Phi29 DNA polymerase mutant with improved thermal stability and use thereof in sequencing

TECHNICAL FIELD

The disclosure belongs to the field of biotechnologies, and particularly relates to a Phi29 DNA polymerase mutant with improved thermal stability and an application thereof in sequencing

BACKGROUND

A Phi29 DNA polymerase, belonging to a family-B DNA polymerase, is a polymerase derived from Bacillus subtilis Phi29 phage. The crystal structure of the Phi29 DNA polymerase shows that, in addition to conserved Palm, Thumb, Finger and Exo domain (having 3′→5′ exonuclease correction activity) of the common family-B DNA polymerase, the Phi29 DNA polymerase also has two other unique domains: TPR1 and TPR2 domains. The TPR2 domain participates in formation of a narrow channel surrounding a downstream DNA strand template, and the dissociation of a double-stranded DNA is forced; at the same time, the Palm, Thumb, TPR1 and TPR2 domains form a circular structure, and tightly bind to the double-stranded DNA formed by an upstream template strand. The structural characteristics of the Phi29 DNA polymerase endow it with a special high sustained synthesis ability, a stronger strand displacement function and a high fidelity. It is often used in applications such as constant temperature amplification such as rolling circle amplification (RCA) of a trace circular DNA or multiple-strand displacement amplification of a genome, for example, a DNA Nanoball Technology of a Beijing Genomics Institute (BGI) gene sequencer and a single-cell whole-gene sequencing technology.

The DNA nanoball technology (DNB Technology) is one of core technologies of the BGI gene sequencer. After a genomic DNA is physically or enzymatically fragmented, a linker is added and it is cyclized into a single-stranded circular DNA, and then the DNA polymerase is replaced with a high-fidelity strand, for example, the Phi29 DNA polymerase or Bst DNA polymerase performs the rolling circle amplification (RCA) on the single-stranded circular DNA, and an amplified product is called as a DNA nanoball (DNB). The nanoball is fixed on an arrayed silicon chip through a DNB loading technology for subsequent on-machine sequencing. Different properties of Phi29 DNA polymerase mutants have different binding abilities to a substrate DNA. The amplified DNA nanoball products are also different in size, uniformity, branch structure, compactness and other characteristics, and then after the DNB is loaded on the silicon chip, the quality of sequencing is also different. Properties of the Phi29 DNA polymerase also affect an amplification effect of a single-cell whole genome amplified by a Multiple Displacement Amplification (MDA) method, and the quality of single-cell sequencing is affected, for example, a Coverage, a Mapping Rate, a mutation detection accuracy and specificity, these are also very concerned parameters in application of a single-cell sequencing scientific research.

The Phi29 DNA polymerase is a medium-temperature polymerase, and may lose activity after being heated at 65° C. for 10 min. Some commercial Phi29 DNA polymerases on the market, due to reasons such as stability, are difficult to meet requirements of kit product development in special applications such as sequencing library amplification and single-cell whole genome amplification because of problems in aspects such as an effective signal site on a chip, an amplification preference, a coverage, and variation detection and evaluation.

SUMMARY

The disclosure provides a protein.

The protein provided by the disclosure is a Phi29 DNA polymerase mutant, and is represented by A) or B) below:

the protein represented by the A) is a protein having DNA polymerase activity that is obtained by modifying at least one amino acid residue in the following 6 sites in a phi29 DNA polymerase amino acid sequence: the 97-th, 123-th, 217-th, 224-th, 515-th, and 474-th sites, herein the rest amino acid residues are not changed; and the protein represented by the B) is a protein having DNA polymerase activity that is derived from the A) by adding a tag sequence to a terminal of the amino acid sequence of the protein represented by the A).

In the above protein, the protein represented by the A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in at least 2 or at least 3 or at least 4 or at least 5 or all of the following 6 sites in the phi29 DNA polymerase amino acid sequence: the 97-th, 123-th, 217-th, 224-th, 515-th, and 474-th sites.

The above modification is to modify only the 6 sites of the 97-th, 123-th, 217-th 224-th, 515-th and 474-th sites in the phi29 DNA polymerase amino acid sequence according to the above sites to be modified, and the rest amino acid residues are not changed except the 6 sites.

In the above protein, the protein represented by the A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in all of the following 5 sites in the phi29 DNA polymerase amino acid sequence: the 97-th, 123-th, 217-th, 224-th, and 515-th sites, herein the rest amino acid residues are not changed. Specifically, it is phi29-337 in an embodiment.

In the above protein, the protein represented by the A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in all of the following 3 sites in the phi29 DNA polymerase amino acid sequence: the 224-th, 515-th and 474-th sites, herein the rest amino acid residues are not changed. Specifically, it is phi29-464 in an embodiment.

In the above protein, the protein represented by the A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in all of the following 3 sites in the phi29 DNA polymerase amino acid sequence: the 97-th, 123-th and 515-th sites, herein the rest amino acid residues are not changed. Specifically, it is phi29-414 in an embodiment.

In the above protein, the protein represented by the A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in all of the following 4 sites in the phi29 DNA polymerase amino acid sequence: the 97-th, 123-th, 217-th and 515-th sites, herein the rest amino acid residues are not changed. Specifically, it is phi29-458 in an embodiment.

In the above protein, the protein represented by the A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in the following 4 sites in the phi29 DNA polymerase amino acid sequence: the 97-th, 123-th, 224-th, and 515-th sites, herein the rest amino acid residues are not changed. Specifically, it is phi29-459 in an embodiment.

In the above protein, the protein represented by the A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in the following 2 sites in the phi29 DNA polymerase amino acid sequence: the 224-th and 474-th sites, herein the rest amino acid residues are not changed. Specifically, it is phi29-463 in an embodiment.

In the above protein, the protein represented by the A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in the following 2 sites in the phi29 DNA polymerase amino acid sequence: the 217-th and 224-th sites, herein the rest amino acid residues are not changed. Specifically, it is phi29-335 in an embodiment.

In the above protein, the modification is amino acid substitution.

In the above protein, the amino acid substitutions in 6 sites of the 97-th, 123-th, 217-th, 224-th, 515-th and 474-th sites are respectively as follows:

a methionine in the 97-th site is substituted with an alanine or a histidine or a lysine or a threonine;

a leucine in the 123-th site is substituted with a lysine or a phenylalanine or an isoleucine or a histidine;

a glycine in the 217-th site is substituted with a glutamic acid;

a tyrosine in the 224-th site is substituted with a lysine;

an isoleucine in the 474-th site is substituted with a lysine; and

a glutamic acid in the 515-th site is substituted with a proline or a glycine.

In the above protein, the protein represented by the A) is a protein having DNA polymerase activity that is obtained by mutating the amino acid residue from M into K in the 97-th site, from L into H in the 123-th site, from E into Pin the 515-th site, from Y into Kin the 224-th site, and from G into K in the 217-th site of the phi29 DNA polymerase amino acid sequence, herein the rest amino acid residues are not changed.

In the above protein, the protein represented by the A) is a protein that is obtained by mutating the amino acid residue from Y into K in the 224-th site, from I into K in the 474-th site, and from E into P in the 515-th site of the phi29 DNA polymerase amino acid sequence.

In the above protein, the protein represented by the A) is a protein that is obtained by mutating the amino acid residue from M into T in the 97-th site, from L into H in the 123-th site, and from E into P in the 515-th site of the phi29 DNA polymerase amino acid sequence.

