 The primer strand (cyan) displays the numbers corresponding to each nucleotide position. The template strand (light green), the incoming nucleotide (magenta), and the two activated metal ions (beige) are also shown.

[0014] Figure 2 .A) Schematic diagram of the wild-type (WT) Phi29 DNApol amino acid residues involved in the interaction with the first 10 nucleotides of the primer strand, derived from the crystal structure (PDB id: 2PYL). The nucleotide numbered 1 is the 3' most terminal, commonly described as the "primer end", and is the nucleotide closest to the enzyme active site. B) This schematic shows the acquisition of new interactions with the primer strand, resulting from the different mutations shown (magenta). The colored arrows indicate whether the interaction involves a phosphodiester (red), a sugar (orange), or a base (green). Mutants T499K and T499R are predicted to interact with the same position of the complementary / template strand (indicated by the dashed arrows).

[0015] Figure 3 Amplification efficiency of WT Phi29 DNApol or designed mutants in combination with TthPrimPol or random primers of different lengths (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)) using 1 ng of human genomic DNA as input in the reaction.

[0016] Figure 4 The balance of exonuclease and polymerase activities of WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K varies with the concentration of supplied deoxynucleotides (dNTPs).

[0017] Figure 5Amplification efficiency of WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K in combination with random primers of varying lengths (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)) using 1 ng of human genomic DNA as input in the reaction. For each primer length N, the columns show, from left to right: WT Phi29 DNApol, mutant K64R, mutant M97K, double mutant K64R / M97K.

[0018] Figure 6 Amplification yields observed in the absence of input DNA under low ionic strength conditions for WT Phi29 DNApol, mutant K64R, mutant M97K, or double mutant K64R / M97K in combination with random synthetic primers of varying sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)). For each primer length N, the columns show, from left to right: WT Phi29 DNApol, mutant K64R, mutant M97K, double mutant K64R / M97K.

[0019] Figure 7 Amplification yields observed in the absence of input DNA under high ionic strength conditions for WT Phi29 DNApol, mutant K64R, mutant M97K, or double mutant K64R / M97K in combination with random synthetic primers of varying sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)). For each primer length N, the columns show, from left to right: WT Phi29 DNApol, mutant K64R, mutant M97K, double mutant K64R / M97K.

[0020] Figure 8 Amplification efficiency of WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K in combination with random primers of varying lengths (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)) under high ionic strength conditions, using 1 ng of human genomic DNA as input. For each primer length N, the columns show, from left to right: WT Phi29 DNApol, mutant K64R, mutant M97K, double mutant K64R / M97K.

[0021] Figure 9Amplification efficiency of WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K in combination with random primers of varying lengths (tetramer (4N), pentamer (5N), and hexamer (6N)) under low and high ionic strength conditions, using different human genomic DNA inputs (1 pg, 10 pg, 100 pg, and 1 ng) in the reaction. For each primer length N, the columns show, from left to right: WT Phi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K.

[0022] Figure 10 Amplification of 1 pg, 10 pg, 100 pg, and 1 ng of human genomic DNA by multiple displacement amplification (MDA) combining Phi29 DNApol variants with TthPrimPol. For each amount of DNA, the columns are shown in order from left to right: WTPhi29 DNApol, mutant K64R, mutant M97K, and double mutant K64R / M97K.

[0023] Figure 11 Estimated coverage values ​​obtained from CovCheck analysis of amplification reactions using hexamers (6N), pentamers (5N), and tetramers (4N) with Phi29 DNApol variants and different human genomic DNA input combinations, in each case achieving conditions where differences in coverage could be observed.

[0024] Figure 12 . Estimated coverage values ​​obtained from CovCheck analysis of amplification reactions performed by TthPrimPol in combination with Phi29 DNApol variants.

[0025] Overview

[0026] Modified DNA polymerases can be used in a variety of applications, such as DNA sequencing, DNA amplification, library preparation, DNA genotyping, and the like. The present invention provides recombinant Phi29 DNA polymerases comprising mutations that confer improved properties, particularly those desired for these or other applications. These amino acid sequence changes can improve the performance of multiple displacement DNA amplification (MDA) by using shorter random synthetic primers, resulting in reduced amplification artifacts, better sequence-dependent hybridization kinetics, and, therefore, improved coverage breadth and uniformity. "Phi29" is sometimes written as "φ29."

[0027] The recombinant Phi29 DNA polymerase comprises one or two mutations from the group consisting of K64R and M97K.

[0028] Detailed description

[0029] I. Definition

[0030] "Isolated" means that the molecule is the predominant species present, i.e., more abundant on a molar basis than any other individual macromolecular species in the composition. Typically, the isolated molecule may constitute greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99% of the macromolecular species present in the composition and is the species of interest for purification. Solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition.

[0031] As used herein, the term "recombinant nucleic acid" refers to a nucleic acid molecule comprising two or more attached nucleotide sequences that are not normally attached to each other in nature.

[0032] As used herein, the term "recombinant cell" refers to a cell, such as an animal, plant, fungal, or microbial (eg, bacterial) cell, that contains a recombinant nucleic acid.

[0033] Terms used to describe sequence relationships between two or more nucleotide sequences or amino acid sequences include "reference sequence," "selected from," "comparison window," "identical," "percentage of sequence identity," "substantially identical," "complementary," and "substantially complementary."

[0034] A "reference sequence" is a defined sequence used as a basis for sequence comparison, and may be a subset of a larger sequence (eg, a complete cDNA, protein, or gene sequence).

[0035] Because two nucleic acids or polypeptides can each contain (1) a sequence that is similar between the two nucleic acids (i.e., only a portion of the complete nucleic acid or polypeptide sequence) or (2) a sequence that is different between the two nucleic acids, sequence comparisons between two (or more) nucleic acids or polypeptides are often performed by comparing the sequences of the two nucleic acids over a "comparison window" to identify and compare local regions of sequence similarity.

[0036] " comparison window " refers to the conceptual fragment of conventional at least 12 continuous nucleotides or 4 continuous amino acid residues compared with reference sequences.Comparison window usually has at least 15 or at least 25 nucleotides or at least 5 or at least 8 amino acid whose length.For the best comparison of two sequences, compared with reference sequences (it does not comprise interpolation or deletion), comparison window can comprise about 20% or interpolation or deletion still less (that is, gap).The best comparison of the sequence for aligning comparison window can be by the computerized tool (Wisconsin Genetics Software Package releases GAP, BESTFIT, FASTA and TFASTA in 7.0, Genetics Computer Group, 575Science Dr.Madison, WI, WI) of algorithm or by checking and selecting the best comparison (that is, causing the highest homology percentage of comparison window) of any one generation of various methods.

[0037] A subject nucleotide or amino acid sequence is "identical" to a reference sequence if the two sequences are identical when aligned for maximum correspondence over the length of the nucleotide or amino acid sequence.

