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WO2020073266A1 - 热稳定性提升的Phi29 DNA聚合酶突变体及其在测序中的应用 - Google Patents

热稳定性提升的Phi29 DNA聚合酶突变体及其在测序中的应用 Download PDF

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WO2020073266A1
WO2020073266A1 PCT/CN2018/109777 CN2018109777W WO2020073266A1 WO 2020073266 A1 WO2020073266 A1 WO 2020073266A1 CN 2018109777 W CN2018109777 W CN 2018109777W WO 2020073266 A1 WO2020073266 A1 WO 2020073266A1
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amino acid
dna polymerase
protein
phi29
sequence
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French (fr)
Inventor
张周刚
刘欢欢
郑越
周玉君
夏军
董宇亮
徐崇钧
章文蔚
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to JP2021546026A priority Critical patent/JP7256280B2/ja
Priority to EP18936847.5A priority patent/EP3854872A4/en
Priority to CN201880096316.1A priority patent/CN112805372B/zh
Priority to PCT/CN2018/109777 priority patent/WO2020073266A1/zh
Priority to CN202311305525.1A priority patent/CN117467642A/zh
Priority to US17/283,981 priority patent/US20210355459A1/en
Publication of WO2020073266A1 publication Critical patent/WO2020073266A1/zh
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology, in particular to Phi29 DNA polymerase mutants with improved thermal stability and their application in sequencing.
  • Phi29 DNA polymerase is a polymerase derived from Phi29 bacteriophage of Bacillus subtilis, which belongs to the group B DNA polymerase.
  • the crystal structure of Phi29 DNA polymerase shows that, in addition to the conservative palm, thumb, finger, and exodomains with general B group DNA polymerase, it has 3 ' ⁇ 5' Dicer correction activity), Phi29 DNA polymerase also has two other unique domains: TPR1 and TPR2 domains.
  • the TPR2 domain participates in the formation of a narrow channel around the downstream template DNA strand, forcing the dissociation of double-stranded DNA; at the same time, the Palm, Thumb, TPR1, and TPR2 domains form a circle-like structure, tightly binding the double strand formed by the upstream template strand Strand DNA.
  • the structural characteristics of Phi29 DNA polymerase endow it with a special high sustained synthesis ability, a strong strand replacement function and high fidelity.
  • Commonly used in constant temperature amplification such as Rolling Amplification (RCA) of small amounts of circular DNA or multiple strand replacement amplification of genome, such as DNA Nanoball Technology of BGI gene sequencer (DNA Nanotechnology) ) And single cell whole gene sequencing technology.
  • DNA nanosphere technology is one of the core technologies of BGI gene sequencer. After the genomic DNA is fragmented by physical or enzymatic methods, a linker is added and circularized into single-stranded circular DNA, and then high-fidelity strand replacement DNA polymerase is used, such as Phi29 DNA polymerase or Bst DNA polymerase to the single-stranded loop The DNA is subjected to rolling circle amplification (RCA), and the amplified product is called DNA nanoball (DNB). The nanospheres are fixed on the arrayed silicon chip through DNB loading technology, and subsequent computer-based sequencing is performed.
  • RCA rolling circle amplification
  • DNB DNA nanoball
  • the phi29 DNA polymerase mutants with different properties have different binding ability to the substrate DNA, and the amplified DNA nanosphere products are also different in size, uniformity, branching structure, tightness and other characteristics, and then DNB is loaded on the silicon chip
  • the quality of post-sequencing is also different.
  • the properties of Phi29 DNA polymerase also affect the amplification effect of the single cell whole genome amplified by MDA (Multiple Displacement Amplification, MDA) method, which in turn affects the quality of single cell sequencing, such as coverage and comparison rate. Rate), variation detection accuracy and specificity, etc., these are also very important parameters in single cell sequencing scientific research applications.
  • MDA Multiple Displacement Amplification
  • Phi29 DNA polymerase is a medium temperature polymerase, which loses its activity when heated at 65 ° C for 10 minutes.
  • Some commercial Phi29 DNA polymerases on the market are due to stability and other reasons, in special applications such as sequencing library amplification, single cell whole genome amplification, etc. because of effective signal sites on the chip, amplification preference, coverage, variation Problems such as detection and evaluation make it difficult to meet the requirements of kit product development.
  • the invention provides a protein.
  • the protein provided by the present invention is a phi29 DNA polymerase mutant, which is A) or B) as follows:
  • the protein shown is the modification of at least one of the 6 amino acid residues in positions 97, 123, 217, 224, 515, and 474 in the amino acid sequence of phi29 DNA polymerase, and the remaining amino acid residues are unchanged, resulting in DNA polymerase activity protein;
  • the protein shown in B) is a protein derived from A) which has a DNA sequence added to the end of the amino acid sequence of the protein shown in A) and has DNA polymerase activity.
