Phi29 DNA polymerase mutants 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] Reference to Related Applications This application claims the benefit of the priority date of U.S. Provisional Application No. 62 / 849,252, filed 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 its entirety.

Background Art

[0003] Background Phi29 DNA polymerase (Phi29 DNApol) is a monomeric enzyme (66 kDa) responsible for the replication of the bacteriophage genome (19,285 bp) by catalyzing both the initiation of protein-primed initiation at the ends of linear dsDNA molecules and the complete elongation of each DNA strand (Blanco and Salas, 1984; 1985). Phi29 DNApol belongs to family B of DNA polymerases (Bernad et al, 1987) and exhibits a common right-hand fold that includes not only the 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 use in numerous DNA amplification and DNA sequencing technologies and platforms: high processive DNA synthesis that allows the enzyme to incorporate over 70,000 nucleotides per DNA binding event in the absence of a processivity factor (Blanco et al, 1989); extremely efficient strand displacement that allows polymerization coupled to the unwinding of double-stranded DNA in the absence of a helicase-type enzyme (Blanco et al, 1989); and a very low error insertion rate (10 -4 ~10 -6 ), combined with efficient proofreading of inserted errors, results in an overall improvement in fidelity to a maximum of one error per 10 6 ~10 8 incorporated nucleotides (Esteban et al, 1993 and 1994).

[0004] Due to these properties, Phi29 DNApol is 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 the combination of Phi29 DNApol with either random synthetic primers (RP), which are mainly hexanucleotides, i.e., hexamers, or DNA primases that can synthesize DNA primers in situ during the amplification reaction (Picher et al, 2016).

[0005] In current sequencing technologies, DNA amplification is frequently required because the amount of DNA obtained from a specific sample (e.g., a single cell) is not sufficient for the sequencing process. Unfortunately, DNA amplification has the risk of introducing errors, generating asymmetries (biases), and even promoting the co-amplification of trace levels of contaminating DNA. Therefore, important parameters that determine the quality of amplification are the absence of contaminants and artifacts in the reaction products, the spread and uniformity of coverage, a low nucleotide error rate, and the ability to revert single nucleotide variants (SNVs), copy number variants (CNVs), and structural variants to their normal state.

[0006] The cause of potential amplification bias in current MDA methods based on random hexamers is the unequal priming resulting from the different rates of sequence-dependent hybridization of the oligonucleotides. More importantly, there is a tendency to generate primer-derived input-independent DNA amplification artifacts caused by the exponential amplification of self-pairing hexamers.

[0007] It has been shown that when using longer primers instead of hexamers and setting the reaction temperature to 40°C, DNA amplification artifacts are significantly reduced (Alsmadi et al, 2009). The most likely reason behind this behavior is that at higher temperatures, the possibility of stable self-pairing of primers decreases, and as a result, their subsequent amplification decreases. However, to perform the amplification reaction at a high temperature of 40°C (10°C higher than the optimal temperature of Phi29 DNApol), a heat-stable or heat-resistant Phi29 DNApol variant is required. In this regard, several mutant Phi29 DNApol have been described as showing improved heat stability (Povilaitis et al, 2016).

Brief Description of the Drawings

[0008] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate exemplary embodiments and, together with the description, further serve to enable those skilled in the relevant 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

Figure 2

Figure 3-1

Figure 3-2

Figure 3-3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9-1

Figure 9-2

Figure 9-3

Figure 10

Figure 11-1

Figure 11-2

Figure 12

[0009] Overview Modified DNA polymerases can be useful in a variety of 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 in amplification artifacts, an improvement in the rate of sequence-dependent hybridization, and therefore 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

[0011] Detailed Description I. Definitions "Isolated" means that a molecule is the predominant 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 comprising 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 that contains 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] Since 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 usually 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 of typically at least 12 contiguous nucleotides or 4 contiguous 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. The comparison window may include up to about 20% addition or deletion (i.e., gap) compared to the reference sequence (without addition or deletion) for optimal alignment of two sequences. The optimal sequence alignment for aligning the comparison window can be performed by a computerized implementation of an algorithm (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 various methods (i.e., the one that yields the highest percentage of homology over the comparison window) is selected.

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

[0019] To calculate the "percentage of sequence identity" between two sequences, compare the two sequences optimally aligned over a comparison window, count the number of positions at which the identical nucleotides or amino acids occur in both sequences to give the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiply the result by 100 to obtain the percentage of sequence identity.

[0020] Unless otherwise specified, the comparison window used to compare two sequences is the length of the shorter sequence.

[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. Alignments are often done by scrutiny and manual alignment.

[0022] A subject nucleotide sequence or amino acid sequence is "substantially identical" to a reference sequence if the subject amino acid sequence or nucleotide sequence has at least 80% sequence identity over a comparison window. Thus, sequences having 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 to the reference sequence are also "substantially identical". Of course, two sequences that are identical to each other are also "substantially identical".

[0023] 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.

[0024] As used herein, a nucleotide sequence is "operably linked" to a transcription regulatory sequence if the transcription regulatory sequence functions in the cell to regulate the transcription of the nucleotide sequence. This includes promoting the transcription of the nucleotide sequence through the interaction between the polymerase and the promoter.