In the above protein, the phi29 DNA polymerase amino acid sequence is (I) or (II) or (III) below:

(I) a protein shown in SEQ ID NO: 2 of a sequence listing;

(II) a protein having more than 90% identity with the protein shown in SEQ ID NO: 2 of the sequence listing and derived from a Bacillus subtilis protein; and

(III) a protein having more than 95% identity with the protein shown in SEQ ID NO: 2 of the sequence listing and derived from a Bacillus subtilis protein.

In the above protein, the stability of the protein is higher than that of the phi29 DNA polymerase.

In the above protein, the stability is thermal stability.

A nucleic acid molecule for encoding the above protein is also within a scope of protection of the disclosure.

An expression cassette, a recombinant vector, recombinant bacteria or a transgenic cell line containing the above nucleic acid molecule is also within a scope of protection of the disclosure.

Use of the above protein in at least one of the following 1)-11) is also within a scope of protection of the disclosure:

1) as a DNA polymerase;

2) catalyzing DNA replication and/or catalyzing DNA amplification;

3) catalyzing rolling circle amplification and/or catalyzing multiple-strand displacement amplification;

4) performing DNA sequencing or RNA sequencing or whole genome sequencing;

5) constructing a RCA library;

6) detecting a genome amplification coverage;

7) preparing a kit for catalyzing the DNA replication and/or catalyzing the DNA amplification;

8) preparing a product for catalyzing the rolling circle amplification and/or catalyzing the multiple-strand displacement amplification;

9) preparing a product for performing the DNA sequencing or the RNA sequencing or the whole genome sequencing;

10) preparing a product for constructing the RCA library; and

11) preparing a product for detecting the genome amplification coverage.

Use of the nucleic acid molecule for encoding the above protein, the expression cassette containing the nucleic acid molecule, the recombinant vector containing the nucleic acid molecule, the recombinant bacteria containing the nucleic acid molecule, or the transgenic containing the nucleic acid molecule in at least one of the following 1)-10) is also within a scope of protection of the disclosure:

1) catalyzing DNA replication and/or catalyzing DNA amplification;

2) catalyzing rolling circle amplification and/or catalyzing multiple-strand displacement amplification;

3) performing DNA sequencing or RNA sequencing or whole genome sequencing;

4) constructing a RCA library;

5) detecting a genome amplification coverage;

6) preparing a product for catalyzing the DNA replication and/or catalyzing the DNA amplification;

7) preparing a product for catalyzing the rolling circle amplification and/or catalyzing the multiple-strand displacement amplification;

8) preparing a product for performing the DNA sequencing or the RNA sequencing or the whole genome sequencing;

9) preparing a product for constructing the RCA library; and

10) preparing a product for detecting the genome amplification coverage.

Another purpose of the disclosure is to provide a method for improving stability of a phi29 DNA polymerase.

The method provided by the disclosure includes the following steps: modifying at least one amino acid residue(s) in the following 6 sites of the phi29 DNA polymerase amino acid sequence: the 97-th, 123-th, 217-th, 224-th, 515-th and 474-th sites, in the modification modes above, herein the rest amino acid residues are not changed, to obtain a protein having DNA polymerase activity.

In the above method, the stability is thermal stability.

The Phi29 DNA polymerase of the disclosure may exist in the form of an independently packaged DNA polymerase product, or may be packaged into a DNA amplification kit or a DNA sequencing kit. The disclosure is capable of, through site-directed mutagenesis and screening technologies to obtain the mutant with the greatly improved thermal stability. Herein the amplification coverage of some mutants in single-cell whole genome amplification is greatly improved, and the accuracy and sensitivity of the mutation detection are improved, and reach a same level of QIAGEN similar products; and other mutants in the disclosure have a good effect on improving a DNB loading effect in application of RCA library amplification.

The recombinant vector is obtained by inserting the nucleic acid molecule into an expression vector.

The expression vector may specifically be a pET28a(+) vector.

The recombinant bacteria are bacteria obtained by introducing the recombinant vector into original bacteria.

The original bacteria may be Escherichia coli.

The E. coli may specifically be Escherichia coli BL21 (DE3).

The transgenic cell line may be obtained by transforming the recombinant vector into recipient cells. The transgenic cell line is a non-plant propagative material.

The stability may specifically be thermal stability. The thermal stability may specifically be thermal stability at 37° C.

The phi29 DNA polymerase may be (I) or (II) or (III) below:

(I) a protein shown in SEQ ID NO: 2 of a sequence listing;

(II) a protein having more than 90% identity with the protein shown in SEQ ID NO: 2 of the sequence listing and derived from a Bacillus subtilis protein; and

(III) a protein having more than 95% identity with the protein shown in SEQ ID NO: 2 of the sequence listing and derived from a Bacillus subtilis protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a construction schematic diagram of a Phi29 gene expression vector.

FIG. 2 is a coverage of a housekeeping gene in a MDA product detected by multiple PCR.

FIG. 3 is data analysis of an on-machine test of RCA library construction.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following embodiments are convenient for understanding the disclosure better, but do not limit the disclosure. Unless otherwise specified, experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, test materials used in the following embodiments are all purchased from conventional biochemical reagent stores. Quantitative tests in the following embodiments are all set to three times of repeated experiments, and results are averaged. For each solution or buffer in the following embodiments, unless otherwise specified, solvents are all water.

pET28a(+) vector: Novagen Company.

Escherichia coli BL21 (DE3): TIANGEN, CB105-02.

Storage buffer: 10 mM Tris-HCI, 100 mM KCI, 1 mM DTT, 0.1 mM EDTA, 0.5% (v/v) Tween20, 0.5% (v/v) NP-40, 50% (v/v) Glycerol, and pH 7. 4@25° C.

141 RCA Primer in the following embodiments: TCCTAAGACCGCTTGGCCTCCGACT (SEQ ID NO:3).

141 Ad ssDNA in the following embodiments: self-made by BGI, a single-stranded loop library of a certain size range, without a fixed sequence (a random library formed by four nucleotides A/T/C/G, and a main band length is 200-300 bp).

In the following embodiments, a DNA molecule shown in SEQ ID NO: 1 of a sequence listing is an encoding gene of a wild-type Phi29 DNA polymerase, and it expresses a protein shown in SEQ ID NO: 2 of the sequence listing, namely the wild-type Phi29 DNA polymerase (represented by WT).

Embodiment 1: Preparation of Phi29 DNA Polymerase Mutant

A Phi29 DNA polymerase mutant is obtained by mutating at least one of the 97-th, 123-th, 217-th, 224-th, 474-th, and 515-th sites in an amino acid sequence of a wild-type Phi29 DNA polymerase. Specifically, a single-site mutation form is shown in Table 1, and a multi-site combination mutation form is shown in Table 2.

I. Construction of a Recombinant Vector for Expressing Phi29 DNA Polymerase Mutant

1. Recombinant Vector for Expressing Phi29 DNA Polymerase Single-Site Mutant

A DNA molecule shown in SEQ ID NO: 2 of a sequence listing is inserted between Ndel and BamHl restriction sites of a pET28a(+) vector (FIG. 1), to obtain a recombinant vector WT for expressing a wild-type Phi29 DNA polymerase.

The recombinant vector WT is served as an original vector, each primer pair shown in Table 1 is used to introduce a site mutation, and each recombinant vector for expressing the Phi29 DNA polymerase mutant is obtained.