[0038] The "percent sequence identity" between two sequences is calculated by comparing the two optimally aligned sequences over a comparison window, determining the number of positions at which the identical nucleotide or amino acid occurs in the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence identity.

[0039] Unless otherwise stated, the comparison window for comparing two sequences is the length of the shorter sequence.

[0040] 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 also often performed by inspection and manual alignment.

[0041] A subject amino acid sequence or nucleotide sequence is "substantially identical" to a reference sequence if the sequence has at least 80% sequence identity over the comparison window. Thus, sequences that have at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with a reference sequence are also "substantially identical." Of course, two sequences that are identical to each other are also "substantially identical."

[0042] As used herein, the term "transcription regulatory sequence" refers to a first nucleotide sequence that regulates the transcription of a second nucleotide sequence to which it is operably linked.

[0043] As used herein, a nucleotide sequence is "operably linked" to a transcriptional regulatory sequence when the transcriptional regulatory sequence functions in a cell to regulate the transcription of the nucleotide sequence. This includes promoting transcription of the nucleotide sequence through interaction between a polymerase and a promoter.

[0044] "Promoter" is a transcriptional regulatory sequence that is at least sufficient to promote the transcription of a nucleotide sequence in a DNA into an RNA transcript. Transcripts transcribed from a promoter typically include sequences from a promoter downstream of the transcription start site, and downstream sequences encoding an amino acid sequence in the case of mRNA. Promoters are the best characterized transcriptional regulatory sequences because they are in a predictable position immediately upstream of the transcription start site. Promoters include sequences that regulate the recognition, binding, and transcription initiation activity of RNA polymerases. These sequences can be cis-acting or can be responsive to trans-acting factors. Depending on the nature of the regulation, promoters can be constitutive or regulated. They are typically described as having two independent fragments: a core promoter region and an extended promoter region.

[0045] The core promoter includes sequences sufficient for RNA polymerase recognition, binding, and transcription initiation. The core promoter includes a transcription initiation site, an RNA polymerase binding site, and other general transcription binding sites, and is where the pre-initiation complex forms and the general transcription machinery assembles. The pre-initiation complex is typically within 50 nucleotides (nt) of the transcription start site (TSS).

[0046] The core promoter also includes sequences for ribosome binding sites that are necessary for translation of mRNA into a polypeptide.

[0047] The promoter region of extension comprises so-called proximal promoter, and it extends to about 250 nucleotides (i.e. -250nt) upstream of the transcription start site. It comprises main regulatory elements such as specific transcription factor binding sites. It has been found that many genes have transcription regulatory elements that are positioned at more upstream. Especially, the fragment of most of transcription regulatory elements that comprise gene can extend to as many as 700nt or more upstream of the transcription start site. In some genes, transcription regulatory sequences have been found at thousands of nucleotides upstream of the transcription start site.

[0048] As used herein, a first nucleotide sequence is "heterologous" to a second nucleotide sequence if the first nucleotide sequence is not found attached, e.g., operably linked, to the second nucleotide sequence in nature. By extension, a polypeptide is "heterologous" to a transcriptional regulatory sequence if it is encoded by a nucleotide sequence that is heterologous to the transcriptional regulatory sequence.

[0049] As used herein, the term "allelic variant" refers to a naturally occurring variation in a gene.

[0050] As used herein, the term "artificial variant" refers to a gene or protein that comprises one or more genetic modifications to a naturally occurring gene or protein.

[0051] As used herein, the term "mutation" as used herein generally refers to a change, variation or polymorphism of a nucleotide sequence compared to the wild type. Such a change, variation or polymorphism can be relative to a reference genome, such as a reference genome in a genomic database. Mutations include, but are not limited to, single nucleotide variations (SNVs), substitutions, insertions or deletions (also collectively referred to as "indels") and duplications.

[0052] II. Introduction

[0053] A new strategy to reduce amplification artifacts and amplification bias arising from sequence-dependent hybridization kinetics can utilize DNA primers shorter than the current gold standard hexamer. This strategy, which requires obtaining Phi29 DNApol variants that can recognize, stably bind, and efficiently use shorter DNA primers, would allow significant improvements to current DNA amplification technologies.

[0054] The availability of the 3D structure of Phi29 DNApol in complex with DNA and the introduced nucleotide (Berman et al., 2007) allowed us to perform a detailed examination of the amino acid residues directly involved in the interaction with the primer strand ( Figure 1 ). These ligands of the primer chain (see Figure 2 A's solution is:

[0055] R96 (phosphodiester interaction between nucleotides 7 and 8 of the primer).

[0056] R306 (phosphodiester interaction between nucleotides 8 and 9 of the primer).

[0057] R308 (phosphodiester interaction between nucleotides 9 and 10 of the primer).

[0058] K498 (interacts with the sugar of the first 3' nucleotide of the primer).

[0059] Y500 (phosphodiester interaction between nucleotides 1 and 2 of the primer).

[0060] K529 (phosphodiester interaction between nucleotides 1 and 2 of the primer).

[0061] Based on these few contacts, Phi29 DNApol establishes direct interactions spanning the first 10 bases of the primer strand, suggesting that this size would confer maximum binding stability to the primer. The lack of contacts in the interval between nucleotides 3 and 6 is quite surprising. Remarkably, current MDA procedures using Phi29 DNApol are based on providing random hexamers, which would be poorly stabilized by base contacts solely with the phosphodiester bonds between the first two nucleotides and with the 3'-terminal nucleotide. Therefore, hexamers do not have the optimal size to serve as initial primers for binding and extension by Phi29 DNApol. It is highly likely that these suboptimal primers were selected to have complementary sequences spaced sufficiently short apart in any DNA sample to achieve efficient and uniform amplification while minimizing self-hybridization artifacts known as primer-dimers.

[0062] 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, but the optimal primer size delivered by TthPrimPol to Phi29 DNApol and the fate of primers that remain shorter than the minimum size required for optimal extension by Phi29 DNApol have not yet been determined.

[0063] Based on this information and considerations, we explored the possibility of generating Phi29 DNApol mutants (variants of the present invention) with improved affinity for short primers (ideally limited to between 4 and 6 nucleotides). To do this, we employed two different approaches: 1) strengthening some existing interactions, and 2) creating new (non-existent) enzyme:DNA ligands in the primer region.

[0064] Such improved variants are expected to be useful in RP-based MDA procedures, potentially reducing primer-dimer artifacts and the formation of amplified chimeras. Furthermore, in the context of TruePrime DNA amplification technology, the use of short primers that can be generated by TthPrimPol may increase amplification efficiency and / or lead to improved coverage.

[0065] Likewise, a detailed analysis of the 3D structure of Phi29 DNApol (Berman et al., 2007) allowed the selection of five amino acid residues as candidates for “gain of function” mutations. These residues are: Lys64 (located in the ExoII motif), Met97 (adjacent to the primer ligand Arg96 of WT Phi29 DNApol), Thr499 (adjacent to the two primer ligands Lys498 and Tyr500 of WT Phi29 DNApol), Thr534 and Lys538 (close to the primer ligand Lys529 of WT Phi29 DNApol). The mutations selected at these residues (summarized in Figure 2 B) is:

[0066] K64R, resulting in the acquisition of a phosphodiester interaction between residues 4 and 5 of the primer strand.