[0025] A "promoter" is at least a sufficient transcriptional regulatory sequence to promote the transcription from a nucleotide sequence of DNA to an RNA transcript. The transcript transcribed from the promoter usually contains a sequence from the promoter downstream of the transcription start site, as well as a downstream sequence encoding an amino acid sequence in the case of mRNA. Because the promoter is at a predictable position immediately upstream of the transcription start site, it is the most well-characterized transcriptional regulatory sequence. A promoter contains sequences that regulate the recognition, binding, and transcriptional initiation activities of RNA polymerase. These sequences can be cis-acting or responsive to trans-acting factors. Promoters can be constitutive or regulated depending on the nature of the regulation. They are often described as having two separate segments, a core promoter region and an extended promoter region.

[0026] The core promoter contains sequences that are sufficient for the recognition, binding, and transcriptional initiation of RNA polymerase. The core promoter includes the transcription start site, the RNA polymerase binding site, and other general transcription binding sites, where a pre-initiation complex is formed and the general transcriptional machinery is assembled. The pre-initiation complex is generally within 50 nucleotides (nt) from the transcription start site (TSS).

[0027] In addition, the core promoter also contains the sequence of the ribosome binding site required for the translation from mRNA to polypeptide.

[0028] The extended promoter region includes the so-called proximal promoter, which extends up to about 250 nucleotides (i.e., -250nt) upstream of the transcription start site. This includes primary regulatory elements such as binding sites for specific transcription factors. Many genes have been found to have transcriptional regulatory elements located further upstream. In particular, a fragment containing most of the transcriptional regulatory elements of a gene can extend more than 700nt upstream from the transcription start site. In certain genes, transcriptional regulatory sequences have been found thousands of nucleotides upstream of the transcription start site.

[0029] As used herein, a first nucleotide sequence is "heterologous" to a second nucleotide sequence if the first nucleotide sequence is not associated in nature with the second nucleotide sequence, for example, is not operably linked. Thus, a polypeptide is "heterologous" to a transcriptional regulatory sequence if the polypeptide is encoded by a nucleotide sequence that is heterologous to the transcriptional regulatory sequence.

[0030] As used herein, the term "allelic variant" refers to a naturally occurring variation of a gene.

[0031] As used herein, the term "artificial variant" refers to a gene or protein to which one or more genetic modifications have been made to a naturally occurring gene or protein.

[0032] As used herein, the term "mutation" generally refers to a modification, variant or polymorphism of a nucleotide sequence as compared to the wild type. Such a modification, variant or polymorphism can be, for example, relative to a reference genome in a genomic database. Mutations include, but are not limited to, single nucleotide variations (SNVs), substitutions, insertions or deletions (collectively also referred to as "indels"), and repeats.

[0033] II. Introduction In a novel strategy for reducing amplification artifacts and amplification bias resulting from the rate of sequence-dependent hybridization, it may be possible to advantageously utilize the use of DNA primers that are shorter than the current gold standard, hexamers. This strategy requires obtaining a Phi29 DNApol variant that recognizes, stably binds to, and efficiently uses shorter DNA primers and could significantly improve current DNA amplification techniques.

[0034] The availability of the 3D structure of Phi29 DNApol complexed with DNA and incoming nucleotides (Berman et al, 2007) has allowed a detailed scrutiny of the amino acid residues directly involved in the interaction with the primer strand (Figure 1). These ligands of the primer strand (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 has established direct interactions that span the first 10 bases of the primer strand, suggesting that such a size confers maximum binding stability to the primer. It is very surprising that there are no contacts between nucleotides 3 and 6. Surprisingly, the current MDA protocol using Phi29 DNApol is based on the provision of random hexamers, which are only marginally stabilized by contacts with the phosphodiester bond between the first two nucleotides and the base of the 3' terminal nucleotide. Therefore, the hexamers are not of optimal size for use as initial primers for Phi29 DNApol to bind and extend. These sub-optimal primers are very likely to have been selected to have complements in any DNA sample at sufficiently short intervals to allow efficient and uniform amplification while minimizing self-hybridization artifacts known as primer dimers.

[0036] On the one hand, the alternative TruePrime DNA amplification technology (Picher et al, 2016) utilizes DNA primase (TthPrimPol) to synthesize DNA primers on demand. However, what the optimal primer size supplied by TthPrimPol to Phi29 DNApol is, and what the fate of these primers that remain shorter than the minimum size required for optimal elongation by Phi29 DNApol is, have not been established so far.

[0037] Based on this information and warning, the inventors explored the possibility of generating Phi29 DNApol variants (variants of the present invention) with improved affinity for short primers, ideally within the range of 4 to 6 nucleotides. For this purpose, the inventors followed two different approaches: 1) strengthening some existing interactions; 2) creating new (not existing) enzyme:DNA ligand in the primer region.