The above method of introducing the site mutation by using each primer pair shown in Table 1 is as follows.

The recombinant vector WT is tested as a template, in a site-directed mutation PCR reaction system (25 pl) containing the following components: 2.5 μl 10× Pfu Reaction Buffer with Mg²⁺, 2 μl dNTP Mix (2.5 mM each), 25 ng pET28a-Phi29 plasmid, 0.5 μl Pfu DNA Polymerase, mutation primers of mutation sites (Table 1) are respectively added, and PCR amplification is performed.

PCR conditions are as follows: 95° C. 3 min, 19 cycle for [95° C. 30s, 53° C. 30s, 68° C. 8 min], 68° C. 10 min, 4° C. forever. After the reaction product is digested with Dpnl, it is transformed into DH5a competent cells, spread out on a kanamycin-resistant LB plate, and then it is incubated overnight in an incubator at 37° C., a monoclonal plasmid is picked out for sequencing, to verify whether a specific amino acid site is successfully mutated.

TABLE 1

Single-site mutation primers

Phi29

DNA

polymerase

mutant
Forward primer
Reverse primer

M97H
CACCATTATTAGCCGCCATGGCCAGT
CAATCATATACCACTGGCCATGGCGGCT

GGTA TATGATTG (SEQ ID NO: 4)
AATA ATGGTG (SEQ ID NO: 5)

M97A
CACCATTATTAGCCGCGCGGGCCAGT
CAATCATATACCACTGGCCCGCGCGGC

GGTA TATGATTG (SEQ ID NO: 6)
TAATA ATGGTG (SEQ ID NO: 7)

M97K
CACCATTATTAGCCGCAAAGGCCAGT
CAATCATATACCACTGGCCTTTGCGGCT

GGTA TATGATTG (SEQ ID NO: 8)
AATA ATGGTG (SEQ ID NO: 9)

L123K
CCGTGATCTATGATAGCAAAAAGAAAC
CGGAAACGGCAGTTTCTTTTTGCTATCA

TGC CGTTTCCG (SEQ ID NO: 10)
TAGA TCACGG (SEQ ID NO: 11)

L123F
CCGTGATCTATGATAGCTTTAAGAAAC
CGGAAACGGCAGTTTCTTAAAGCTATCA

TGC CGTTTCCG (SEQ ID NO: 12)
TAGA TCACGG (SEQ ID NO: 13)

L123I
CCGTGATCTATGATAGCATTAAGAAAC
CGGAAACGGCAGTTTCTTAATGCTATCA

TGC CGTTTCCG (SEQ ID NO: 14)
TAGA TCACGG (SEQ ID NO: 15)

L123H
CCGTGATCTATGATAGCCATAAGAAAC
CGGAAACGGCAGTTTCTTATGGCTATCA

TGC CGTTTCCG (SEQ ID NO: 16)
TAGA TCACGG (SEQ ID NO: 17)

E515P
GGATGGCAAACTGGTTCCGGGCAGC
CATCCGGGCTGCCCGGAACCAGTTTGC

CCGGA TG (SEQ ID NO: 18)
CATCC (SEQ ID NO: 19)

Phi29-192
GATAAAGAAGTGCGCAAAGCGTATCG
GCCACCGCGATACGCTTTGCGCACTTC

(Y224K)
CGGT GGC (SEQ ID NO: 20)
TTTATC (SEQ ID NO: 21)

Phi29-103
GACCCTGAGCCTGGAACTGGATAAAG
GCACTTCTTTATCCAGTTCCAGGCTCAG

(G217E)
AAGT GC (SEQ ID NO: 22)
GGTC (SEQ ID NO: 23)

phi29-85
GATGTGATCAAAGATTTTGTGGACCC
GTTTTTTCGGGTCCACAAAATCTTTGAT

(I474K)
GAAA AAAC (SEQ ID NO: 24)
CACA TC (SEQ ID NO: 25)

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant M97H is only that: the 289-291-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “ATG” into “CAT”. The mutated DNA molecule encodes the mutant. M97H. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant M97H is only that the amino acid residue in the 97-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M into H.

Compared with the recombinant vector WI, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant M97A is only that the 289-291-th nucleotides of the DNA molecule shown in SEQ ID NO. 1 of the sequence listing are mutated from “ATG” into “GCG”. The mutated DNA molecule encodes, the mutant M97A Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant M97A is only that the amino acid residue in the 97-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M into A.

Compared with the recombinant vector WI, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant M97K is only that the 289-291-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “ATG” into “AAA”. The mutated DNA molecule encodes the mutant M97K. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant M97K is only that the amino acid residue in the 97-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M into K.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant L123K is only that: the 367-369-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “CTG” into “AAA”. The mutated DNA molecule encodes the mutant L123K. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant L123K is only that the amino acid residue in the 123-th site of the wild-type Phi29 DNA polymerase amino acid, sequence is mutated from L into K.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant L123F is only that: the 367-369-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “CTG” into “TTT”. The mutated DNA molecule encodes, the mutant L123F. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant L123F is only that the amino acid residue in the 123-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from L into F.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant L123I is only that: the 367-369-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “CTG” into “ATT”. The mutated DNA molecule encodes the mutant L123I. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant L 123I is only that the amino acid residue in the 123-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from L into I.

Compared with the recombinant vector WT, a different from a recombinant vector for expressing a Phi29 DNA polymerase mutant L123H is only that: the 367-369-th nucleotides of the DNA molecule shown in SEQ ID NO 1 of the sequence listing are mutated from “CTG” into “CAT”. The mutated DNA molecule encodes the mutant L123H. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant L123H is only that the amino acid residue in the 123-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from L into H.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant E515P is only that: the 1543-1545-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GAA” into “CCG”. The mutated DNA molecule encodes the mutant E515P. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant E515P is only that the amino acid residue in the 515-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from E into P.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant Y224K is only that: the 670-672-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “TAT” into “AAA”. The mutated DNA molecule encodes the mutant Y224K. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant Y224K is only that the amino acid residue in the 224-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from Y into K.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant G217E is only that: the 649-651-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GGC” into “GAA”. The mutated DNA molecule encodes the mutant G217E. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant G217E is only that the amino acid residue in the 217-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from G into E.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant 1474K is only that: the 1420-1422-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “ATT” into “TTT”, The mutated DNA molecule encodes the mutant I474K. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant 1474K is only that the amino acid residue in the 474-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from I into K.