  • the protein shown in A) is a combination of at least 2 or at least 3 or at least 4 or at least 5 of the six positions 97, 123, 217, 224, 515 and 474 in the amino acid sequence of phi29 DNA polymerase Or all amino acid residues are modified to obtain a protein with DNA polymerase activity.
  • the above modification is to modify only the six positions 97, 123, 217, 224, 515, and 474 in the amino acid sequence of phi29 DNA polymerase according to the above-mentioned need to be modified, except that the remaining amino acid residues at these six positions are unchanged.
  • the protein shown in A) is the modification of all the amino acid residues in the five positions 97, 123, 217, 224, and 515 in the amino acid sequence of phi29 DNA polymerase, and the remaining amino acid residues are unchanged.
  • the DNA polymerase active protein is specifically phi29-337 in the examples.
  • the protein shown in A) is the modification of all the amino acid residues in the three positions 224, 515 and 474 in the amino acid sequence of phi29 DNA polymerase, and the remaining amino acid residues are unchanged, resulting in DNA polymerase activity.
  • the specific protein is phi29-464 in the examples.
  • the protein shown in A) is the modification of all the amino acid residues in the three positions 97, 123 and 515 in the amino acid sequence of phi29 DNA polymerase, and the remaining amino acid residues are unchanged to obtain DNA polymerase activity Protein, specifically phi29-414 in the examples;
  • the protein shown in A) is the modification of all the amino acid residues in the four positions 97, 123, 217, and 515 in the amino acid sequence of phi29 DNA polymerase, and the remaining amino acid residues are unchanged, resulting in DNA polymerization Enzymatically active protein, specifically phi29-458 in the examples;
  • the protein shown in A) is the modification of amino acid residues 97, 123, 224, and 515 in the amino acid sequence of phi29 DNA polymerase, and the remaining amino acid residues are unchanged, resulting in DNA polymerase activity.
  • the protein shown in A) is the modification of the amino acid residues at positions 224 and 474 in the amino acid sequence of phi29 DNA polymerase, and the remaining amino acid residues remain unchanged to obtain a protein with DNA polymerase activity.
  • the protein shown in A) is the amino acid residues at positions 217 and 224 in the amino acid sequence of phi29 DNA polymerase, and the remaining amino acid residues are unchanged to obtain a protein with DNA polymerase activity. It is phi29-335 in the example.
  • the modification is amino acid substitution.
  • amino acid substitutions at the six positions 97, 123, 217, 224, 515 and 474 are as follows:
  • Methionine at position 97 is replaced with alanine or histidine or lysine or threonine;
  • Leucine at position 123 was replaced with lysine or phenylalanine or isoleucine or histidine;
  • the glutamic acid at position 515 was replaced with proline or glycine.
  • the protein shown in A) is the mutation of the 97th amino acid residue in the amino acid sequence of phi29 DNA polymerase from M to K, and the mutation of the 123rd position from L to H, and the 515th position from E to P, position 224 was mutated from Y to K, position 217 was mutated from G to E, and the remaining amino acid residues were unchanged, resulting in a protein with DNA polymerase activity.
  • the protein shown in A) is the mutation of the 224th amino acid residue in the amino acid sequence of phi29 DNA polymerase from Y to K, and the mutation of 474 from I to K, and the mutation of 515 from E to P.
  • the protein shown in A) is the mutation of the 97th amino acid residue in the amino acid sequence of phi29 DNA polymerase from M to T, and the mutation of the 123rd position from L to H, and the 515th position from E to P.
  • amino acid sequence of the phi29 DNA polymerase is as follows (I) or (II) or (III):
  • the stability of the protein is higher than that of the phi29 DNA polymerase.
  • the stability is thermal stability.
  • Nucleic acid molecules encoding the above proteins are also within the scope of the present invention.
  • Expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the above nucleic acid molecules are also within the scope of protection of the present invention.
  • nucleic acid molecule 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 cell line containing the nucleic acid molecule are as follows 1) -10) The application in at least one of them is also within the scope of protection of the present invention:
  • Another object of the present invention is to provide a method for improving the stability of phi29 DNA polymerase.
  • the method provided by the present invention includes the steps of: modifying at least one of the amino acid residues at positions 6 of 97, 123, 217, 224, 515, and 474 in the amino acid sequence of phi29 DNA polymerase according to the modification method described above, The remaining amino acid residues remain unchanged, resulting in a protein with DNA polymerase activity.
  • the stability is thermal stability.
  • the Phi29 DNA polymerase of the present invention can exist in the form of independently packaged DNA polymerase products, or can be packaged into a DNA amplification kit or a DNA sequencing kit.
  • the invention obtains mutants with greatly improved thermal stability through fixed-point mutation and screening technology. Some of these mutants have greatly improved the coverage of amplification in single-cell whole genome amplification, and the accuracy and sensitivity of mutation detection have been improved to reach the same level as QIAGEN ’s similar products; other mutants in the present invention are In the application of RCA library amplification, it has a good improvement effect on the loading effect of DNB.