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

[0039] Again, through a detailed analysis of the 3D structure of Phi29 DNApol (Berman et al, 2007), five amino acid residues were selected as candidates for "gain of function" mutations. These residues are as follows: Lys64 (in the ExoII motif), Met97 (adjacent to Arg96, which is the primer ligand of WT Phi29 DNApol), Thr499 (adjacent to Lys498 and Tyr500, which are the two primer ligands of WT Phi29 DNApol), Thr534, and Lys538 (proximal to Lys529, which is the primer ligand of WT Phi29 DNApol). The mutations selected with these residues (summarized in Figure 2B) were as follows: · K64R, which results in the acquisition of an interaction with the phosphodiester between residues 4 and 5 of the primer strand. · K64KG; K64KK; L63LG; L63LH, which are +1 insertion mutations flanking Lys64 and are designed to be consistent with the heterogeneity observed in different ExoII motifs of B-family DNA polymerases. These changes are also predicted to acquire an interaction with residues 4 and 5 of the primer strand. · R96K, which is predicted to weaken the interaction with the phosphodiester bond between residues 7 and 8 of the primer. · M97K, which results in the acquisition of an interaction with the nitrogenous base of nucleotide 5 of the primer strand. · M97R, which results in the acquisition of an interaction with the bases of amino acid residues 4 and 5 of the primer strand. · T499K, which results in the acquisition of an interaction with the sugar of amino acid residue 5 of the template strand. · T499R, which results in the acquisition of an interaction with the sugars of amino acid residues 4 and 5 of the template strand. · K529R, which results in the acquisition of a dual interaction with the phosphodiester bond between residues 1 and 3 of the primer strand. · T534K, which results in the acquisition of an interaction with the sugar of amino acid residue 4 of the primer strand. · T534R, which results in the acquisition of an interaction with the phosphodiester between residues 3 and 4 of the primer strand. · Results in the acquisition of an interaction with the phosphodiester between residues 2 and 3 of the primer strand, namely K538R.

[0040] The mutants designed to enhance the affinity of Phi29 DNApol for short primers were expressed and purified according to the standard protocol for obtaining WT Phi29 DNApol. It is not possible to predict whether the specific acquisition of the interaction with the primer strand caused by the introduced mutations will adversely affect Phi29 DNApol functions such as translocation and processivity, or DNA amplification techniques using appropriate (TthPrimPol) and random primers.

[0041] III. Nucleic Acids, Expression Constructs, Recombinant Cells, and Mutant Polymerase Polypeptides A. Nucleic Acids Provided herein are nucleic acids having a nucleotide sequence encoding a mutant Phi29 polymerase with improved primer recognition. The nucleotide sequence of wild-type Phi29 polymerase is shown in SEQ ID NO: 1. Nucleic acids encoding the sequence of mutant Phi29 polymerase have 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.

[0042] B. Expression Constructs Also provided herein are expression constructs comprising a transcriptional regulatory sequence operably linked to the nucleotide sequence encoding the mutant Phi29 polymerase described herein. This expression construct can be in the form of a plasmid or any other form suitable for expression in the cell of interest.

[0043] C. Recombinant Cells Also provided herein are recombinant cells comprising the expression constructs described herein. In certain embodiments, the cells are bacterial cells. Such recombinant cells are useful for replicating the nucleic acid molecules of the present disclosure and for producing the mutant Phi29 polymerase of the present disclosure. The mutant Phi29 polymerase can be produced by culturing recombinant cells comprising the expression construct. The transcriptional regulatory sequences used can include constitutive promoters.

[0044] D. Mutant Phi29 Polymerase Also provided herein is a mutant Phi29 polymerase with improved primer recognition. The mutant Phi29 polymerase of the present disclosure has an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 1 (also deposited as UniProtKB - P03680) and comprises one or both of the amino acid substitutions K64R and M97K.

[0045] Polymerases having substantially identical amino acid sequences can be based on sequences found in nature, such as allelic variants, provided that they comprise one or both of the amino acid substitutions K64R and M97K. Such variants can have up to 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 substitution, addition, or deletion, or up to 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 or fewer amino acid substitutions, additions, or deletions compared to the wild - type sequence SEQ ID NO: 1, provided that one 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. Usage This specification provides a method for performing primer extension and / or nucleic acid polymerization using the mutant Phi29 polymerase described herein. The method of primer extension is useful for nucleic acid replication, amplification, and sequencing.

[0048] Primer extension includes hybridization of a primer nucleic acid molecule to a template and a subsequent polymerization reaction catalyzed by a polymerase that adds nucleotides to the 3'-end of the primer. The primer can be added exogenously to the reaction mixture or can be generated by a primase / polymerase. A primase is an enzyme that catalyzes the synthesis of an oligonucleotide called a primer that is complementary to a nucleic acid template. One such primase is, for example, TthPrimPol.

[0049] For nucleic acid amplification, synthetic primers are generally used. Such primers typically range in length from about 6 to about 25 nucleotides. When attempting to amplify a specific sequence, the primer can have a sequence complementary to the target sequence. For whole genome amplification or other non-directed amplification methodologies, random primers can be used. Random primers typically consist of a collection or set of oligonucleotides, with each base present at each position of the oligonucleotides of one or more primers within the set. 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 Amplification of nucleic acids, such as by the 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 various applications, such as nucleic acid cloning, manipulation or sequencing, DNA-based functional and phylogenetic analysis of genes, detection and diagnosis of diseases, and even scientific investigations (forensics) and paternity testing.