2. Construction of Recombinant Vector for Expressing Phi29 DNA Polymerase Multi-Site Mutant

The recombinant vector WT is served as an original vector, each primer pair shown in. Table 1 is used to introduce a site mutation successively, to obtain each recombinant vector for expressing a Phi29 DNA polymerase mutant. A specific mutation form is shown in Table 2:

TABLE 2

Phi29 DNA polymerase
Phi29 DNA polymerase

mutant number
mutant name

phi29-337
M97K-L123H-E515P-Y224K-G217E

phi29-414
M97T-L123K-E515P

phi29-456
M97K-L123H-E515P

Phi29-464
Y224K-I474K-E515P

phi29-458
M97K-L123H-E515P-G217E

phi29-459
M97K-L123H-E515P-Y224K

Phi29-463
Y224K-I474K

Phi29-335
Y224K-G217E

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant M97K-L123H-E515P-Y224K-G217E is only that: the 289-291-th nucleotides of the DNA molecule shown in SEQ ID NO 1 of the sequence listing are mutated from “ATG” into “AAA”, the 367-369-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “CTG” into “CAT” the 1543-1545-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GAA” into “COG”, the 670-672-th nucleotides of the DNA molecule shown in SEQ ID NO 1 of the sequence listing are mutated from “TAT” into “AAA”, the 649-651-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GGC” into “GAA”. The mutated DNA molecule encodes the mutant M97K-L123H-E515P-Y224K-G217E. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant M97K-L123H-E515P-Y224K-G217E is only that the amino acid residue in the 97-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M into K, and the 123-th site is mutated from L into H, the 515-th site is mutated from E into P, the 224-th site is mutated from Y into K, and the 217-th site is mutated from G into E.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant M97T-L123K-E515P is only that the 289-291-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “ATG” into “ACC”, the 367-369-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “CTG” into “AAA”, the 1543-1545-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GAA” into “CCG”. The mutated DNA molecule encodes the mutant M97T-L123K-E515P. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant M97T-L123K-E515P is only that the amino acid residue in the 97-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M into T, and the 123-th site is mutated from L into K, and the 515-th site is mutated from E into P.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant M97K-L123H-E515P is only that: the 289-291-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “ATG” into “AAA”, the 367-369-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “CTG” into “CAT”, the 1543-1545-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GAA” into “CCG”. The mutated DNA molecule encodes the mutant M97T-L123H-E515P. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant M97K-L123H-E515P is only that the amino acid residue in the 97-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M into T, and the 123-th site is mutated from L into H, and the 515-th site is mutated from E to P.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant Y224K-I474K-E515P is only that: the 670-672-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “TAT” into “AAA”, the 1420-1422-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “ATT” into “TTT”, the 1543-1545-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GAA” into “CCG”. The mutated DNA molecule encodes the mutant Y224K-I474K-E515P. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant Y224K-I474K-E515P is only that the amino acid residue in the 224-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from Y into K, and the 474-th site is mutated from I into K, and the 515-th site is mutated from E into P.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant M97K-L123H-E515P-G217E is only that: the 289-291-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “ATG” into “AAA”, the 367-369-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “CTG” into “CAT”, the 1543-1545-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GAA” into “CCG”, and the 649-651-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GGC” to “GAA”. The mutated DNA molecule encodes the mutant M97K-L123H-E515P-G217E. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant M97K-L123H-E515P-G217E is only that the amino acid residue in the 97-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M into K, the amino acid residue in the 123-th site is mutated from L into H, and the 515-th site is mutated from E into P, and the 217-th site is mutated from G into E.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant M97K-L123H-E515P-Y224K is only that: the 289-291-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “ATG” into “AAA”, the 367-369-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “CTG” into “CAT”, the 1543-1545-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “GAA” into “CCG”, and the 670-672-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “TAT” into “AAA”. The mutated DNA molecule encodes the mutant M97K-L123H-E515P-G217E. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant M97K-L123H-E515P-G217E is only that the amino acid residue in the 97-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M into K, the amino acid residue in the 123-th site is mutated from L into H, and the 515-th site is mutated from E into P, and the 224-th site is mutated from Y into K.

Compared with the recombinant vector WT, a difference from a recombinant vector for expressing a Phi29 DNA polymerase mutant Y224K-1474K is only that: the 670-672-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “TAT” into “AAA”, the 1420-1422-th nucleotides of the DNA molecule shown in SEQ ID NO: 1 of the sequence listing are mutated from “ATT” into “TTT”. The mutated DNA molecule encodes the mutant Y224K-1474K. Compared with the wild-type Phi29 DNA polymerase, a difference from the mutant Y224K-I474K is only that the amino acid residue in the 224-th site of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from Y into K, and the amino acid residue in the 474-th site is mutated from I into K.

II. Construction of Recombinant Bacteria for Expressing Phi29 DNA Polymerase Mutant

The recombinant vector INT and each recombinant vector for expressing the Phi29 DNA polymerase mutant constructed in step I are respectively introduced into Escherichia coli BL21 (DE3), to obtain recombinant bacteria for expressing the wild-type Phi29 DNA polymerase and each recombinant bacterium for expressing the Phi29 DNA polymerase mutant.

III. Induced Expression of Recombinant Bacteria

The recombinant bacteria for expressing the wild-type Phi29 DNA polymerase and each recombinant bacterium for expressing the Phi29 DNA polymerase mutant obtained in step II are taken, and sequentially induced and purified, to obtain the wild-type Phi29 DNA polymerase fused with an His₆tag at an N-terminal and each Phi29 DNA polymerase mutant fused with an His₆tag at an N-terminal.

The wild-type Phi29 DNA polymerase fused with the His₆tag at the N-terminal and each Phi29 DNA polymerase mutant fused with the His₆tag at the N-terminal are successively named as a wild-type Phi29 DNA polymerase with the His₆tag, a Phi29 DNA polymerase mutant M97H with the His₆tag, a Phi29 DNA polymerase mutant M97A with the His₆tag, a Phi29 DNA polymerase mutant M97K with the His₆tag, a Phi29 DNA polymerase mutant L123K with the His₆tag, a Phi29 DNA polymerase mutant L123F with the His₆tag, a Phi29 DNA polymerase mutant L123I with the His₆tag, a Phi29 DNA polymerase mutant L123H with the His₆tag, a Phi29 DNA polymerase mutant E515P with the His₆tag, a Phi29 DNA polymerase mutant E515G with the His₆tag, a Phi29 DNA polymerase mutant Y224K with the His₆tag, a Phi29 DNA polymerase mutant G217E with the His₆tag, a Phi29 DNA polymerase mutant 1474K with the His₆tag, a Phi29 DNA polymerase mutant M97K-L123H-E515P-Y224K-G217E with the His₆tag, a Phi29 DNA polymerase mutant M97T-L123K-E515P with the His₆tag, a Phi29 DNA polymerase mutant Y224K-1474K-E515P with the His₆tag, a Phi29 DNA polymerase mutant M97K-L123H-E515P-G217E with the His₆tag, a Phi29 DNA polymerase mutant M97K-L123H-E515P-Y224K with the His₆tag, a Phi29 DNA polymerase mutant Y224K-1474K with the His₆tag, and a Phi29 DNA polymerase mutant Y224K-G217E with the His₆tag.

1. Specific steps of induction are as follows:

(1) Live bacteria

The recombinant bacteria are seeded into 3 ml of a liquid LB medium containing Kana resistance, and cultured overnight.

(2) Transfer

After the step (1) is completed, the above bacterial solution is transferred to 2 ml of the liquid LB medium containing the Kana resistance according to a 1/100 volume, and shake-cultured at 37° C. and 220 rpm until OD_600nm=0.6 (in a practical application, OD_600nm=0.4−0.8 is acceptable).

(3) Induction

After the step (2) is completed, IPTG with a final concentration of 0.5 mM is added to a system, and shake-cultured at 16° C. and 220 rpm for 12 h.

(4) Bacteria collection

After the step (3) is completed, it is centrifuged at 4° C. and 8000 rpm for 5 min, and bacteria are collected.