[0067] K64KG, K64KK, L63LG, L63LH, as +1 insertion mutations flanking Lys64, were designed to be consistent with the heterogeneity observed in different ExoII motifs of B-family DNA polymerases. These changes are also expected to confer interactions with residues 4 and 5 of the primer strand.

[0068] R96K, predicted to weaken the interaction with the phosphodiester bond between residues 7 and 8 of the primer.

[0069] M97K, resulting in a gain of interaction with the nitrogen base of nucleotide 5 of the primer strand.

[0070] M97R, resulting in gain of base interactions with amino acid residues 4 and 5 of the primer strand.

[0071] T499K, resulting in gain of interaction with the sugar at amino acid residue 5 of the template chain.

[0072] T499R, resulting in gain of interaction with the sugars at amino acid residues 4 and 5 of the template chain.

[0073] K529R, resulting in acquisition of a double interaction with the phosphodiester bond between residues 1 and 3 of the primer strand.

[0074] T534K, resulting in gain of interaction with the sugar at amino acid residue 4 of the primer strand.

[0075] T534R, resulting in the acquisition of a phosphodiester interaction between residues 3 and 4 of the primer strand.

[0076] K538R, resulting in the acquisition of a phosphodiester interaction between residues 2 and 3 of the primer strand.

[0077] The mutants shown were designed to increase the affinity of Phi29 DNApol for short primers and were expressed and purified according to standard protocols to obtain WT Phi29 DNApol. It is unpredictable whether the acquisition of any specific interaction with the primer strand caused by the introduced mutations will adversely affect the properties of Phi29 DNApol such as translocation, processivity, or proper (TthPrimPol) and random-primed DNA amplification techniques.

[0078] III. Nucleic Acids, Expression Constructs, Recombinant Cells, and Mutant Polymerase Polypeptides

[0079] A. Nucleic acid

[0080] Provided herein are nucleic acids having nucleotide sequences encoding mutant Phi29 polymerases with improved primer recognition. The nucleotide sequence of wild-type Phi29 polymerase is provided in SEQ ID NO: 1. The nucleic acid encoding the sequence of the mutant Phi29 polymerase has one or both of the mutations K64R and M97K. In some embodiments, the nucleotide sequence encoding one or both of these mutations is substantially identical to the sequence of SEQ ID NO: 1.

[0081] B. Expression Constructs

[0082] Also provided herein are expression constructs comprising a transcriptional regulatory sequence operably linked to a nucleotide sequence encoding a mutant Phi29 polymerase described herein. The expression construct can take the form of a plasmid or any other form suitable for expression in a target cell.

[0083] C. Recombinant cells

[0084] Also provided herein are recombinant cells comprising the expression constructs described herein. In certain embodiments, the cells are bacterial cells. Such recombinant cells can be used to replicate the nucleic acid molecules of the present disclosure and to produce mutant Phi29 polymerases of the present disclosure. Mutant Phi29 polymerases can be produced by culturing recombinant cells comprising the expression constructs. The transcriptional regulatory sequences used can comprise constitutive promoters.

[0085] D. Mutant Phi29 polymerase

[0086] Also provided herein are mutant Phi29 polymerases with improved primer recognition.The mutant Phi29 polymerases of the present disclosure have an amino acid sequence substantially identical to that of SEQ ID NO: 1 (also deposited as UniProtKB-P03680) and comprising one or both of K64R and M97K amino acid substitutions.

[0087] Polymerases having substantially the same amino acid sequence can be based on naturally occurring sequences, such as allelic variants, as long as they include one or both of the K64R and M97K amino acid substitutions. Such variants can have up to, or no more than, any of 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 of SEQ ID NO: 1, also as long as one or both of the K64R and M97K amino acid substitutions are present.

[0088] Preferably, the amino acid sequence of the DNA polymerase of the present invention is at least 80% identical to 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 is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Still 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.

[0089] IV. How to use

[0090] Provided herein are methods for primer extension and / or nucleic acid polymerization using the mutant Phi29 polymerases described herein. Primer extension methods can be used for nucleic acid replication, amplification, and sequencing.

[0091] Primer extension involves hybridization of a primer to a nucleic acid molecule template, followed by a polymerase-catalyzed polymerization reaction that adds nucleotides to the 3' end of the primer. Primers can be added to the reaction mixture exogenously, or they can be produced by an initiator / polymerase. An initiator is an enzyme that catalyzes the synthesis of an oligonucleotide complementary to the nucleic acid template, called a primer. One such initiator is TthPrimPol.

[0092] Synthetic primers are generally used for nucleic acid amplification. The length of such primers is generally between about 6 and about 25 nucleotides. When a specific sequence is to be increased, primers can have a sequence complementary to the target sequence. For whole genome amplification or other non-directional amplification method purposes, random primers can be used. Random primers generally comprise a set (collection) or group (set) of oligonucleotides, wherein each base is present in each position of the oligonucleotide of one or more primers in the group. In some cases, one or more positions (such as 1,2,3,4,5,6,7,8,9 or 10) can be filled by a combination of fixed bases or two or three bases.

[0093] A. Amplification

[0094] Nucleic acid amplification, for example by the polymerase chain reaction (PCR) as described by Mullis (US 5,656,493), is an indispensable technique in medicine and biological research. It has been successfully used in a variety of applications, such as cloning, manipulation or sequencing of nucleic acids, DNA-based functional and phylogenetic analysis of genes, detection and diagnosis of diseases, and forensic and paternity testing.

[0095] B. Rolling Circle Amplification

[0096] Rolling circle amplification is a method for amplifying a covalently closed DNA molecule, such as a single-stranded covalently closed DNA molecule. A template DNA molecule is primed with a primer, such as a primer provided by an priming enzyme / polymerase. The DNA polymerase performs primer extension around the closed DNA molecule. The polymerase displaces the hybridized copy and continues to extend the polynucleotide around the template to produce a series of amplified products.

[0097] C. Multiple Displacement Amplification (MDA)

[0098] Multiple displacement amplification (MDA) is a kind of isothermal, non-PCR-based DNA amplification method, wherein from the initiation and extension of template, ssDNA chain is produced, and this chain can be continuously re-initiated and replicated by strand displacement synthesis, produces the DNA structure of many branches.After the initial denaturation of double-stranded DNA sample, multiple strand displacement (MDA) amplification produces many branched chain structure, because DNA synthesis can continuously initiate and extend from many positions of amplified molecules, without the need for the denaturation of other rounds.When new primers extend into the branched chain region from a DNA molecule template, branched chains are replaced by each other.MDA is further described in WO2011 / 047307A1 (" Multiple Displacement Amplification ") announced on April 21, 2011 for example.MDA can be simply described as: the isothermal polymerization of multiple initiation sites extension primers on the ssDNA template (self-generated ssDNA templates) of self generation.