[0051] B. Rolling Circle Amplification Rolling circle amplification is a method for amplifying covalently closed circular DNA molecules, such as single-stranded covalently closed circular DNA molecules. The template DNA molecule is primed with a primer, such as a primer provided by primase / polymerase. The DNA polymerase performs primer extension with respect to the primer along the closed circular DNA molecule. The polymerase replaces the hybridized copy and continues the extension of the polynucleotide along the template to generate a concatenated amplification product.

[0052] C. Multiple Displacement Amplification (MDA) Multiple displacement amplification (MDA) is a PCR-based isothermal DNA amplification method; in this method, ssDNA strands are generated by priming and extension from a template, which are continuously re-primed and copied by strand displacement synthesis to generate a multi-branched DNA structure. After initially denaturing a double-stranded DNA sample, DNA synthesis can be continuously primed and extended from many positions on the amplified molecules without the need for further denaturation rounds, so multiple displacement amplification (MDA) results in a multi-branched structure. As new primers are extended into regions branched from a single DNA molecule template, the branched strands replace 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 follows: 「Isothermal polymerization that extends primers at multiple priming sites on a self-generated ssDNA template」.

[0053] In certain embodiments, MDA utilizes random trimers, tetramers, pentamers, hexamers, heptamers, or octamers as primers to promote amplification at multiple sites on the initial template and its amplified copies. 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) random synthetic primers and / or DNA-dependent primase / polymerase, such as TthPrimPol; 2) a modified DNA polymerase having strand displacement activity, such as Phi29 DNApol; 3) dNTPs. In certain embodiments, the dNTP substrates are unmodified. In other embodiments, the dNTPs can be modified by the attachment of a labeling group, such as 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. These three reagents are combined to facilitate multiple displacement amplification (MDA) of a given DNA that is multiply primed by either a random synthetic primer or a primase / polymerase and extended by a DNA polymerase. Further, combinations of random synthetic primers and / or primase / polymerase and DNA polymerase can perform multiple strand displacement amplification via priming of the amplified molecules by the primase / polymerase and / or random oligonucleotide primers and primer extension by the DNA polymerase.

[0055] 1. DNA polymerase having strand displacement activity Amplification methods such as MDA can use a DNA polymerase having strand displacement activity, such as a polymerase that exhibits a stronger binding to single-stranded DNA than to double-stranded DNA. Strand displacement activity can be useful for displacing the hybridized strand of a DNA molecule while extending the primer position.

[0056] Examples of DNA polymerases with strand displacement activity useful in the methods disclosed herein include, for example, Phi29 DNApol. Phi29 DNApol can be obtained commercially, for example, from New England Biolabs (Ipswich, MA, USA), ThermoFisher Scientific (Waltham, MA, USA), and Expedeon (Cambridge, UK). Phi29 DNApol combines essentially high processivity and strand displacement ability associated with DNA polymerization and can generate DNA fragments longer than 70 kb from a single enzyme:DNA binding event (Blanco et al., 1989). Due to such potential, Phi29 DNApol can replicate DNA templates containing secondary structures such as hairpin loops. This enzyme also has 3'→5' exonuclease proofreading activity (Blanco and Salas, 1985; Garmendia et al., 1992), resulting in a fidelity up to 1000 times higher compared to methods based on Taq DNA polymerase.

[0057] 2. Deoxyribonucleoside triphosphates Primer creation and primer extension can be achieved simply by providing deoxynucleotide substrates, such as dNTPs, in combination with a special DNA primase / polymerase, such as TthPrimPol, that can synthesize DNA primers (Picher et al, 2016), and a processive DNA polymerase, such as Phi29 DNApol. Generally, these contain the four standard bases A, T, G, C. However, in certain embodiments, non-natural nucleotides such as inosine may be included. In certain embodiments, the nucleotides can have labels for detecting or capturing the polynucleotide into which they are incorporated.

[0058] D. DNA Sequencing Currently, there are many different sequencing technologies, which are generally classified into "first-generation sequencing", "second-generation sequencing" (often referred to as "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 technologies that can obtain many sequences simultaneously using advanced technical (optical) detection methods for base positions. An overview of existing methods is described in (Metzker, 2010).

[0060] Third-generation or single-molecule sequencing (SMS) technologies 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, the term "high-throughput sequencing" refers to sequencing thousands of nucleic acid molecules simultaneously or almost simultaneously. Examples of 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 (SMRT) sequencing (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 thereto. However, the amplified molecules themselves may be subject to amplification of specific amplicons. Amplified molecules corresponding to the exome can be isolated using sequence capture with baits for gene sequences in the genome. An amplified transcriptome can be generated for sequencing by reverse transcribing mRNA into double-stranded cDNA.

[0063] V. Kits Also provided herein are kits for use in practicing the methods described herein. As used herein, the term "kit" refers to a collection of items intended for use together.

[0064] The specific kits disclosed herein contain 2, 3, 4, 5, 6, 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 above elements. The kit can include a container for holding the reagents. The container can itself be placed in a shipping container. The container can be transmitted by hand or by a common carrier such as the national postal system or a delivery service such as FedEx. The kit can also include a container for transporting the collected blood to a central facility, such as a box or a bag. The kit can also typically include instructions for use and software for data analysis and interpretation.