2. An AKTA Pure purification system of GE Company is used for purification, and specific steps are as follows:

(1) The bacteria obtained in the step 1 are taken, shaken and uniformly mixed with resuspension buffer (20 mM Tris-HCI, 500 mM NaCI, 20 mM Imidazole, 5% Glycerol, and pH 7.9 @25° C.), it is ultrasonically crushed on ice, and then centrifuged at 4° C. and 12000 rpm for 30 min, and supernatant is collected.

(2) The supernatant obtained in the step (1) is taken, and a nickel column affinity chromatography (HisTrap FF 5 ml pre-packed column) is used for purification. Specific steps are as follows: firstly it is equilibrated with 10 column volumes of Buffer A; then a sample is loaded; then it is rinsed with 20 column volumes of the Buffer A; then it is eluted with 15 column volumes of eluent and post-column solution with a target protein is collected (the eluent is formed by Buffer A and Buffer B, and in an elution process, a volume fraction of the Buffer B is linearly increased from 0% to 100%, and correspondingly a volume fraction of the Buffer A is linearly decreased 100% to 0%).

Buffer A: 20 mM Tris-HCI, 500 mM NaCI, 20 mM Imidazole, 5% (vlv) Glycerol; pH 7.9@ 25° C.

Buffer B: 20 mM Tris-HCI, 500 mM NaCI, 500 mM Imidazole, 5% (vlv) Glycerol; pH7.9@ 25° C.

(3) The post-column solution obtained in the step (2) is taken, and a strong anion column chromatography (HiTrap Q HP 5 ml pre-packed column) is used for purification. Specific steps are as follows: firstly it is equilibrated with 10 column volumes of mixed buffer formed by Buffer A with a volume fraction of 59% and Buffer B with a volume fraction of 41%; then a sample is loaded; and after a protein peak appears, flow-through solution is collected (collection is performed after a UV detection value raises to 20 mAu, and the collection is stopped after the UV detection value drops to 50 mAu).

Buffer A: 20 mM Tris-HCI, 150 mM NaCI, 5% (v/v) Glycerol, pH7.5@ 25° C.

Buffer B: 20 mM Tris-HCI, 1 M NaCI, 5% (v/v) Glycerol, pH7.5@ 25° C.

(4) The flow-through solution obtained in the step (3) is taken, and a cation exchange chromatography (HiTrap SP HP pre-packed column) is used for purification, to obtain protein sample solution of which a purity is greater than 95%. Specific steps are as follows: firstly it is equilibrated with 10 column volumes of Buffer A; then a sample is loaded; then it is rinsed with 15 column volumes of the Buffer A; then it is eluted with 10 column volumes of eluent (the eluent is formed by Buffer A and Buffer B, and in an elution process, a volume fraction of the Buffer B is linearly increased from 0% to 50%, and correspondingly a volume fraction of the Buffer A is linearly decreased 100% to 50%) and post-column solution with a target protein is collected (collection is performed after a UV detection value raises to 50 mAu, and the collection is stopped after the UV detection value drops to 100 mAu).

Buffer A: 20 mM Tris-HCI, 150 mM NaCI, 5% (v/v) Glycerol, pH7.5@ 25° C.

Buffer B: 20 mM Tris-HCI, 1 M NaCI, 5% (vlv) Glycerol, pH7.5@ 25° C.

(5) The target protein collected in the step (4) is taken and transferred to a dialysis bag, after dialysis in dialysis buffer overnight, protein solution in the dialysis bag is collected, and other reagents are added, to obtain target protein solution with a protein concentration of 1 mg/ml. The concentrations of other components in the target protein solution are as follows: 10 mM Tris-HCI (pH7.4@ 25° C.), 100 mM KCI, 1 mM DTT, 0.1 mM EDTA, 0.5%(v/v)NP-40, 0.5% (v/v) Tween20, 50% (vlv) Glycerol.

Dialysis buffer: 23.75 mM Tris-HCI (pH7.4@ 25° C.), 237.5 mM KCI, 2.375 mM DTT, 0.2375 mM EDTA, 5% (v/v) Glycerol.

Embodiment 2: Polymerization Activity Test of Wild-Type Phi29 DNA Polymerase and Mutant

The wild-type Phi29 DNA polymerase solution with the His₆tag and the Phi29 DNA polymerase mutant solution with the His₆tag prepared in Embodiment 1 are taken as enzyme solution to be tested.

1. Each enzyme solution to be tested is diluted to 5000 times in a gradient by using storage buffer, adequately and uniformly mixed in a vortex shaker, and it is standing on ice for 5 min, to obtain each solution to be tested.

A pre-reaction system is uniformly mixed in a PCR tube, and placed in a PCR instrument, the following procedures are performed: 95° C. 1 min, 65° C. 1 min, 40° C. 1 min, and a hot lid temperature is set to 102° C.

Pre-reaction system (80.8 μl): 50 mM Tris-HCI (pH7.5), 4 mM DTT, 10 mM (NH₄)₂SO₄, 10 mM MgCl₂, 50 nM dNTP Mixture, 2 pM 141 RCA Primer and 18 ng single-stranded circular DNA template 141 Ad ssDNA.

2. After the step 1 is completed, the PCR tube is taken out and placed on ice while the temperature reaches 4° C. 1 μl of each solution to be tested is respectively added in a test group, and 1 μl of the storage buffer is added in a negative control group. Then, the vortex shaker is used to shake and mix uniformly, it is placed in the PCR instrument after being transitorily centrifuged for 5 s in a centrifuge, and the following procedures are performed: 30° C. 60 min, and a hot lid temperature is set to 65° C.

3. After the step 2 is completed, 5 μL of 0.5 M EDTA solution is added to stop a reaction, and it is shaken and mixed uniformly.

4. A Qubit ssDNAAssay Kit (Q10212, INVITROGEN) is used and operated according to instructions. A Qubit fluorometer 3.0 is used to detect a concentration of DNB (DNA Nano ball) in a reaction product.

Enzyme activity of enzyme solution to be tested=ΔDNB×5000÷37·38.

Note: ΔDNB is a difference value between concentration average values of the reaction products in the systems after the reactions of the test group and the negative control group are terminated, 5000 is a dilution factor, and 37.38 is a slope of a functional relationship between the enzyme activity and the ΔDNB.

Enzyme activity results of some enzyme solution to be tested are shown in Table 3.

TABLE 3

Control
Experimental

Enzyme activity

group
group

of prepared

average
average
ΔDNB
enzyme solution

Polymerase
value
value
(ng/μl)
(U/μl)

WT
0.46
0.96
0.51
67.6

E515P
0.37
0.73
0.37
49.3

L123K
0.4
0.77
0.37
50.1

M97A
0.4
0.75
0.36
48

G217E
0.42
0.97
0.55
73.8

Y224K
0.38
0.8
0.42
56.1

1474K
0.48
1.06
0.57
15.4

Embodiment 3: Thermal Stability Test of Wild-Type Phi29 DNA Polymerase and Mutant

37° C. 10 min experiment:

The target protein solution (the wild-type Phi29 DNA polymerase solution with the His₆tag and the Phi29 DNA polymerase mutant solution with the His₆tag) prepared in Embodiment 1 is taken, and divided into two parts, and they are respectively treated as follows:

First part: it is placed in a metal bath preheated to 37° C. for 10 min, and centrifuged at 4° C. and 13000 rpm for 1 min, and supernatant is collected; and then, the supernatant is taken, and diluted to 1000 times of a volume with the storage buffer, and the vortex shaker is used to adequately mix uniformly, and then it is standing on ice for 5 min, to obtain solution 1 to be tested.