[0099] In certain embodiments, MDA employs random trimers, tetramers, pentamers, hexamers, heptamers or octamers as primers to initiate amplification at multiple sites of the initial template and its amplified copies. In certain embodiments of the disclosed methods, initiation is achieved using a DNA priming enzyme / polymerase such as TthPrimPol.

[0100] In certain embodiments, the amplification of double-stranded linear polynucleotides includes the use of: 1) random synthetic primers and / or DNA-guided primases / polymerases, such as TthPrimPol; 2) modified DNA polymerases with chain displacement activity, such as Phi29 DNApol; 3) dNTPs. In certain embodiments, the dNTP substrate is unmodified. In other embodiments, the dNTP can be modified by attaching a labeling group (e.g., a fluorescent molecule). As used herein, the term "label" refers to a chemical moiety attached to a molecule (such as a nucleic acid molecule). Detectable labels include, for example, fluorescent labels, luminescent labels, enzyme labels, colorimetric labels such as colloidal gold or colored glass or plastic beads and radioactive labels. In combination, these three reagents promote multiple displacement amplification (MDA) of a given DNA, by random synthetic primers or by multiple priming of a primase / polymerase, and by extension of a DNA polymerase. Furthermore, a combination of random synthetic primers and / or a primase / polymerase and a DNA polymerase can achieve multiple strand displacement amplification by priming the amplified molecules with the primase / polymerase and / or random oligonucleotide primers and extending the primers with a DNA polymerase.

[0101] 1. DNA polymerase with strand displacement activity

[0102] Amplification methods such as MDA can employ a DNA polymerase with strand displacement activity, such as a polymerase that binds strongly to single-stranded DNA (e.g., preferentially to double-stranded DNA). The strand displacement activity can be used to displace hybridized strands of a DNA molecule while simultaneously extending the primer position.

[0103] DNA polymerases with strand displacement activity that can be used in the methods disclosed herein include, for example, Phi29 DNApol. 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 has inherent high processivity and strand displacement ability coupled to DNA polymerization, capable of generating DNA fragments longer than 70 kb from a single enzyme: DNA binding event (Blanco et al., 1989). This potential enables Phi29 DNApol to replicate DNA templates containing secondary structures such as hairpin loops. The enzyme also has 3'→5' exonuclease proofreading activity (Blanco and Salas, 1985; Garmendia et al., 1992) and provides up to 1000-fold higher fidelity than Taq DNA polymerase-based methods.

[0104] 2. Deoxyribonucleoside triphosphates

[0105] Primer generation and primer extension can be accomplished by combining a specialized DNA priming enzyme / polymerase (e.g., TthPrimPol, which synthesizes DNA primers) (Picher et al., 2016) with a DNA extension polymerase (e.g., Phi29 DNApol), requiring only deoxyribonucleotide substrates, such as dNTPs. Typically, these include the four standard bases, A, T, G, and C. However, in certain embodiments, non-natural nucleotides, such as inosine, may be included. In certain embodiments, nucleotides may be labeled for detection or capture of the polynucleotides into which they are incorporated.

[0106] D.DNA sequencing

[0107] To date, there are many different sequencing technologies, which are generally divided into "first-generation sequencing," "second-generation sequencing" (often referred to as "next-generation sequencing" or NGS), and "third-generation sequencing," which is also called single-molecule sequencing (SMS). First-generation sequencing mainly refers to the methods of Maxam and Gilbert (Maxam and Gilbert, 1977) or Sanger (Sanger et al., 1977; Sanger and Coulson, 1978), of which only the latter is used today.

[0108] Second generation or next generation sequencing refers to techniques that use advanced (optical) techniques to detect base positions and generate many sequences simultaneously. An overview of current methods is given in (Metzker, 2010).

[0109] Third-generation or single-molecule sequencing (SMS) technologies do not require prior amplification, and the template is not a clone or pool of DNA, but a single molecule, the sequence of which is typically copied / read and recorded online in “real time” as a result of polymerase activity (Sam et al., 2011; Thompson and Milos, 2011).

[0110] As used herein, the term "high-throughput sequencing" refers to sequencing thousands of nucleic acid molecules simultaneously or almost simultaneously. The platform for high-throughput sequencing includes but is not limited to massively parallel tag sequencing (MPSS), Polony sequencing, 454 pyrophosphate sequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing (Complete Genomics / BGI Shenzhen), Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing (PacBio) and nanopore DNA sequencing (e.g., Oxford Nanopore).

[0111] Method described herein can be used for but not limited to whole genome sequencing, exon group sequencing and amplicon sequencing.However, the amplified molecule itself can be carried out the amplification of specific amplicon.The sequence capture of the bait for the gene sequence in the genome can be used to separate the molecule of the representative exon of amplification.By reverse transcription of mRNA into double-stranded cDNA, the transcriptome of amplification can be produced to order-check.

[0112] V. Kit

[0113] Also provided herein are kits for performing the methods disclosed herein. As used herein, the term "kit" refers to Figure 1 A collection of items to be used together.

[0114] Certain kits disclosed herein include 2, 3, 4, 5, 6, or 7 elements selected from the group consisting of: (1) a PrimPol enzyme (e.g., TthPrimPol); (2) a DNA polymerase (e.g., Phi29 DNApol); (3) random trimers; (4) random tetramers; (5) random pentamers; (6) random heptamers; (7) random octamers; (8) random primers; (9) dNTPs; (10) a reaction buffer; and (11) a buffer for use with any of the foregoing elements. The kit may include a container for holding the reagents. The container itself may be placed in a shipping container. The container may be shipped by hand delivery or by a common carrier such as a national postal system or a delivery service such as FedEx. The kit may also include a container, such as a box or bag, for shipping the collected blood to a central facility. The kit may also typically include instructions for use and software for data analysis and interpretation.

[0115] Exemplary embodiments

[0116] 1. A Phi29-type DNA polymerase comprising one or both of a K64R or M97K mutation.

[0117] 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.

[0118] 3. A method for replicating, amplifying or sequencing a template DNA, comprising contacting the DNA with a reaction mixture comprising at least: a) a DNA polymerase according to any one of embodiments 1 to 2, b) a buffer, c) magnesium chloride, d) a primer, and e) nucleoside triphosphates.

[0119] 4. A kit for implementing the method according to embodiment 3, comprising: a) the DNA polymerase according to any one of embodiments 1 to 2, b) a buffer, and c) magnesium chloride.

[0120] 5. A kit for implementing the method according to embodiment 3, comprising a DNA polymerase according to any one of embodiments 1 to 2, and one or more of the following: (a) PrimPol enzyme (e.g., TthPrimPol); (b) random trimers; (c) random tetramers; (d) random pentamers; (e) random heptamers; (f) random octamers; (g) dNTPs; (h) reaction buffer; (i) a buffer for use with any of the foregoing elements.