[0065] Exemplary embodiments 1. A Phi29 DNA polymerase comprising one or both of the mutations K64R or M97K. 2. A Phi29 DNA polymerase having an amino acid sequence that has at least 80% identity with SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 3. A method for replicating, amplifying, or sequencing template DNA, the method comprising contacting the DNA with a reaction mixture comprising at least (a) the DNA polymerase according to embodiment 1 or 2, (b) a buffer, (c) magnesium chloride, (d) a primer, and (e) nucleoside triphosphates wherein the method comprises the step of contacting the DNA with the reaction mixture. 4. A kit for performing the method according to embodiment 3, the kit comprising (a) the DNA polymerase according to embodiment 1 or 2, (b) a buffer, and (c) magnesium chloride. 5. A kit for carrying out the method according to embodiment 3, comprising the DNA polymerase according to embodiment 1 or 2, (a) A PrimPol enzyme (e.g., TthPrimPol), (b) Random trimer, (c) Random tetramer, (d) Random pentamer, (e) Random heptamer, (f) Random octamer, (g) dNTP, (h) Reaction buffer, (i) A buffer for use together with any of the above elements The kit, comprising one or more of the above. 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 to SEQ ID NO: 1 and comprising one or both of the amino acid substitutions K64R and M97K. 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. 8. The Phi29-type DNA polymerase according to embodiment 6, having in addition to one or both of the amino acid substitutions K64R and M97K, 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 or fewer amino acid substitutions, additions or deletions. 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 with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to 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 DNA polymerase has the sequence of 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 DNA polymerase has one or both of the amino acid substitutions K64R and M97K, and 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 1 or fewer amino acid substitutions, additions, or deletions. 12. A recombinant nucleic acid comprising a transcriptional regulatory sequence operably linked to the Phi29 DNA polymerase according to any one of embodiments 9 to 11. 13. The recombinant nucleic acid according to embodiment 12, wherein the transcriptional regulatory sequence comprises a bacterial or mammalian promoter. 14. The recombinant nucleic acid according to embodiment 12, which is contained in a vector selected from plasmid vectors, viral vectors, cosmids, and transposons. 15. The recombinant nucleic acid according to embodiment 14, comprising a cloning site arranged with respect to the nucleotide sequence encoding the Phi29 DNA polymerase such that the transcriptional regulatory sequence inserted into the cloning site is operably linked to the nucleotide sequence encoding the Phi29 DNA polymerase. 16. A recombinant cell comprising the recombinant nucleic acid according to any one of embodiments 12 to 15. 17. (a) contacting a nucleic acid template molecule with the Phi29 DNA polymerase according to any one of embodiments 1, 2, and 6 to 8 and reagents sufficient for primer extension; and (b) performing primer extension with the polymerase using the nucleic acid template A method comprising. 18. The method according to embodiment 17, wherein the reagents sufficient for primer extension comprise an oligonucleotide primer. 19. The method according to embodiment 18, wherein the oligonucleotide primer comprises one or more of a trimer, a tetramer, a pentamer, a hexamer, an octamer, a nonamer, or a decamer. 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 5-25 nucleotide link. 22. The method according to embodiment 17, wherein the reagent sufficient for primer extension comprises a primase / polymerase (e.g., TthPrimPol). 23. The method according to embodiment 17, wherein the primer extension is performed at any temperature of about 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 comprises (1) multiple displacement amplification ("MDA") or (2) rolling circle amplification. 26. The method according to embodiment 17, wherein the primer extension comprises a multiple annealing and looping-based amplification cycle (MALBAC). 27. The method according to any one of embodiments 17-26, wherein the Phi29 type DNA polymerase comprises both substitution K64R and M97K.

Examples

[0066] Example 1: Screening to Detect Which Mutants Can Use Shorter Random Synthesis Primers Compared to WT Phi29 DNApol in a Multiple Displacement Amplification Reaction Figure 3 shows the amplification of 1 ng of human genomic DNA by multiple displacement amplification (MDA) using a Phi29 DNApol variant in combination with TthPrimPol or random synthetic primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N) or octamer (8N)).

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

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

[0069] Mutant T499R showed behavior similar to WT Phi29 DNApol.

[0070] Finally, mutants K64R and M97K showed significant improvements compared to WT Phi29 DNApol. Both mutants were the only ones able to use the tetramer, while WT Phi29 DNApol and the remaining mutants did not show amplification yields.