Second part: it is diluted to 5000 times of a volume with the storage buffer, and adequately mixed uniformly with the vortex shaker, and then it is standing on ice for 5 min, to obtain solution 2 to be tested.

It is performed according to the steps 1 to 4 of Embodiment 2.

Unheated enzyme activity (U1)=ΔDNB×5000÷37·38; ΔDNB is a difference value between concentration average values of reaction products in the systems after the reactions of the test group (second part) and the negative control group are terminated;

Heated enzyme activity (U2)=ΔDNB×1000÷37·38; ΔDNB is a difference value between concentration average values of reaction products in the systems after the reactions of the test group (first part) and the negative control group are terminated;

5000 and 1000 are dilution factors respectively, and 37.38 is a slope of a functional relationship between the enzyme activity and the ΔDNB;

an enzyme activity loss ratio is calculated according to the activities of the unheated and heated enzyme solution,

Enzyme activity loss ratio (%)=(U1-U2)÷U1×100%;

U1 is the activity of the unheated enzyme solution, and U2 is the activity of the heated enzyme solution.

−20° C. experiment: a difference from the 37° C. 10 min experiment is only that the 10 min in the 37° C. metal bath is replaced with the 10 min in a refrigerator at −20° C.

The specific enzyme activity and the enzyme activity loss ratio of each mutant are shown in Table 4. Compared with the wild type (WT), the enzyme activity loss ratios of the mutants in examples are all improved to a certain extent; and a numeral value of the enzyme activity loss ratio is larger, the enzyme is more unstable, and the thermal stability is worse.

TABLE 4

Note

Diluted
Original

Original

enzyme
enzyme
Specific

enzyme

Heat
Average

solution
solution
enzyme
Enzyme
Enzyme
solution

treatment
value
ΔDNB
activity
activity
activity
activity
concentration
dilution

Mutation site
condition
(ng/μl)
(ng/μl)
(U)
(U/μl)
(U/ug)
loss ratio
(mg/ml)
factor

Phi29-WT
−20°
C.
1.12
0.74
0.02
59.2
74
98.60%
0.8
3000

(Wild type)
37°
C.

10
min
0.68
0.3
0.008
0.8

100

NC
0.38

Phi29-Enzymatics

(Commercial enzyme

of Enzymatics Company)

−20°
C.
1.04
0.57
0.015
76.7
63.9
85.40%
1.2
5000

37°
C.

10
min
0.55
0.08
0.002
11.2

5000

NC
0.46

Phi29-337
−20°
C.
1.87
1.41
0.038
18.8
104.6
7.80%
0.18
500

(M97K-L123H-
37°
C.

E515P-Y224K-
15
min
1.76
1.3
0.035
17.4

500

G217E)
NC
0.46

Phi29-414
−20°
C.
1.13
0.734
0.020
19.6
67.7
12.26%
0.29
1000

(M97T-L123K-
37°
C.

E515P)
10
min
1.04
0.644
0.017
17.2

1000

NC
0.396

Phi29-456
−20°
C.
1.14
0.697
0.018
36.9
60.2
35.19%
0.613
2000

(M97K-L123H-
37°
C.

E515P)
10
min
2.25
1.807
0.048
23.9

500

NC
0.443

Phi29-464
−20°
C.
2.05
1.633
0.027
53.72
57.15
63.67%
0.94
2000

(Y224K-I474K-
37°
C.

E515P)
10
min
2.79
2.373
0.039
19.51

500

NC
0.417

Phi29-36
−20°
C.
1.46
1.04
0.017
51.5
51.5
70.20%
1
3000

(E515P)
37°
C.

10
min
1.35
0.93
0.015
15.3

1000

NC
0.42

Phi29-458
−20°
C.
0.936
0.583
0.016
31.2
31.2
70.97%
1
2000

(M97K-L123H-
37°
C.

E515P-G217E)
10
min
1.03
0.677
0.018
9.1

500

NC
0.353

Phi29-459
−20°
C.
0.825
0.472
0.013
25.3
25.3
72.35%
1
2000

(M97K-L123H-
37°
C.

E515P-Y224K)
10
min
0.875
0.522
0.014
7.0

500

NC
0.353

Phi29-463
−20°
C.
1.81
1.347
0.04
36.04
72.07
80.91%
0.5
1000

(Y224K-I474K)
37°
C.

10
min
1.32
0.857
0.02
6.88

300

NC
0.463

Phi29-103
−20°
C.
0.97
0.55
0.015
73.8
70.3
85.20%
1.05
5000

(G217E)
37°
C.

10
min
0.5
0.08
0.002
10.9

5000

NC
0.42

Phi29-335
−20°
C.
1.94
1.51
0.041
81
85.3
86.70%
0.95
2000

(Y224K-G217E)
37°
C.

10
min
1.23
0.8
0.022
10.8

500

NC
0.43

Phi29-192
−20°
C.
1.43
1.05
0.028
56.1
56.1
87.30%
1
2000

(Y224K)
37°
C.

10
min
1.71
1.33
0.036
7.1

200

NC
0.38

Phi29-85
−20°
C.
0.682
0.2205
0.027
29.5
36.9
88.66%
0.8
5000

(I474K)
37°
C.
0.586

10
min
5
0.125
0.039
3.3

1000

NC
0.4615

In the above table, NC is a background value, and a blank is because there is no data calculation processing.

Embodiment 4: Genomic Amplification Coverage Detection

In order to detect a coverage to which a sample is extended before on-machine sequencing, a multiple PCR mode is used to amplify a multiple housekeeping gene in an MDA product.

A specific operating process is as follows: a NA12878 reference genome sample (Coriell Institute) is added to 200 μl of a PCR tube, a total volume is 4 μl, and if it is not enough, PBS is added to supplement to 4 μl. 3 μl of Buffer D2 (QIAGEN REPLI-g Single Cell Kit) is added to the PCR tube, a pipette tip should be attached to a tube wall, and is not inserted under a liquid surface, and centrifuging is performed without shaking. After being centrifuged, it is put on a PCR instrument at 65° C. for 10 min. After it is finished, it is placed on ice, 3 μl of neutralization buffer (QIAGEN REPLI-g Single Cell Kit) is added to the PCR tube, the pipette tip should be attached to the tube wall, and is not inserted under the liquid surface, the centrifuging is performed without shaking. 39 μl of MDA reaction buffer (10× phi29 Reaction Buffer 5 μl, 25 mM dNTP 2μl 1, mM N8Primer 2μl, 100× BSA 0.5 μl, 10% F68 0.05 μl, and H₂O 29.75 μl) is added to the PCR tube, 1 μl of phi29-337, phi29-456, phi29-458, phi29-36, phi29-335, phi29-414, and phi29-464 prepared in Embodiment 1 and 1 μl of the Phi29 DNA polymerase carried by the QIAGEN REPLI-g Single Cell Kit are respectively added. The pipette tip should be attached to the tube wall, and is not inserted below the liquid surface, the centrifuging is performed without shaking. After being centrifuged, it is put on the PCR instrument at 30° C. for 8 h and 65° C. for 3 min. After a reaction is finished, the shaking and centrifuging are performed, and a concentration is measured with Qubit BR. After the concentration is measured, a multiple housekeeping gene PCR detection is performed. 50-200 ng of a DNA is taken, a negative control (N) and a positive control (P) should be set, the negative is water, the positive is a NA12878 reference genome without amplification, and an experimental group is set with 3 different samples. Multiple PCR primers and procedures are shown in Table 5 below:

TABLE 5

Multiple PCR primer and PCR reaction system

Primer name
Primer sequence

18-F
GGCATCGCTTAGACTCTATGTG (SEQ ID NO: 26)

18-R
CTGCCCTTGGCCTAACTAACCT (SEQ ID NO: 27)

12-F
GTTCCTCAAGTAGCTCCACGAG (SEQ ID NO: 28)

12-R
CGTTAGACTCTGGATCTGGCGT (SEQ ID NO: 29)

16-F
CCAGCCAGTTCATGCGTCGGTG (SEQ ID NO: 30)

16-R
CCTGACAACTCGCAAGTAGCAC (SEQ ID NO: 31)

17-F
GCTCAATGGGGTACTTCAGGT (SEQ ID NO: 32)

17-R
GTGGACTTACGTAAAAGGCCC (SEQ ID NO: 33)

19-F
TGCTCTGTATGTGAAGATGCCA (SEQ ID NO: 34)

19R
TTCCAGGTAAATCCAGCCCAGG (SEQ ID NO: 35)

3-F
CAGCCAGTCGGCATCATCCAAC (SEQ ID NO: 36)

3-R
GAAAGCCGGATTGCGGTAACAT (SEQ ID NO: 37)

8-F
GGATAGCTCTGCCAGGGGAGAG (SEQ ID NO: 38)

8-R
TCGTCGCAGTAGAAATACGGCT (SEQ ID NO: 39)

22-F
AGATGTCAGGCACGTAGCTCAG (SEQ ID NO: 40)

22-R
GGCACGTTGGCGTTTACGATGA (SEQ ID NO: 41)

Reaction component
Volume

10x PCR Buffer
3 μl

dNTP (2.5 mM)
3.2 μl

Primer Mix
3 μl

DNA (50 ng/μl)
1 μl

100x BSA (NEB)
0.2 μl

rTaq (5 U/μl)
0.4 μl

ddH20
19.2 μl

Total
30 μl

PCR procedure: 95° C. 5 min, 35 cycles ×[94° C. 30 s, 60° C. 50 s, 72° C. 1 min], 72° C. 5 min, 12° C. holder.

Results are shown in FIG. 2, compared with the wild-type WT, the amplification coverage of the phi29-337, phi29-456, phi29-458, phi29-36, and phi29-414 is greatly improved, and 8 housekeeping gene products may at least be amplified for 6 or more. For the concentrations of the amplified products, the phi29-337, phi29-456, phi29-458, phi29-36, and phi29-414 are higher than the wild-type WT and a phi29 commercial product from Enzymatics Company.

Embodiment 5: Application effect of Wild-Type Phi29 DNA Polymerase and Phi29 DNA Polymerase Mutant in Single-Cell Whole Genome Sequencing

In order to test effects of a wild-type Phi29 DNA polymerase and a mutant in single-cell genome sequencing, library construction is performed respectively on products of a NA12878 trace gDNA (200pg) amplified by the wild-type Phi29 DNA polymerase and the Phi29 DNA polymerase mutant prepared in Embodiment 1, after that, high-depth sequencing is performed; at the same time, the same NA12878 trace gDNA (200 pg) amplified by a commercial Qiagen single-cell amplification kit is used for control.

Analysis and mutation detection analysis are performed on sequencing data, a NA12878 standard mutation data set is used to evaluate, and a difference between each mutant and the Qiagen kit is compared. Specific data analysis results are shown in Table 6 and Table 7.

It may be seen that the representation of the mutants Phi29-337, Phi29-456, Phi29-335, and Phi29-414 are comparable to that of the wild-type WT and the QIAGEN kit in mapping rate and coverage, but the representation of the wild-type WT is the worst in terms of duplication rate parameters, the mutants listed in the table are all slightly better than the wild type, herein the representation of the 337 mutant is best, and is comparable to the QIAGEN (REPLI-g Single Cell Kit) kit. At the same time, in aspects of mutation detection evaluation parameters snp_Precision, snp_Sensitivity, and snp_-measure, the representation of the 337 mutant is best, and slightly better than the QIAGEN kit, the wild-type WT and the 456, 335, 414 mutants are worse than the QIAGEN kit; and in aspects of parameters indel_Precision, indel_Sensitivity and indel_F-measure, the representation of the 337 mutant is also the best, and data is better than the QIAGEN kit.

TABLE 6

Sequencing data analysis of wild-type Phi29 and mutant in single-cell sequencing

Mutant number
QIAGEN
Phi29-WT
Phi29-36
Phi29-335
Phi29-337

ReadLength
100
100
100
100
100

Read_raw
1.12E+09
7.96E+08
1.07E+09
1.01 E+09
1.08E+09

Q30_clean
88.67%
87.09%
86%
87.67%
87.07%

Depth_clean
28.48
19.96
28.95
25.56
28.47

Mapping rate
99.93%
99.85%
99.95%
99.91%
99.93%

Duplicate rate
3.71%
5.07%
4.56%
4.85%
3.87%

Coverage
98.01%
97.20%
94.52%
97.16%
98.33%

Genome depth CV
1.01
2.21
1.36
1.7
1

GC depth CV
0.35
0.34
0.35
0.33
0.36

snp_Precision
0.9222
0.8993
0.9004
0.8747
0.9383

snp_Sensitivity
0.92
0.8621
0.8199
0.8466
0.9349

snp_F-measure
0.9211
0.8803
0.8582
0.8604
0.9366

indel_Precision
0.3466
0.1229
0.072
0.1405
0.7437

indel_Sensitivity
0.7249
0.5759
0.5076
0.5925
0.7819

indel_F-measure
0.469
0.2025
0.126
0.2271
0.7623

TABLE 7

Sequencing data analysis of wild-type Phi29

and mutant in single-cell sequencing

Mutant number
Phi29-414
Phi29-456
Phi29-458

ReadLength
100
100
100

Read_raw
1.14E+09
1.28E+09
1.25E+09

Q30_clean
87.90%
88.54%
88.02%

Depth_clean
28.98
32.99
32.74

Mapping rate
99.91%
99.91%
99.96%

Duplicate rate
4.67%
4.51%
4.31%

Coverage
97.03%
97.60%
95.79%

Genome depth CV
1.09
1.01
1.06

GC depth CV
0.35
0.35
0.36

snp_Precision
0.9183
0.9379
0.9335

snp_Sensitivity
0.8898
0.9165
0.8707

snp_F-measure
0.9038
0.9271
0.901

indel_Precision
0.1318
0.1327
0.075

indel_Sensitivity
0.6275
0.6667
0.5502

indel_F-measure
0.2179
0.2214
0.132

(Note:

QIAGEN is a control of a commercial REPLI-g Single Cell Kit)

Embodiment 6: Detection of RCA Library Construction On-Machine Testing Effect

Synthesized with the thermal stability results in Table 4, the embodiment selects several mutants with improved thermal stability, and performs an on-machine test in a BGISEQ-500 sequencer, application effects of the different mutants in DNA sequencing are detected with reference to a standard of the BGISEQ-500 sequencer. Reagents used in the whole test are a complete set of a PE50V2.0 kit produced by the BGI, an E. coil Ad153 standard library produced by the BGI and a Qubit ssDNA Assay reagent produced by Invitrogen. The reagents used below are all included in the PE50V2.0 kit except the library and QubitssDNA Assay, and a PE50V2.0 reagent tank written below only refers to reagents used in the on-machine test.