[0121] 6. A Phi29-type DNA polymerase, wherein the Phi29-type DNA polymerase has an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99%, 99.5% sequence identity to SEQ ID NO: 1, and wherein the Phi29-type DNA polymerase comprises one or both of K64R and M97K amino acid substitutions.

[0122] 7. The Phi29-type DNA polymerase according to embodiment 6, having the sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0123] 8. The Phi29-type DNA polymerase of embodiment 6, having no more than 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 in addition to one or both of the K64R and M97K amino acid substitutions.

[0124] 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% or 99%, 99.5% sequence identity to SEQ ID NO: 1, and wherein the Phi29-type DNA polymerase comprises one or both of a K64R and M97K amino acid substitution.

[0125] 10. The isolated nucleic acid molecule of embodiment 9, wherein the Phi29-type DNA polymerase has the sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0126] 11. The isolated nucleic acid molecule of embodiment 9, wherein the Phi29-type DNA polymerase has no more than 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 in addition to one or both of the amino acid substitutions K64R and M97K.

[0127] 12. A recombinant nucleic acid comprising a transcriptional regulatory sequence operably linked to the Phi29-type DNA polymerase of any one of embodiments 9-11.

[0128] 13. The recombinant nucleic acid of embodiment 12, wherein the transcriptional regulatory sequence comprises a bacterial or mammalian promoter.

[0129] 14. The recombinant nucleic acid of embodiment 12, which is contained in a vector selected from the group consisting of a plasmid vector, a viral vector, a cosmid and a transposon.

[0130] 15. The recombinant nucleic acid of embodiment 14, comprising a cloning site positioned relative to the nucleotide sequence encoding the Phi29-type DNA polymerase such that a transcriptional regulatory sequence inserted into the cloning site becomes operably linked to the nucleotide sequence encoding the Phi29-type DNA polymerase.

[0131] 16. A recombinant cell comprising the recombinant nucleic acid of any one of embodiments 12-15.

[0132] 17. A method comprising: a) contacting a nucleic acid template molecule with the Phi29-type DNA polymerase of any one of embodiments 1, 2, 6-8 and reagents sufficient for primer extension; and b) performing primer extension using the nucleic acid template with the polymerase.

[0133] 18. The method of embodiment 17, wherein the reagents sufficient for primer extension comprise oligonucleotide primers.

[0134] 19. The method of embodiment 18, wherein the oligonucleotide primer comprises one or more of a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer, a nonamer, or a decamer.

[0135] 20. The method of embodiment 19, wherein the primers are random primers.

[0136] 21. The method of embodiment 18, wherein the oligonucleotide primer has a linkage of between 5 and 25 nucleotides.

[0137] 22. The method of embodiment 17, wherein the reagents sufficient for primer extension include a primase / polymerase (eg, TthPrimPol).

[0138] 23. The method of embodiment 17, wherein primer extension is performed at about or above any of the following temperatures: 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.

[0139] 24. The method of embodiment 17, wherein the template nucleic acid molecule is present in an amount of no greater than 1 ng, 100 pg, 10 pg, or 1 pg.

[0140] 25. The method of embodiment 17, wherein primer extension comprises (i) multiple displacement amplification ("MDA") or (2) rolling circle amplification.

[0141] 26. The method of embodiment 17, wherein primer extension comprises multiple annealing and looping-based amplification cycles (MALBAC).

[0142] 27. The method of any one of embodiments 17-26, wherein the Phi29-type DNA polymerase comprises both K64R and M97K substitutions. Example

[0143] Example 1: Screening to detect which mutants can use shorter random synthetic primers than WTPhi29 DNA pol in multiple displacement amplification reactions

[0144] Figure 3 Shown is the amplification of 1 ng of human genomic DNA by multiple displacement amplification (MDA) combining Phi29 DNApol variants with TthPrimPol or random synthetic primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)).

[0145] like Figure 3 As shown, WT Phi29 DNApol efficiently amplifies human genomic DNA using pentamers, hexamers, heptamers, and octamers, as well as TthPrimPol. Trimers and tetramers are not suitable for amplification.

[0146] From the set of Phi29 DNApol variants generated, six of them (K538R, T534K, T534R, L63LH, K64KG and K64KK) were completely inactive in MDA, regardless of primer size or alternative use of TthPrimPol. Another group of mutants (K529R, M97R, R96K, L63LG and T499K) showed worse amplification performance than WT Phi29 DNApol, indicating lower amplification yields and / or limitations in the use of certain primer sizes. For example, mutant M97R was able to efficiently use pentamers and hexamers, while heptamers and octamers did not trigger amplification. Similarly, mutant R96K could only use hexamers from a set of randomly synthesized primers. Remarkably, the insertion mutant L63LG was able to amplify DNA with pentamers, hexamers, heptamers and octamers, but did not produce any amplified material in combination with TthPrimPol. In contrast, mutant T499K only slightly amplified DNA in the presence of TthPrimPol, while none of the randomly synthesized primers promoted MDA.

[0147] The mutant T499R exhibited behavior roughly similar to that of WT Phi29 DNApol.

[0148] Finally, mutants K64R and M97K showed significant improvement over WT Phi29 DNApol. Only the two were able to use tetramers, while WT Phi29 DNApol and the remaining mutants did not show any amplification yield.

[0149] Two "gain of function" mutations were introduced into the same polypeptide to generate the double mutant K64R / M97K, which is well characterized compared to WTPhi29 DNApol and the single mutants K64R and M97K, as shown in the Examples below.

[0150] Example 2: In the Phi29 DNApol mutant M97K and the double mutant K64R / M97K, the polymerase activity is superior to the exonuclease activity.

[0151] Figure 4 Shown is a dynamic balance analysis between 3'-5' exonuclease and 5'-3' polymerization activities of the most relevant inventive mutants (K64R, M97K, and the double mutant K64R / M97K) relative to WT Phi29 DNApol. DNA duplexes formed by hybridization of a 5'-labeled primer (5'GATCACAGTGAGTAC, SEQ ID NO: 5) and a template (5'AGAAGTGTATCTGGTACTCACTGTGATC, SEQ ID NO: 6) were used to analyze the coupling between DNA synthesis and DNA degradation as a function of dNTP concentration (0 nM, 10 nM, 25 nM, 50 nM, 100 nM, and 500 nM). In the absence of dNTPs, exonuclease degradation of the primer ends was observed. The degradation pattern reflects the level of exonuclease activity of the inventive variants relative to WT Phi29 DNApol. As dNTP concentration increases, exonuclease activity is gradually overcome by 5'-3' polymerization, and net dNTP incorporation can be observed as an increase in the size of the labeled primer, defining the dNTP concentration required for efficient primer extension for each mutant. Figure 4 As observed in Figure 2, mutant K64R showed a roughly similar Pol / Exo equilibrium to that exhibited by the WT enzyme, reaching the 28-mer position at 25 nM dNTPs. On the other hand, mutant M97K and the double mutant K64R / M97K reached the same position (28-mer) at the lowest dNTP concentration tested (10 nM), indicating that these mutants have superior polymerase activity to exonuclease.