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

[0072] Example 2: In Phi29 DNApol mutants M97K and double mutant K64R / M97K, polymerase activity is dominant over exonuclease activity Figure 4 shows the analysis of the dynamic equilibrium between 3'-5' exonuclease activity and 5'-3' polymerization activity of the optimal mutants (K64R, M97K and double mutant K64R / M97K) of the present invention with respect to WT Phi29 DNApol. 5'-labeled primer TIFF2025108504000001.tif4128 was used as the template Using DNA duplexes formed by hybridizing to TIFF2025108504000002.tif4128, the coupling of DNA synthesis and DNA degradation was analyzed as a function of dNTP concentrations (0, 10, 25, 50, 100, and 500 nM). In the absence of dNTP, exonuclease degradation at the primer ends is observed. This degradation pattern reflects the level of exonuclease activity of the variants of the present invention relative to WT Phi29 DNApol. As the concentration of dNTP increases, 5'-3' polymerization gradually exceeds exonuclease activity; net dNMP incorporation is observed as an increase in the size of the labeled primer, defining the concentration of dNTP required to obtain efficient elongation of the primer for each variant. As seen in Figure 4, variant K64R exhibits a Pol / Exo balance similar to that shown by the WT enzyme and reached the 28mer position at 25 nM dNTP. On the other hand, variants M97K and double mutant K64R / M97K reached the same position (28mer) at the lowest dNTP concentration tested (10 nM), indicating that the polymerase activity of these variants is dominant over exonuclease.

[0073] Example 3: Variants of the present invention (K64R, M97K, and K64R / M97K) can use shorter random synthesis primers compared to WT Phi29 DNApol in multiple displacement amplification reactions Figure 5 shows the amplification of 1 ng of human genomic DNA by multiple displacement amplification (MDA) combining selected Phi29 DNApol variants (K64R, M97K, and double mutant K64R / M97K) with random synthesis primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)). The amplification yields shown are the average values of two independent experiments including 3 replicates for each condition. The standard deviation from the two experiments is shown.

[0074] As can be seen in Figure 5, none of the enzymes tested were able to efficiently amplify genomic DNA using random synthetic trimers. 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 seen with WT Phi29 DNApol. Variant K64R showed the lowest amplification yield (2.7 μg), variant 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 with the double mutant represents the 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 use random pentamers to initiate amplification. Again, the double mutant K64R / M97K gave the highest yield (more than 20 μg of amplified DNA), clearly outperforming the single variants and the WT enzyme.

[0077] A similar comparison pattern was seen for random hexamers, but in all cases the amplification yields were higher.

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

[0079] In the case of octamers, both single mutants of 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 robust and efficient amplification values were confirmed regardless of the length of the random synthetic primer used to initiate amplification.

[0080] Example 4: Influence of ionic strength on background amplification observed in the absence of input DNA in a non-template control (NTC) Figure 6 shows the amplification yields observed in the absence of input DNA when combining WT Phi29 DNApol or selected variants of the present invention (K64R, M97K, and K64R / M97K) 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 not only showed significant amplification yields in the absence of input DNA when using pentamers and hexamers, but also showed significant amplification yields when using tetramers in the case of the double mutant (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 that different amplification mechanisms are involved. 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 in the M97K single mutant and the K64R / M97K double mutant, the stability of primer dimers may be increased under the tested conditions.

[0082] Figure 7 shows the amplification yields observed in the absence of input DNA but with increased ionic strength conditions 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 completely disappeared for all variants and all primer sizes.

[0083] Example 5: High ionic strength conditions enhance the robustness and efficiency of the double mutant K64R / M97K against DNA amplification using random primers of different lengths. Figure 8 shows 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) with WT Phi29 DNApol or selected variants of the invention (K64R, M97K, and K64R / M97K) in combination with random synthetic primers of different sizes (trimer (3N), tetramer (4N), pentamer (5N), hexamer (6N), heptamer (7N), or octamer (8N)). The amplification yields shown are the average values of two independent experiments including three replicates per condition. The standard deviation from the two experiments is shown.

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

[0085] In contrast to what was observed under previous conditions (see Figure 5), the tetramer was efficiently used only by the M97K single mutant and the K64R / M97K double mutant, and the single mutant K64R yielded only a slight amount close to 1 μg. Notably, the amplification yields observed with the M97K single mutant and the K64R / M97K double mutant 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] In the case of the pentamer and hexamer, the M97K single mutant and the K64R / M97K double mutant showed similar results and clearly exceeded the amplification yields obtained with the WT enzyme or the K64R variant. As shown in the absence of ammonium sulfate, WT Phi29 DNApol showed a higher yield than the K64R variant.

[0087] In the case of the heptamer, only the double mutant K64R / M97K maintained the amplification yield obtained with shorter random synthetic primers and / or obtained in the absence of ammonium sulfate. Both the WT Phi29 DNApol and the K64R variant significantly decreased the yield and showed the same value under these conditions. The yield obtained with the M97K mutant also decreased compared to the previous conditions.

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

[0089] The double mutant K64R / M97K of Phi29 DNApol preserves amplification performance under both low and high ionic strength conditions, which may be the result of a gain of function obtained from additional contacts between the enzyme and the nitrogenous bases 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 strength conditions.

[0090] Example 6: The double mutation K64R / M97K results in highly sensitive amplification of trace 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 displacement amplification (MDA) combining WT Phi29 DNApol or selected variants of the present invention (K64R, M97K and K64R / M97K) with random synthetic primers of different sizes (tetramer (4N), pentamer (5N), or hexamer (6N)) under low ionic strength (20 mM KCl; 57 mM NaCl) or high ionic strength (20 mM KCl; 57 mM NaCl; 45 mM (NH4)2SO4) conditions.