  • 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 the starting bacteria.
  • the starting bacteria may be E. coli.
  • the E. coli may specifically be E. coli BL21 (DE3).
  • the transgenic cell line may be obtained by transforming the recombinant vector into a recipient cell.
  • the transgenic cell line is non-plant propagation material.
  • the stability may specifically be thermal stability.
  • the thermal stability may specifically be thermal stability at 37 ° C.
  • the phi29 DNA polymerase may specifically be as follows (I) or (II) or (III):
  • Figure 1 is a schematic diagram of the construction of Phi29 gene expression vector.
  • Figure 2 is the multiplex PCR detection of housekeeping gene coverage in MDA products.
  • Figure 3 shows the analysis of the test data of the RCA database building.
  • pET28a (+) vector Novagen Corporation.
  • E. coli BL21 (DE3): TIANGEN, CB105-02.
  • Storage buffer 10mM Tris-HCl, 100mM KCl, 1mM DTT, 0.1mM EDTA, 0.5% (v / v) 20, 0.5% (v / v) NP-40, 50% (v / v) Glycerol; pH7.4@25°C.
  • 141AdssDNA in the following examples: BGI self-made, a single-stranded loop library of a certain size range, no fixed sequence (random library composed of A / T / C / G four nucleotides, the main band length is 200- 300bp).
  • the DNA molecule shown in sequence 1 of the sequence table is a gene encoding wild-type Phi29 DNA polymerase, and expresses the protein shown in sequence 2 of the sequence table as wild-type Phi29 DNA polymerase (denoted by WT).
  • Phi29 DNA polymerase mutant is to mutate at least one of the amino acid sequences 97, 123, 217, 224, 474 and 515 of the wild-type Phi29 DNA polymerase.
  • the specific single point mutation form is shown in Table 1.
  • the mutation forms are shown in Table 2.
  • each primer pair shown in Table 1 was used to introduce point mutations to obtain each recombinant vector expressing the Phi29 DNA polymerase mutant.
  • the PCR conditions are: 95 °C 3min, 19 cycles for [95 °C 30s, 53 °C 30s, 68 °C 8min], 68 °C 10min, 4 °C forever.
  • the reaction product was transformed into DH5a competent cells, coated with kanamycin-resistant LB plates, and cultured at 37 ° C in an incubator overnight.
  • the monoclonal plasmid was picked and sequenced to verify whether the specific amino acid site was successfully mutated.
  • the recombinant vector expressing Phi29 DNA polymerase mutant M97H differs only in that the nucleotides 289-291 of the DNA molecule shown in sequence 1 of the sequence listing are mutated from "ATG” to "CAT ".
  • the mutated DNA molecule encodes mutant M97H.
  • the mutant M97H differs only in that the 97th amino acid residue of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M to H.
  • the recombinant vector expressing Phi29 DNA polymerase mutant M97A differs only in that the nucleotides 289-291 of the DNA molecule shown in sequence 1 of the sequence listing are mutated from "ATG” to "GCG” ".
  • the mutated DNA molecule encodes mutant M97A.
  • mutant M97A differs only in that the 97th amino acid residue in the amino acid sequence of wild-type Phi29 DNA polymerase is mutated from M to A.
  • the recombinant vector expressing Phi29 DNA polymerase mutant M97K differs only in that the nucleotides 289-291 of the DNA molecule shown in sequence 1 of the sequence listing are mutated from "ATG” to "AAA ".
  • the mutated DNA molecule encodes mutant M97K.
  • mutant M97K differs only in that the 97th amino acid residue in the amino acid sequence of wild-type Phi29 DNA polymerase is mutated from M to K.
  • the recombinant vector expressing Phi29 DNA polymerase mutant L123K differs only in that the nucleotides 367-369 of the DNA molecule shown in sequence 1 of the Sequence Listing are mutated from "CTG" to "AAA ".
  • the mutated DNA molecule encodes mutant L123K.
  • the difference of the mutant L123K is only the mutation of the amino acid residue 123 in the amino acid sequence of the wild-type Phi29 DNA polymerase from L to K.
  • the recombinant vector expressing Phi29 DNA polymerase mutant L123F differs only in that the nucleotides 367-369 of the DNA molecule shown in sequence 1 of the Sequence Listing are mutated from “CTG” to “TTT ".
  • the mutated DNA molecule encodes mutant L123F.
  • mutant L123F differs only in that the amino acid residue at position 123 of the amino acid sequence of wild-type Phi29 DNA polymerase is mutated from L to F.
  • the recombinant vector expressing Phi29 DNA polymerase mutant L123I differs only in that the nucleotides 367-369 of the DNA molecule shown in sequence 1 of the Sequence Listing are mutated from "CTG” to "ATT ".
  • the mutated DNA molecule encodes mutant L123I.