1. DNB Preparation

DNB preparation buffer, a standard library, DNB polymerase mixed solution and DNB polymerase mixed solution II are taken out from a refrigerator at −20° C. and melted on an ice box, after being completely dissolved, they are placed on a vortex shaker, shaken and continuously mixed uniformly for 5 s, and transiently centrifuged in a handheld centrifuge for 3 s, then it is placed on the ice box for later use. Molecular-grade water and DNB stop buffer are taken out from a refrigerator at 4° C., and placed on the ice box for later use. In a labeled eight-connected tube, 20 μl of the DNB preparation buffer and 6 ng of the ssDNA (E. coli standard substance library) are successively added, and supplemented to 40 μl with Nuclease Free Water. The eight-connected tube is placed in the vortex shaker and continuously mixed uniformly for 5 s, and transiently centrifuged for 3 s in the handheld centrifuge. The above eight-connected tube is placed in the PCR instrument, and reaction conditions are set or checked: 95° C. 1 min, 65° C. 1 min, 40° C. 1 min, and 4° C. for maintaining (a hot lid temperature is set to 103° C.). After a reaction is completed, the PCR eight-connected tube is taken, and placed on the ice box. After a lid temperature of the eight-connected tube drops to a room temperature, it is placed in the handheld centrifuge and transiently centrifuged for 3 s, and immediately placed on the ice box. Each of the following components is successively added into the eight-connected tube: 40 μl of reaction solution in the eight-connected tube after denaturation, 40 μl of the DNB polymerase mixed solution, and 4 pl of the DNB polymerase mixed solution II. The eight-connected tube is placed in the vortex shaker and continuously mixed uniformly for 5 s, and transiently centrifuged for 3 s in the handheld centrifuge. It is immediately placed in the PCR instrument or a water bath kettle to start a reaction. Reaction conditions are set or checked: 30° C. 20min, and 4° C. for maintaining (a hot lid does not need to be installed). While the above RCA reaction is completed, the eight-connected tube is taken and placed on the ice box, and 20 pl of the DNB stop buffer is immediately added. A pipette with a 100 μl wide-mouth pipette tip is used to slowly blow and mix uniformly for 5-8 times (one-suction and one-blow are counted as 1 time). After being mixed uniformly, it is stored at 4° C. for later use. A Qubit ssDNA Assay Kit and a Qubit Fluorometer instrument are used to measure a concentration of the above DNB preparation product. While the concentration is 8ng/μl-40ng/μl, it is judged to be qualified, and it is reserved for a subsequent experiment.

2. DNB Loading

After the DNB is prepared, the DNB needs to be loaded on a chip, namely DNB loading.

A sample loading reagent plate V2.1 is taken and placed at a room temperature for melting, shaken and mixed uniformly. After being transiently centrifuged, it is placed on the ice box for later use. DNB loading buffer II is taken, and shaken uniformly. After being transiently centrifuged, it is placed on the ice box for later use. Firstly, the chip and the sample loading reagent plate V2.1 are placed on BGIDL-50. Secondly, 35 pl of the DNB loading buffer II is taken and added to a PCR tube containing 100 μl of the DNB, and a wide-mouth sucker is used to gently mix uniformly for 15 times. Then, the above mixed solution is placed in a designated DNB placement area of a loading system, a DNB loading program (Sample load 2.0) is selected, and loading is started. Finally, after the loading is completed, it is incubated at the room temperature for 30 min, and then stored at 2-8° C. for later use.

3. On-Machine Test

For the wild-type phi29 polymerase or mutant to be tested, an on-machine sequencing test is performed on the BGISEQ-500 sequencer, one chip and one BGISEQ-500RS high-throughput sequencing reagent tank (PE50V2.0) are used. Before the on-machine test, a sequencing reagent tank II, dNTPs mixed solution (V3.0) and dNTPs mixed solution II (V2.0) are firstly thawed and melted, and placed in a refrigerator or ice box at 4° C. for later use; a sequencing enzyme is shaken and mixed uniformly, and placed on the ice box for later use. Firstly, a No. 5 well reagent is prepared, namely 1 ml of a pipette is used to pipette 1150 pl of the DNA polymerase mixed solution for adding to a No. 5 well and pipette 1150 μl of the dNTPs mixed solution (V3.0) for adding to the No. 5 well, and the pipette is used to blow and mix uniformly for 10-15 times. Secondly, a No. 6 well reagent is prepared, namely 1 ml of the pipette is used to pipette 890 μl of the DNA polymerase mixed solution for adding to a No. 6 well and pipette 890 μl of the dNTPs mixed solution II (V2.0) for adding to the No. 6 well, and the pipette is used to blow and mix uniformly for 10-15 times. Then, a No. 14 well reagent is prepared, namely 5 ml of a pipette is used to pipette all No. 14 well reagents, 2.8 ml of the No. 14 well reagent is taken and mixed uniformly with 400 μl of the phi29 polymerase mutant, and added to a No. 14 well. The prepared reagent tanks are assembled. Finally, the on-machine test is performed, namely the sequencer is started and washing is performed, the reagent tank is put into a designated position of the sequencer, and actual preloading is performed in an operating order. After the preloading is finished, the chip prepared in the above step (2) is installed, and related sequencing information is written, namely the sequencing is started. After the end, the chip, the reagent tanks and a washing instrument are taken out. One cycle is tested in the embodiment.

4. Data Analysis

After the sequencing is finished, an analysis report is downloaded, a previously specified standard is compared, and the performance of the phi29 DNA polymerase mutant is judged.

Each of comparison parameters is explained as follows: Effective Spots Rates (ESR), and basecall information content (Bic) may be used as a DNB ratio of basecalling; fit (crosstalk fit score) reflects a crosstalk situation, after correction, the intensity distribution of each base is more concentrated, a fit value thereof is higher; a Signal to Noise Ratio (SNR) takes the calculation of a single DNB SNR as an example, base A (max intensity) is served as a signal, CGT is a background, and a variance of the CGT intensity is a noise; Rho Intensity (RHO) is an intensity value of corrected intensity removing normalization, and it is intuitively interpreted as an intensity value of the original intensity of dye after correction.

Results are shown in FIG. 3 (FIG. A is comparison of BIC, FIT, ESR parameters of each mutant; FIG. B is comparison of SNR parameters of each mutant; and FIG. C is comparison of RHO parameters of each mutant), it may be seen that synthesized with the analysis of the above various parameter indexes, the best of the tested mutants is 464, and the second is 463.

INDUSTRIAL APPLICATION

On the basis of the disclosure, the mutants with the good effects may be further screened through performing saturation mutations on the amino acids in the mutation sites of the disclosure; or on the basis of the mutants of the disclosure, and other amino acids except the amino acid sites contained in the disclosure are mutated, to obtain a similar effect. The disclosure may also be applied in the technical fields including food detection, virus detection, RNA detection, single-cell sequencing and the like, and it may also be used to develop third and fourth generation sequencer technologies.

Phi29 DNA polymerase mutant with improved thermal stability and use thereof in sequencing

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