[0152] Example 3: The mutants of the present invention (K64R, M97K and K64R / M97K) can use shorter random synthetic primers than WT Phi29 DNApol in multiple displacement amplification reactions

[0153] Figure 5 Shown is the amplification of 1 ng of human genomic DNA by multiple displacement amplification combining selected variants of Phi29 DNApol (K64R, M97K, and the double mutant K64R / M97K) with random synthetic primers of varying sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N). The amplification yields shown are the average of two independent experiments, each including three replicates per condition. The standard deviation of the two experiments is shown.

[0154] like Figure 5 As observed in Figure 2, none of the enzymes tested were able to efficiently amplify genomic DNA using randomly synthesized trimers. Only the double mutant K64R / M97K showed a yield close to 1 μg.

[0155] Three variants of the invention were able to trigger amplification using tetramers, whereas no amplification was observed with WT Phi29 DNApol. Variant K64R exhibited the lowest amplification yield (2.7 μg), mutant M97K exhibited a slightly higher yield (3.8 μg), and the double mutant K64R / M97K exhibited a much higher yield (12.9 μg). The highest yield observed in the double mutant suggests a synergistic effect of two mutations in the same polypeptide.

[0156] WT Phi29 DNApol and the three variants of the invention were able to efficiently use random pentamers to initiate amplification. Likewise, the double mutant K64R / M97K produced the highest yield (over 20 μg of amplified DNA), significantly outperforming the performance of the single variants and the WT enzyme.

[0157] Similar comparable patterns were observed in the case of random hexamers, although the amplification yield was higher in all cases.

[0158] Using random heptamers, WT Phi29 DNApol maintained the same yield relative to that obtained using hexamers, whereas the three inventive variants tended to decrease amplification efficiency, generating DNA levels similar to those obtained using random pentamers.

[0159] In the case of the octamer, both the K64R and M97K single mutants showed lower amplification yields than WT Phi29 DNApol. On the other hand, the double mutant K64R / M97K clearly outperformed WT Phi29 DNApol as it appeared in all conditions tested, confirming its robust and efficient amplification value independent of the length of the randomly synthesized primers used to initiate amplification.

[0160] Example 4: Effect of ionic strength on background amplification observed in the absence of input DNA in the no template control (NTC).

[0161] Figure 6 Shown are the amplification yields observed in the absence of input DNA when WT Phi29 DNApol or selected variants of the invention (K64R, M97K, and K64R / M97K) were combined with random synthetic primers of varying sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)).

[0162] Under the low ionic strength conditions tested (20 mM KCl; 57 mM NaCl), both the M97K single mutant and the K64R / M97K double mutant showed significant amplification yields using pentamers and hexamers, and in the case of the double mutant also using tetramers, in the absence of input DNA (see Figure 6 However, the amplification yield was significantly lower than that obtained using DNA (1 ng) as input under the same conditions (see Figure 5 ), suggesting that different amplification mechanisms are involved. The ability of Phi29 DNApol to amplify primer-dimers in the absence of input DNA is well known in the art (Alsmadi et al., 2009), and under the conditions tested, primer-dimer stability may be enhanced in the M97K single mutant and the K64R / M97K double mutant.

[0163] Figure 7 Shown are the amplification yields observed in the absence of input DNA but with increased ionic strength by the addition of ammonium sulfate [(NH4)2SO4]. In the presence of ammonium sulfate (45 mM), the amplification levels observed in the absence of input DNA were completely eliminated in all variants and all primer sizes.

[0164] Example 5: High ionic strength conditions enhance the robustness and efficiency of DNA amplification using random primers of different lengths for the double mutant K64R / M97K.

[0165] Figure 8Shown is the amplification of 1 ng of human genomic DNA by multiple displacement amplification (MDA) under high ionic strength conditions (20 mM KCl; 57 mM NaCl; 45 mM (NH4)2SO4) by combining WT Phi29 DNApol or selected variants of the invention (K64R, M97K, and K64R / M97K) with random synthetic primers of varying sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N). The amplification yields shown are the average of two independent experiments, each including three replicates under each condition. The standard deviation of the two experiments is depicted.

[0166] like Figure 8 As observed in Figure 2, none of the tested enzymes were able to efficiently amplify genomic DNA using randomly synthesized trimers. Only the double mutant K64R / M97K showed a yield close to 600 ng.

[0167] Compared to the situation observed under the previous conditions (see Figure 5 ) In contrast, tetramers were only efficiently utilized by the M97K single mutant and the K64R / M97K double mutant, with the single mutant K64R producing only a small yield of approximately 1 μg. Notably, the amplification yields observed for the M97K single mutant and the K64R / M97K double mutant were increased compared to the amplification yields obtained in the absence of ammonium sulfate (from 4 μg to 12 μg and from 13 μg to 16 μg, respectively).

[0168] In the case of pentamers and hexamers, the M97K single mutant and the K64R / M97K double mutant showed similar results, significantly better than the amplification yields obtained with the WT enzyme or the K64R variant. As shown in the absence of ammonium sulfate, WTPhi29 DNApol showed higher yields than the K64R variant.

[0169] In the case of the heptamer, only the double mutant K64R / M97K maintained the amplification yields obtained with shorter random synthesis primers and / or in the absence of ammonium sulfate. Both WT Phi29 DNApol and the K64R variant had significantly reduced yields, showing equivalent values ​​under these conditions. The yields obtained with the M97K mutant were also reduced compared to the previous conditions.

[0170] Finally, the octamer was efficiently utilized only by the double mutant K64R / M97K, while the other three enzymes showed very low amplification yields.

[0171] The Phi29 DNApol double mutant K64R / M97K maintains full amplification performance under both low and high ionic strength conditions, likely as a result of gain of function brought about by additional contacts of the enzyme with the nitrogen base from primer nucleotide 5 and the phosphodiester bond between nucleotides 4 and 5 (see Figure 2 These additional contacts enable the enzyme to expertly stabilize primers of varying sizes under conditions of varying ionic strength.

[0172] Example 6: The double mutation K64R / M97K leads to very sensitive amplification of minute amounts of DNA regardless of the size of the primers tested.

[0173] Figure 9 Shown is the amplification of different amounts of human genomic DNA (1 pg, 10 pg, 100 pg, and 1 ng) by multiple displacement amplification (MDA) under low (20 mM KCl; 57 mM NaCl) or high (20 mM KCl; 57 mM NaCl; 45 mM (NH4)2SO4) ionic strength conditions, in which the WT Phi29 DNApol or selected variants of the invention (K64R, M97K, and K64R / M97K) are combined with random synthetic primers of different sizes (tetramer (4N), pentamer (5N), or hexamer (6N)).