  • mutant L123I differs only in that the amino acid residue at position 123 of the amino acid sequence of wild-type Phi29 DNA polymerase is mutated from L to I.
  • the recombinant vector expressing Phi29 DNA polymerase mutant L123H differs only in that the nucleotides 367-369 of the DNA molecule shown in sequence 1 of the Sequence Listing are mutated from "CTG” to "CAT ".
  • the mutated DNA molecule encodes mutant L123H.
  • mutant L123H differs only in that the amino acid residue at position 123 of the amino acid sequence of wild-type Phi29 DNA polymerase is mutated from L to H.
  • the recombinant vector expressing Phi29 DNA polymerase mutant E515P differs only in that the nucleotides 1543-1545 of the DNA molecule shown in sequence 1 of the Sequence Listing are mutated from "GAA" to "CCG ".
  • the mutated DNA molecule encodes mutant E515P.
  • mutant E515P differs only in that the amino acid residue at position 515 of the amino acid sequence of wild-type Phi29 DNA polymerase is mutated from E to P.
  • the recombinant vector expressing Phi29 DNA polymerase mutant Y224K differs only in that the nucleotides 670-672 of the DNA molecule shown in sequence 1 of the Sequence Listing are mutated from "TAT" to "AAA ".
  • the mutated DNA molecule encodes mutant Y224K.
  • mutant Y224K differs only in that the amino acid residue at position 224 in the amino acid sequence of wild-type Phi29 DNA polymerase is mutated from E to K.
  • the recombinant vector expressing Phi29 DNA polymerase mutant G217E differs only in that the 649-651 nucleotides of the DNA molecule shown in sequence 1 of the Sequence Listing are mutated from "GGC” to "GAA” ".
  • the mutated DNA molecule encodes mutant G217E.
  • the mutant G217E differs only in that the amino acid residue at position 217 of the amino acid sequence of the wild-type Phi29 DNA polymerase is mutated from G to E.
  • the recombinant vector expressing Phi29 DNA polymerase mutant I474K differs only in that the nucleotides 1420-1422 of the DNA molecule shown in sequence 1 of the sequence listing are mutated from "ATT" to "TTT” ".
  • the mutated DNA molecule encodes mutant I474K.
  • the difference of mutant I474K is only the mutation of amino acid residue 474 of wild-type Phi29 DNA polymerase amino acid sequence from I to K.
  • the recombinant vector expressing Phi29 DNA polymerase mutant M97K-L123H-E515P-Y224K-G217E differs only in that the nucleotides 289-291 of the DNA molecule shown in sequence 1 of the sequence listing Mutation from “ATG” to "AAA”, nucleotides 367-369 of the DNA molecule shown in sequence 1 of the sequence list From "CTG” to "CAT”, DNA molecules shown in the sequence 1 of sequence list 1543 The -1545th nucleotide was mutated from "GAA” to "CCG", the DNA molecule shown in sequence 1 of the sequence list.
  • the 670-672th nucleotide was mutated from “TAT” to "AAA", sequence 1 of the sequence list.
  • the nucleotides 649-651 of the DNA molecule shown are mutated from “GGC” to "GAA”.
  • the mutated DNA molecule encodes the mutant M97K-L123H-E515P-Y224K-G217E.
  • the mutant M97K-L123H-E515P-Y224K-G217E differs only in that the amino acid residue 97 of the wild-type Phi29 DNA polymerase amino acid sequence was mutated from M to K, and the 123rd position Mutation from L to H, position 515 from E to P, position 224 from Y to K, and position 217 from G to E.
  • the recombinant vector expressing Phi29 DNA polymerase mutant M97T-L123K-E515P differs only in that the nucleotides 289-291 of the DNA molecule shown in sequence 1 of the Sequence Listing are selected from "ATG” Mutation to "ACC", nucleotides 367-369 of the DNA molecule shown in sequence 1 of the sequence table from "CTG” to "AAA”, 1543-1545 nucleus of the DNA molecule shown in sequence 1 of the sequence table
  • the glucuronide is mutated from "GAA" to "CCG".
  • the mutated DNA molecule encodes the mutant M97T-L123K-E515P.
  • the mutant M97T-L123K-E515P differs only in that the 97th amino acid residue of the wild-type Phi29 DNA polymerase amino acid sequence was mutated from M to T, and the 123rd position was mutated from L Into K, the 515th position is mutated from E to P.
  • the recombinant vector expressing Phi29 DNA polymerase mutant M97K-L123H-E515P differs only in that the nucleotides 289-291 of the DNA molecule shown in sequence 1 of the Sequence Listing are selected from "ATG” Mutation to "AAA”, nucleotides 367-369 of the DNA molecule shown in sequence 1 of the sequence list From "CTG” to "CAT”, nucleus 1543-1545 of the DNA molecule shown in sequence 1 of the sequence list
  • the glucuronide is mutated from "GAA" to "CCG".