[0091] Under low ionic strength conditions (upper panel of Figure 9), the double mutant K64R / M97K gives the most consistent and highest amplification yields under all conditions tested.

[0092] In the case of the random synthetic tetramer, as shown previously (see Figure 5), WT Phi29 DNApol was unable to amplify any of the DNA inputs tested. Variant K64R yielded a detectable amount only with 1 ng of DNA input and lacked the sensitivity to amplify smaller amounts of DNA. In contrast, both the M97K variant and the M97K / K64R variant efficiently amplified the DNA inputs tested, and the double mutant yielded a higher amount in all cases.

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

[0094] In the case of the random synthetic hexamer, all the enzymes were able to efficiently use them to initiate the amplification of each DNA input tested and showed a significant amplification yield in every case. The three variants of the present invention showed a higher amplification yield when analyzing a small DNA input, exceeding WT Phi29 DNApol. Similar to the case of the pentamer, the double mutant M97K / K64R showed the highest amplification efficiency among the three variants of the present invention regardless of the DNA input amount.

[0095] As shown previously (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 effect of this artifact due to primer-dimer amplification.

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

[0097] In the case of random synthetic pentamers under high ionic strength conditions, the M97K variant and the M97K / K64R variant showed the best performance in terms of sensitivity and efficiency, showing higher amplification yields compared to low ionic strength conditions with the same DNA input amount. Increasing the ionic strength of the reaction decreased the amplification efficiency of the variant K64R when testing limited amounts (1 pg and 10 pg) of DNA, but the efficiency was comparable (100 pg) or higher (1 ng) at the other two input amounts. Surprisingly, under these conditions, WT Phi29 DNApol outperformed the K64R variant in all cases.

[0098] In the case of randomly synthesized hexamers 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 showed lower yields for the lowest inputs (1 pg and 10 pg), but increased yields for 100 pg and 1 ng DNA inputs; this indicates a decrease in sensitivity. Variants K64R and WT Phi29 DNApol showed similar behavior. As in the case of pentamers, under these conditions, in all cases, WT Phi29 DNApol resulted in higher amplification yields than the K64R variant.

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

[0100] Example 7: Amplification efficiency and sensitivity are not altered by using the variants of the invention when the primer is generated by TthPrimPol Figure 10 shows the amplification of 1, 10, 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 the DNA primase TthPrimPol (Picher et al, 2016) that can synthesize primers for Phi29 DNApol during the reaction.

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

[0102] Example 8: Selected variants of the invention (K64R, M97K and K64R / M97K) improve amplification coverage measured by CovCheck technology With the CovCheck technology, it is possible to perform 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 verified by comparing the CovCheck coverage values with the actual coverage obtained by 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 corresponds to 5-fold the amount of the human diploid genome and can be the minimum amount that ensures sufficient copies of each chromosome are available for amplification. Below this level, due to the random distribution of molecules within the purified DNA sample, specific regions or entire chromosomes may not be present in the input for amplification, and as a result, regions that are not covered in the amplification products will occur due to the absence of the template rather than amplification failure.

[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 the 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 random synthetic hexamers, the amplification coverage is improved when using 3 variants of the present invention compared to the coverage values obtained with WT Phi29 DNApol.

[0106] In the case of random synthetic pentamers, all enzymes showed coverage values of over 90% under these conditions. Therefore, no significant difference was observed. However, the M97K variant showed perfect coverage in 6 repeats tested and stood out.

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

[0108] When using the enzyme (TthPrimPol) to create DNA primers for Phi29 DNApol, 30 pg (equivalent to 5 genomes) of human genomic DNA was used as input to the amplification reaction, and the estimated coverage values obtained from CovCheck analysis of amplification reactions performed by combining TthPrimPol with WT Phi29 DNApol or the variants of the present invention are shown in Figure 12. The coverage values are the average of 12 independent reactions per condition. CovCheck analysis also reveals the improvement of amplification coverage when using the variants of the present invention and supports their advantage of enhancing the uniformity of amplification products relative to the original DNA input.

[0109] References (incorporated herein by reference) TIFF2025108504000003.tif134158TIFF2025108504000004.tif239159TIFF2025108504000005.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 in a mandatory sense (i.e., must). The terms "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., it includes 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 1, at least 2, or at least 3". In certain embodiments, an invention that "comprises" various elements may also "consist essentially of" these elements. The term "consisting essentially of" refers to including the recited elements and other elements that do not materially affect the basic and novel characteristics of the combination according to the claim.

[0111] The description and drawings of this specification are not intended to limit the invention to the specific forms disclosed, but on the contrary, the invention should be understood to cover all modifications, equivalents, and alternatives within the spirit and scope of the invention as defined by the appended claims. Further modifications and alternative aspects of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description and the drawings should be construed as illustrative only and are for the purpose of teaching those skilled in the art a general way of implementing the invention. It should be understood that the forms of the invention shown and described herein should be regarded as examples of various aspects. Substitutions of elements and materials illustrated and described herein, reversing and omitting parts and processes, and using certain features of the invention independently can all be apparent to those skilled in the art after benefiting from the description of the invention. Changes can be made to the elements described herein without departing from the spirit and scope of the invention as recited in the appended claims. The headings used herein are for organizational purposes only and are not used to limit the scope of the description.