[0174] Under low ionic strength conditions ( Figure 9 , upper panel), the double mutant K64R / M97K produced the most consistent and highest amplification yields in all conditions tested.

[0175] In the case of random tetramer synthesis, as shown previously (see Figure 5 ), WT Phi29 DNApol was unable to amplify any of the DNA inputs tested. The K64R variant produced detectable yields at only 1 ng of DNA input, lacking the sensitivity to amplify lower amounts of DNA. In contrast, both the M97K and M97K / K64R mutants efficiently amplified the DNA inputs tested, with the double mutant producing higher yields in all cases.

[0176] In the case of randomly synthesized pentamers, all enzymes were able to use them to initiate amplification, but exhibited varying 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 reasonable efficiency under all conditions tested. The double mutant M97K / K64R exhibited the highest amplification efficiency of the three variants of the present invention, regardless of the amount of DNA input, and thus exhibited the best sensitivity.

[0177] In the case of randomly synthesized hexamers, all enzymes were able to efficiently use them to initiate amplification of every DNA input tested, demonstrating significant amplification yields in each case. When analyzing low amounts of DNA input, the three variants of the present invention outperformed WT Phi29 DNApol, demonstrating higher amplification yields. As with the pentamers, the double mutant M97K / K64R exhibited the highest amplification efficiency of the three variants of the present invention, regardless of DNA input amount.

[0178] As shown previously (see Figure 6 ), when using pentamers and hexamers, the M97K and M97K / K64R variants showed significant amplification yields in the absence of input DNA (no template control, NTC). Therefore, the same sensitivity analysis was performed under high ionic strength conditions (20mM KCl; 57mM NaCl; 45mM (NH4)2SO4) to prevent this artifact effect from primer-dimer amplification.

[0179] Under conditions of high ionic strength (see Figure 9 , lower panel), randomly synthesized tetramers showed similar usage patterns among the variants tested compared to low ionic strength conditions, although amplification yields were increased in most cases. The exception to this rule was the M97K variant, which showed lower yields at 1 pg and 10 pg DNA input.

[0180] Under conditions of random pentamer synthesis at high ionic strength, the M97K and M97K / K64R variants exhibited the best performance in terms of sensitivity and efficiency, demonstrating higher amplification yields compared to low ionic strength conditions with the same DNA input amount. When limiting amounts of DNA were tested (1 pg and 10 pg), increasing the reaction ionic strength resulted in reduced amplification efficiency for the K64R variant, while the other two inputs showed similar efficiencies (100 pg) or higher (1 ng). Surprisingly, in all cases, WT Phi29 DNApol outperformed the K64R variant under these conditions.

[0181] In the case of random hexamers synthesized under high ionic strength conditions, the double mutant K64R / M97K was the only variant that increased the yield observed for all DNA inputs compared to the results obtained under low ionic strength conditions. The single mutant M97K exhibited lower yields at the lowest inputs (1 pg and 10 pg), while yields increased at 100 pg and 1 ng DNA inputs, indicating reduced sensitivity. Variant K64R and WT Phi29 DNApol exhibited similar behavior. As with the pentamers, in all cases, WT Phi29 DNApol produced higher amplification yields under these conditions than the K64R variant.

[0182] In conclusion, during amplification reactions with all the tested DNA primers, the double mutant K64R / M97K showed the best performance in terms of amplification efficiency and sensitivity under both low and high ionic strength conditions.

[0183] Example 7: When the primers are generated by TthPrimPol, the amplification efficiency and sensitivity are not changed by the use of the variants of the present invention

[0184] Figure 10 Shown is the amplification of 1 pg, 10 pg, 100 pg, and 1 ng of human genomic DNA by multiple displacement amplification (MDA) combining WT Phi29 DNApol or selected variants of the invention (K64R, M97K, and K64R / M97K) with TthPrimPol, a DNA primase capable of synthesizing primers during the reaction of Phi29 DNApol (Picher et al., 2016).

[0185] like Figure 10 As observed in Figure 5, there were no significant yield differences between WT Phi29 DNA pol and the tested variants of the invention, which resulted in similar sensitivity and efficiency levels in this setting.

[0186] Example 8: Selected variants of the invention (K64R, M97K and K64R / M97K) improve amplification coverage measured by CovCheck technology

[0187] CovCheck technology allows for coverage analysis of whole-genome amplification using a PCR panel consisting of 24 different primer pairs that amplify a small portion of each human chromosome. CovCheck technology was validated by comparing CovCheck coverage values ​​with the true coverage obtained by low-pass whole-genome sequencing, achieving excellent correlation values ​​(https: / / www.expedeon.com / products / genomics / dna-rna-products / covcheck-pcr-kits / ).

[0188] To analyze the amplification coverage obtained for each variant, a limited amount of input material was selected: 30pg of human genomic DNA. This amount of DNA is equivalent to 5 human diploid genomes, and this may be the minimum amount to ensure that there are enough copies of each chromosome for amplification. Below this level, due to the random distribution of molecules in the purified DNA sample, certain regions or complete chromosomes may be missing in the input for amplification, resulting in regions not being covered by amplification material due to lack of template, rather than due to amplification failure.

[0189] Figure 11 Shown are estimated coverage values ​​obtained from CovCheck analysis of amplification reactions using combinations of hexamers, pentamers, and tetramers with WT Phi29 DNApol or selected variants of the invention and 30 pg of human genomic DNA input. Coverage values ​​are the average of 6 independent reactions per condition.

[0190] In the case of randomly synthesized hexamers, when using the three variants of the invention, the amplification coverage was improved compared to the values ​​obtained with WTPhi29 DNApol.

[0191] In the case of random pentamers, all enzymes showed coverage values ​​above 90% under these conditions. Therefore, no significant differences could be observed. However, the M97K variant stood out with perfect coverage in the 6 replicates tested.

[0192] In the case of randomly synthesized tetramers, only the M97K and M97K / K64R variants produced amplified DNA, which is consistent with the amplification sensitivity shown when WTPhi29 DNApol and the K64R variant were combined with tetramers ( Figure 9 CovCheck analysis showed excellent amplification coverage (99%) in both cases, demonstrating the benefit of using the variants of the present invention in combination with primers that are as short as possible in order to maximize amplification coverage and consistency, preventing amplification bias and sequence loss.

[0193] When using enzyme (TthPrimPol) to make DNA primers for Phi29 DNApol, Figure 12Estimated coverage values ​​obtained by CovCheck analysis of amplification reactions performed by TthPrimPol in combination with WT Phi29 DNApol or variants of the present invention using 30 pg (5 genome equivalents) of human genomic DNA as input are shown. Coverage values ​​are the average of 12 independent reactions per condition. CovCheck analysis also revealed an improvement in amplification coverage when the variants of the present invention were used, supporting the advantages of enhancing the consistency of the amplified material relative to the original DNA input.

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[0216] WO 2011 / 047307A1,“Multiple Displacement Amplification”,April 21,2011.