  • the mutated DNA molecule encodes the mutant M97T-L123H-E515P.
  • the mutant M97K-L123H-E515P differs only in that the 97th amino acid residue of the wild-type Phi29 DNA polymerase amino acid sequence is mutated from M to T, and the 123rd position is mutated from L It becomes H, and the 515th position is mutated from E to P.
  • the recombinant vector expressing Phi29 DNA polymerase mutant Y224K-I474K-E515P differs only in that: the 670-672th nucleotides of the DNA molecule shown in sequence 1 of the sequence table are selected from "TAT” Mutation to "AAA”, nucleotides 1420-1422 of the DNA molecule shown in sequence 1 of the sequence list from "ATT" to "TTT", nucleotides 1543-1545 of the DNA molecule shown in sequence 1 of the sequence list The glucuronide is mutated from "GAA" to "CCG". The mutated DNA molecule encodes mutant Y224K-I474K-E515P.
  • the mutant Y224K-I474K-E515P differs only in that the wild-type Phi29 DNA polymerase amino acid sequence 224th amino acid residue is mutated from Y to K, and the 474th position is mutated by I Into K, the 515th position is mutated from E to P.
  • the recombinant vector expressing Phi29 DNA polymerase mutant M97K-L123H-E515P-G217E differs only in that the nucleotides 289-291 of the DNA molecule shown in sequence 1 of the sequence listing are ATG "is mutated to” AAA ", nucleotides 367-369 of the DNA molecule shown in sequence 1 of the sequence table are changed from” CTG "to” CAT ", 1543-1545 are DNA molecules shown in sequence 1 of the sequence table.
  • the nucleotide at position 649 is mutated from “GAA” to “CCG”, and the nucleotides at positions 649-651 of the DNA molecule shown in sequence 1 of the Sequence Listing are mutated from “GGC” to "GAA”.
  • the mutated DNA molecule encodes the mutant M97K-L123H-E515P-G217E.
  • the mutant M97K-L123H-E515P-G217E differs only in the mutation of the 97th amino acid residue of the wild-type Phi29 DNA polymerase amino acid sequence from M to K, and the 123rd amino acid residue From L to H, and the 515th position is mutated from E to P, and the 217th position is mutated from G to E.
  • the recombinant vector expressing Phi29 DNA polymerase mutant M97K-L123H-E515P-Y224K differs only in that the nucleotides 289-291 of the DNA molecule shown in sequence 1 of the sequence listing are ATG "is mutated to” AAA ", nucleotides 367-369 of the DNA molecule shown in sequence 1 of the sequence table are changed from” CTG "to” CAT ", 1543-1545 are DNA molecules shown in sequence 1 of the sequence table.
  • the nucleotide at position 670 is mutated from “GAA” to “CCG”, and the nucleotides at positions 670-672 of the DNA molecule shown in sequence 1 of the Sequence Listing are mutated from “TAT” to "AAA”.
  • the mutated DNA molecule encodes the mutant M97K-L123H-E515P-G217E.
  • the mutant M97K-L123H-E515P-G217E differs only in the mutation of the 97th amino acid residue of the wild-type Phi29 DNA polymerase amino acid sequence from M to K, and the 123rd amino acid residue From L to H, and the 515th position from E to P, and the 224th position from Y to K.
  • the recombinant vector expressing Phi29 DNA polymerase mutant Y224K-I474K differs only in that the nucleotides 670-672 of the DNA molecule shown in sequence 1 of the sequence list are mutated from "TAT” to "AAA", the nucleotides 1420-1422 of the DNA molecule shown in Sequence 1 of the Sequence Listing are mutated from "ATT" to "TTT”.
  • the mutated DNA molecule encodes mutant Y224K-I474K.
  • mutant Y224K-I474K differs only in the mutation of wild-type Phi29 DNA polymerase amino acid sequence at position 224 from Y to K, and at position 474 amino acid residue from I to K.
  • the recombinant vector WT constructed in Step 1 and each recombinant vector expressing Phi29 DNA polymerase mutant were introduced into E. coli BL21 (DE3) to obtain recombinant bacteria expressing wild-type Phi29 DNA polymerase and each recombinant expressing Phi29 DNA polymerase mutant. bacteria.
  • Wild-type N-terminal fusion the DNA polymerase and Phi29 respective N-terminal His 6 tag fused to the DNA polymerase Phi29 mutant His 6 tag sequence was named the DNA polymerase Phi29 wild type His 6 tag, the His6 tag having Phi29 DNA polymerase mutant M97H, His6 tag Phi29 DNA polymerase mutant M97A, His6 tag Phi29 DNA polymerase mutant M97K, His6 tag Phi29 DNA polymerase mutant L123K, His6 tag Phi29 DNA polymerization Enzyme mutant L123F, His6 tag Phi29 DNA polymerase mutant L123I, His6 tag Phi29 DNA polymerase mutant L123H, His6 tag Phi29 DNA polymerase mutant E515P, His6 tag Phi29 DNA polymerase mutation E515G, His6 tag Phi29 DNA polymerase mutant Y224K, His6 tag Phi29 DNA polymerase mutant G217E, His6 tag Phi29 DNA polymerase mutant I474K, His6 tag Phi29
  • the recombinant bacteria were inoculated into 3 ml of liquid LB medium containing Kana resistance, and cultured overnight.