[0112] All publications, patents, and patent applications cited in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0113] Sequence Listing Italicized amino acids are not expressed in some aspects. SEQ ID NO 1: Wild-type Phi29 DNA polymerase (UniProtKB - P03680) TIFF2025108504000006.tif215138TIFF2025108504000007.tif240140TIFF2025108504000008.tif47138SEQ ID NO 2: K64R Phi29 DNApol mutant TIFF2025108504000009.tif175138TIFF2025108504000010.tif240138TIFF2025108504000011.tif87140SEQ ID NO 3: M97K Phi29 DNApol variant TIFF2025108504000012.tif136138TIFF2025108504000013.tif240138TIFF2025108504000014.tif126140SEQ ID NO 4: K64R / M97K Phi29 DNApol double variant TIFF2025108504000015.tif87138TIFF2025108504000016.tif245138TIFF2025108504000017.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 DNA polymerase comprising one or both of the mutations K64R or M97K.

2. A Phi29 DNA polymerase having an amino acid sequence with at least 80% identity to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

3. A method for replicating, amplifying, or sequencing template DNA, the method comprising contacting the DNA with a reaction mixture comprising at least (a) the DNA polymerase according to claim 1 or 2, (b) a buffer, (c) magnesium chloride, (d) a primer, and (e) nucleoside triphosphates wherein the DNA is contacted with the reaction mixture.

4. A kit for performing the method according to claim 3, the kit comprising (a) the DNA polymerase according to claim 1 or 2, (b) a buffer, and (c) magnesium chloride.

5. A kit for performing the method according to claim 3, the kit comprising the DNA polymerase according to claim 1 or 2 and (a) a PrimPol enzyme (e.g., TthPrimPol), (b) a random trimer, (c) a random tetramer, (d) a random pentamer, (e) a random heptamer, (f) a random octamer, (g) dNTP, (h) a reaction buffer, (i) a buffer for use together with any of the above elements wherein the kit comprises one or more of the foregoing.

6. A Phi29 DNA polymerase having an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 1 and comprising one or both of the amino acid substitutions K64R and M97K.

7. The Phi29 DNA polymerase according to claim 6, having the sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

8. The Phi29 DNA polymerase according to claim 6, having in addition to one or both of the amino acid substitutions K64R and M97K, 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 or fewer amino acid substitutions, additions, or deletions.

9. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a Phi29 DNA polymerase, wherein the Phi29 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 wherein the Phi29 DNA polymerase comprises one or both of the amino acid substitutions K64R and M97K, said isolated nucleic acid molecule.

10. The isolated nucleic acid molecule according to claim 9, wherein the Phi29 DNA polymerase has the sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

11. The isolated nucleic acid molecule according to claim 9, wherein the Phi29 DNA polymerase has one or both of the amino acid substitutions K64R and M97K, and in addition 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 1 or fewer amino acid substitutions, additions, or deletions.

12. A recombinant nucleic acid comprising a transcriptional regulatory sequence operably linked to a Phi29 DNA polymerase according to any one of claims 9 to 11.

13. The recombinant nucleic acid according to claim 12, wherein the transcriptional regulatory sequence comprises a bacterial or mammalian promoter.

14. The recombinant nucleic acid according to claim 12, contained in a vector selected from plasmid vectors, viral vectors, cosmids, and transposons.

15. The recombinant nucleic acid according to claim 14, comprising a cloning site arranged with respect to the nucleotide sequence encoding the Phi29 DNA polymerase such that a transcriptional regulatory sequence inserted into the cloning site is operably linked to the nucleotide sequence encoding the Phi29 DNA polymerase.

16. A recombinant cell comprising the recombinant nucleic acid according to any one of claims 12 to 15.

17. (a) contacting a nucleic acid template molecule with a Phi29 DNA polymerase according to any one of claims 1, 2, and 6 to 8 and reagents sufficient for primer extension; and (b) performing primer extension with the polymerase using the nucleic acid template comprising a method.

18. The method according to claim 17, wherein the reagent sufficient for the primer extension comprises an oligonucleotide primer.

19. The method according to claim 18, wherein the oligonucleotide primer comprises one or more of a trimer, a tetramer, a pentamer, a hexamer, an octamer, a nonamer, or a decamer.

20. The method according to claim 19, wherein the primer is a random primer.

21. The method according to claim 18, wherein the oligonucleotide primer has a link of 5 to 25 nucleotides.

22. The method according to claim 17, wherein the reagent sufficient for the primer extension comprises a primase / polymerase (e.g., TthPrimPol).

23. The method according to claim 17, wherein the primer extension is performed at any temperature of about 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 claim 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 claim 17, wherein the primer extension comprises (1) multiple displacement amplification ("MDA") or (2) rolling circle amplification.

26. The method according to claim 17, wherein the primer extension comprises a multiple annealing and looping-based amplification cycle (MALBAC).

27. The method according to any one of claims 17 to 26, wherein the Phi29-type DNA polymerase comprises both substitution K64R and M97K.

Images