[0217] Garmendia C, Bernad A, Esteban JA, Blanco L, Salas M. The bacteriophagephi 29 DNA polymerase, a proofreading enzyme. J Biol Chem. 1992 Feb 5;267(4):2594-9.

[0218] As used herein, unless otherwise specified, the following meanings apply. The wording "may" is used in a permissive sense (i.e., meaning possible), rather than a mandatory sense (i.e., meaning must). The words "include / comprising (include)", "including / comprising (including)" and "includes (includes)" etc. mean including / including but not limited to. The singular forms "a", "a" and "the" include plural referents. Thus, for example, reference to "an element" includes a combination of two or more elements, although other terms and words, such as "one or more" are used for one or more elements. Unless otherwise specified, the term "or" is non-exclusive, i.e., encompasses both "and" and "or". The term "any one of..." between a modifier and a sequence means each member in the modifier modification sequence. Thus, for example, the wording "at least 1, 2 or 3 any one of" means "at least 1, at least 2 or at least 3". In certain embodiments, an invention that "includes" various elements can also be "essentially composed of" these elements. The term "consisting essentially of" means comprising the recited elements and other elements that do not materially affect the basic and novel characteristics of the claimed combination.

[0219] It should be understood that this specification and the accompanying drawings are not intended to restrict the present invention to the specific form disclosed, but on the contrary, it is intended to cover all modifications, equivalents and alternative forms that fall within the spirit and scope of the present invention as defined by the appended claims. Based on this specification, further modifications and alternative embodiments of the various aspects of the present invention will be obvious to those skilled in the art. Therefore, this specification and the accompanying drawings should only be interpreted as illustrative, and are for the purpose of teaching those skilled in the art to carry out the general mode of the present invention. It should be understood that the form of the present invention shown and described herein should be considered as an example of an embodiment. The elements and materials illustrated and described herein can be replaced, components (part) and processes can be reversed or omitted, and certain features of the present invention can be used independently, all of which will be obvious to those skilled in the art who understand the benefits of this specification. Without departing from the spirit and scope of the present invention as described in the appended claims, the elements described herein may be changed. The titles used herein are only for the purpose of organization and are not meant to limit the scope of this specification.

[0220] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0221] Sequence Listing

[0222] Italicized amino acids are not expressed in some embodiments

[0223] SEQ ID NO 1: Wild-type Phi29 DNA polymerase (UniProtKB-P03680)

[0224]

[0225]

[0226]

[0227] SEQ ID NO 2: K64R Phi29 DNApol mutant

[0228]

[0229]

[0230] SEQ ID NO 3: M97K Phi29 DNApol mutant

[0231]

[0232]

[0233] SEQ ID NO 4: K64R / M97K Phi29DNApol double mutant

[0234]

[0235] Sequence Listing <110> 4Beth Biological Co., Ltd. <120> PHI29 DNA polymerase mutants with improved primer recognition <130> 4BA17083PCT <140> US62 / 849,252 <141> 2020-05-17 <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 Phi29 DNA pol 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'-labeled primer of DNA duplex <400> 5 gatcacagtg agtac 15 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> DNA duplex template <400> 6 agaagtgtat ctggtactca ctgtgatc 28

Claims

1. A Phi29-type DNA polymerase, wherein the amino acid sequence of the Phi29-type DNA polymerase is shown in SEQ ID NO: 2 or SEQ ID NO:

4.

2. A method for replicating, amplifying or sequencing a template DNA, the method comprising contacting the template DNA with a reaction mixture comprising at least: a) a Phi29-type DNA polymerase represented by the amino acid sequence of SEQ ID NO: 4, b) buffer, c) magnesium chloride, d) a random primer tetramer, pentamer, hexamer, heptamer, or octamer, and e) Nucleoside triphosphates.

3. A kit for implementing the method according to claim 2, comprising: a) a Phi29-type DNA polymerase represented by the amino acid sequence of SEQ ID NO: 4, b) a buffer, and c) magnesium chloride.

4. A kit for implementing the method according to claim 2, comprising a Phi29-type DNA polymerase represented by the amino acid sequence of SEQ ID NO: 4, and one or more of the following: (a) Random primer tetramer; (b) Random primer pentamer; (c) random primed heptamer; (d) random primer octamer; (e) dNTPs; (f) reaction buffer; (g) A buffer for use with any one of (a) to (f).

5. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a Phi29-type DNA polymerase, wherein the amino acid sequence of the Phi29-type DNA polymerase is shown in SEQ ID NO: 2 or SEQ ID NO:

4.

6. A recombinant nucleic acid comprising a transcriptional regulatory sequence operably linked to the isolated nucleic acid molecule of claim 5.

7. The recombinant nucleic acid of claim 6, wherein the transcriptional regulatory sequence comprises a bacterial promoter or a mammalian promoter.

8. The recombinant nucleic acid according to claim 6, which is contained in a vector selected from the group consisting of a plasmid vector, a viral vector, a cosmid and a transposon.

9. The recombinant nucleic acid according to claim 8, comprising a cloning site positioned relative to the nucleotide sequence encoding the Phi29-type DNA polymerase such that a transcriptional regulatory sequence inserted into the cloning site becomes operably linked to the nucleotide sequence encoding the Phi29-type DNA polymerase.

10. A recombinant cell comprising the recombinant nucleic acid according to any one of claims 6 to 9.

11. A method comprising: a) contacting a nucleic acid template molecule with the Phi29-type DNA polymerase according to claim 1 and a reagent sufficient for primer extension; as well as b) performing primer extension using the nucleic acid template molecule and the Phi29 type DNA polymerase.

12. The method of claim 11, wherein the reagents sufficient for primer extension comprise oligonucleotide primers.

13. The method according to claim 12, wherein when the amino acid sequence of the Phi29-type DNA polymerase is as shown in SEQ ID NO: 2, the oligonucleotide primer comprises one or more of a tetramer, a pentamer or a hexamer.

14. The method according to claim 12, wherein when the amino acid sequence of the Phi29-type DNA polymerase is as shown in SEQ ID NO: 4, the oligonucleotide primer comprises one or more of a trimer, a tetramer, a pentamer, a hexamer, a heptamer or an octamer.

15. The method of claim 13 or 14, wherein the oligonucleotide primers are random primers.

16. The method of claim 12, wherein the oligonucleotide primer has a linkage of between 5 and 25 nucleotides.

17. The method of claim 11, wherein the reagents sufficient for primer extension comprise a primase / polymerase.

18. The method of claim 11, wherein the primer extension is performed at or above any of the following temperatures: 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.

19. The method of claim 11, wherein the nucleic acid template molecule is present in an amount of no greater than 1 ng, 100 pg, 10 pg, or 1 pg.

20. The method of claim 11, wherein the primer extension comprises (1) multiple displacement amplification ("MDA") or (2) rolling circle amplification.

21. The method of claim 11, wherein the primer extension comprises multiple annealing and circularization-based amplification cycles (MALBAC).

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