  • IPTG was added to the system with a final concentration of 0.5 mM, and cultured with shaking at 16 ° C and 220 rpm for 12 h.
  • step (3) centrifuge at 4 ° C and 8000 rpm for 5 min to collect cells.
  • step 1 Take the bacterial cells obtained in step 1, shake and mix with resuspending buffer (20mM Tris-HCl, 500mM NaCl, 20mM Imidazole, 5% Glycerol; pH 7.9@25°C), then sonicate on ice, then Centrifuge at 12,000 rpm at 4 ° C for 30 minutes, and collect the supernatant.
  • resuspending buffer (20mM Tris-HCl, 500mM NaCl, 20mM Imidazole, 5% Glycerol; pH 7.9@25°C
  • step (2) Take the supernatant obtained in step (1) and use nickel column affinity chromatography (HisTrap FF 5ml prepacked column) for purification. Specific steps: first equilibrate with 10 column volumes of Buffer A; then load the sample; then rinse with 20 column volumes of Buffer A; then elute with 15 column volumes of eluent and collect the column with the target protein After the solution (the eluent is composed of Buffer A and Buffer B, during the elution process, the volume fraction of Buffer B increases linearly from 0% to 100%, and the corresponding volume fraction of Buffer A decreases linearly from 100% to 0% ).
  • Buffer A 20mM Tris-HCl, 500mM NaCl, 20mM Imidazole, 5% (v / v) Glycerol; pH7.9@25°C.
  • Buffer B 20mM Tris-HCl, 500mM NaCl, 500mM Imidazole, 5% (v / v) Glycerol; pH7.9@25°C.
  • step (3) Take the post-column solution obtained in step (2) and use strong anion column chromatography (HiTrap QHP 5ml prepacked column) for purification. Specific steps: first equilibrate with 10 column volumes of a mixed buffer consisting of 59% Buffer A and 41% Buffer B; then load the sample; collect the flow-through (UV) after the protein peak appears (The detection value is raised to 20mAu and collected immediately, and the UV detection value is reduced to 50mAu, and the collection is stopped).
  • strong anion column chromatography HiTrap QHP 5ml prepacked column
  • Buffer A 20mM Tris-HCl, 150mM NaCl, 5% (v / v) Glycerol, pH7.5@25°C.
  • Buffer B 20mM Tris-HCl, 1M NaCl, 5% (v / v) Glycerol, pH7.5@25°C.
  • the flow-through liquid obtained in step (3) is purified by cation exchange chromatography (HiTrap SP SP pre-packed column) to obtain a protein sample solution with a purity greater than 95%.
  • Buffer A 20mM Tris-HCl, 150mM NaCl, 5% (v / v) Glycerol, pH7.5@25°C.
  • Buffer B 20mM Tris-HCl, 1M NaCl, 5% (v / v) Glycerol, pH7.5@25°C.
  • step (4) Take the target protein collected in step (4) into a dialysis bag. After dialysis in dialysis buffer overnight, collect the protein solution in the dialysis bag and add other reagents to obtain the target protein solution with a protein concentration of 1 mg / ml .
  • concentration of other components in the target protein solution is as follows: 10mM Tris-HCl (pH7.4@25°C), 100mM KCl, 1mM DTT, 0.1mM EDTA, 0.5% (v / v) NP-40, 0.5% (v / v) Tween20, 50% (v / v) Glycerol.
  • Dialysis buffer 23.75mM Tris-HCl (pH7.4@25°C), 237.5mM KCl, 2.375mM DTT, 0.2375mM EDTA, 5% (v / v) Glycerol.
  • the wild-type Phi29 DNA polymerase solution with His 6 tag and the Phi29 DNA polymerase mutant solution with His 6 tag prepared in Example 1 were used as the enzyme solution to be tested.
  • the pre-reaction system was mixed in a PCR tube, placed in a PCR instrument, and the following procedures were performed: 95 ° C for 1 min, 65 ° C for 1 min, 40 ° C for 1 min, and the hot lid temperature was set to 102 ° C.
  • Pre-reaction system 80.8 ⁇ l: 50 mM Tris-HCl (pH 7.5), 4 mM DTT, 10 mM (NH 4 ) 2 SO 4 , 10 mM MgCl 2 , 50 nM dNTP Mixture, 2pM 141RCA Primer and 18ng single-stranded circular DNA template 141Ad ssDNA.
  • step 2 After completing step 1, remove the PCR tube and place it on ice when the temperature reaches 4 ° C. Add 1 ⁇ l of each test solution to the test group, and add 1 ⁇ l of storage buffer to the negative control group. Then use a vortex shaker to mix well. After centrifuging for 5 seconds in a short centrifuge, place it in the PCR instrument and perform the following procedure: 30 °C for 60min.
  • the hot lid temperature is set to 65 ° C.
  • step 3 After completing step 2, add 5 ⁇ L 0.5M EDTA solution to stop the reaction, and mix by shaking.
  • the enzyme activity of the enzyme solution to be tested ⁇ DNB ⁇ 5000 ⁇ 37.38.
  • ⁇ DNB is the difference between the average concentration of the reaction products in the system after the termination of the reaction between the test group and the negative control group, 5000 is the dilution factor, and 37.38 is the slope of the functional relationship between enzyme activity and ⁇ DNB.
  • the first part placed in a metal bath preheated to 37 ° C for 10 min, centrifuged at 4 ° C and 13000 rpm for 1 min, and collected the supernatant; then, the supernatant was taken, diluted with storage buffer to 1000 times the volume, and vortexed Mix thoroughly with a vortex shaker, then let stand on ice for 5 minutes to obtain solution 1 to be tested.
  • Second part Dilute to 5000 times volume with storage buffer, mix thoroughly with vortex shaker, then let stand on ice for 5min to obtain solution 2 to be tested.
  • Unheated enzyme activity (U1) ⁇ DNB ⁇ 5000 ⁇ 37.38; ⁇ DNB is the difference between the average concentration of the reaction products in the system after the test group (second part) and the negative control group terminate the reaction;
  • Enzyme activity loss ratio (%) (U1-U2) ⁇ U1 ⁇ 100%;
  • U1 is the activity of the enzyme solution without heat treatment
  • U2 is the activity of the enzyme solution after heat treatment
  • the specific enzyme activity and enzyme activity loss of each mutant are shown in Table 4. Compared with the wild type (WT), the enzyme mutants have a certain increase in enzyme activity loss ratio; the larger the enzyme activity loss ratio value, the more unstable the enzyme and the worse the thermal stability.
  • NC is the background value
  • blank space is because there is no data calculation processing.
  • the specific operation steps are as follows: add NA12878 reference genome sample (Coriell Institute) to a 200ul PCR tube, the total volume is 4ul, if not enough, make up to 4ul with PBS. Add 3ul Buffer D2 (QIAGEN REPLI-g Single Cell Kit) to the PCR tube, the pipette tip should be attached to the wall of the tube, do not insert it under the liquid surface, centrifuge, and do not shake. After centrifugation, put it on the PCR instrument at 65 °C for 10min.
  • PCR program 95 °C 5min, 35cycles x [94 °C 30s, 60 °C 50s, 72 °C 1min], 72 °C 5min, 12 °C holder.
  • the Phi29 DNA polymerase wild-type and Phi29 DNA polymerase mutants prepared in Example 1 were used to amplify the product of NA12878 trace gDNA (200pg)
  • the library was built, followed by high-depth sequencing; at the same time, the same NA12878 microgDNA (200 pg) amplified by a commercial Qiagen single-cell amplification kit was used for control.
  • mutants Phi29-337, Phi29-456, Phi29-335, and Phi29-414 perform similarly to wild-type WT and QIAGEN kits in mapping, coverage, but wild-type WT performs the worst in terms of duplication rate parameters
  • wild-type WT performs the worst in terms of duplication rate parameters
  • the mutants listed in the table are slightly better than the wild type, and the 337 mutant performs best, which is comparable to the QIAGEN (REPLI-g Single Cell Kit) kit.
  • the mutation detection evaluation parameters snp_Precision, snp_Sensitivity, snp_F-measure and aspect, the 337 mutant performed best, slightly better than the QIAGEN kit, wild-type WT and 456, 335, 414 mutants are worse than the QIAGEN kit;
  • the indel_Sensitivity and indel_F-measure parameters are also the best for the 337 mutant, and the data is better than the QIAGEN kit.
  • 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 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
  • BGISEQ-500 Sequencer standard The reagents used for the entire test were the complete PE50V2.0 kit produced by BGI and the E.coil Ad153 standard library produced by BGI and the QubitssDNA Assay reagent produced by Invitrogen. The following reagents are included in the PE50V2.0 kit except the library and QubitssDNA Assay, and the PE50V2.0 reagent tank described below refers only to the reagents used for on-board testing.
  • the temperature of the hot lid is set to 103 ° C.
  • the eight PCR tubes were taken out and placed on an ice box. After the lid temperature of the eight tubes was lowered to room temperature, they were placed in a palm centrifuge and briefly centrifuged for 3 seconds, and immediately placed on the ice box.
  • the following components are added to the eight-connected tube in sequence: 40 ul of denatured reaction solution in the eight-connected tube, 40 ul of DNB polymerase mixed solution, and 4 ul of DNB polymerase mixed solution II. Place the eight-connected tube in a vortex and continue to mix for 5s, and centrifuge briefly in a palm centrifuge for 3s. Immediately place in PCR machine or water bath to start the reaction. Set or check the reaction conditions: 30 °C 20min, 4 °C to maintain (no need to set hot cover). When the above RCA reaction is completed, remove the eight tubes and place them on an ice box, and immediately add 20 ⁇ L of DNB stop buffer.
  • the DNB After the DNB is prepared, the DNB needs to be loaded onto the chip, that is, the DNB is loaded.
  • the phi29 polymerase mutant or wild type to be tested was tested on the BGISEQ-500 sequencer by using on-chip sequencing, using a chip and a BGISEQ-500RS high-throughput sequencing reagent tank (PE50V2.0). Before the on-board test, first defrost and thaw the sequencing reagent tank II, dNTPs mixed solution (V3.0) and dNTPs mixed solution II (V2.0), and put them in a refrigerator or ice box at 4 °C for standby; Put it on the ice box and set aside.
  • ESR Effective Spots Rates
  • Bic basecall information
  • fit crosstalk fitscore
  • SNR Signal to Noise Ratio
  • SNR Signal to Noise Ratio
  • base A maximum Intensity
  • CGT CGT Intensity
  • RHO Ra Intensity
  • the mutants with good effects can be further selected by saturation mutation of the amino acid at the mutation site of the present invention; or on the basis of the mutant of the present invention, the amino acid positions included in the present invention can be further selected. Other amino acids outside the point are mutated to obtain a similar effect.
  • the technical fields that this law may also apply include food testing, virus testing, RNA testing, single cell sequencing, etc., and may also be used to develop third- and fourth-generation sequencer technologies.

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Abstract

提供一种热稳定性提升的Phi29 DNA聚合酶突变体及其在测序中的应用，所述Phi29 DNA聚合酶突变体是如下A）或B）：A）所示的DNA聚合酶突变体为将Phi29 DNA聚合酶氨基酸序列中第97、123、217、224、515和474这6位中至少一位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白；B）所述的DNA聚合酶突变体为将A）所示蛋白的氨基酸序列末端添加标签序列且具有DNA聚合酶活性的由A）衍生的蛋白质。

Description

热稳定性提升的Phi29 DNA聚合酶突变体及其在测序中的应用 技术领域
本发明属于生物技术领域，尤其涉及热稳定性提升的Phi29 DNA聚合酶突变体及其在测序中的应用。
背景技术
Phi29 DNA聚合酶是源于枯草芽孢杆菌(Bacillus subtilis)Phi29噬菌体的聚合酶，该酶属于B族DNA聚合酶。Phi29 DNA聚合酶的晶体结构显示，除了具有一般B族DNA聚合酶的保守的手掌(Palm)、拇指(Thumb)、手指(Finger)和外切结构域(Exo domain，具有3’→5’外切酶校正活性)，Phi29 DNA聚合酶还拥有另外两个独特的结构域：TPR1和TPR2结构域。TPR2结构域参与形成围绕下游模板DNA链的狭小通道，迫使双链DNA的解离；与此同时，Palm、Thumb、TPR1和TPR2结构域形成圆圈状结构，紧密地结合在上游模板链形成的双链DNA上。Phi29 DNA聚合酶的结构特征赋予了其特殊的高持续合成能力、较强的链置换功能和高保真度。常应用于微量的环状DNA的滚环扩增(Rolling Circle Amplification,RCA)或基因组的多重链置换扩增等恒温扩增等应用，例如华大基因测序仪的DNA纳米球技术(DNA Nanoball Technology)和单细胞全基因测序技术。
DNA纳米球技术(DNB Technology)是华大基因测序仪的核心技术之一。基因组DNA经过物理法或酶法片段化处理后，加上接头并环化成单链环状DNA，随后使用高保真的链置换DNA聚合酶，如Phi29 DNA聚合酶或Bst DNA聚合酶对单链环状DNA进行滚环扩增(Rollingcircle amplification,RCA)，扩增产物即称为DNA纳米球(DNAnanoball,DNB)。纳米球经过DNB装载技术固定在阵列化的硅芯片上，进行后续的上机测序。不同性质的phi29 DNA聚合酶突变体与底物DNA的结合能力不同，扩增的DNA纳米球产物在大小、均一性、分支结构、紧密度等特性上也有所不同，进而DNB装载至硅芯片上后测序的质量也不一样。Phi29 DNA聚合酶的性质同样也影响MDA(Multiple Displacement Amplification,MDA)方法扩增的单细胞全基因组的扩增效果，进而影响单细胞测序的质量，比如覆盖度(Coverage)、比对率(Mapping Rate)、变异检测准确度和特异性等等，这些也是单细胞测序科研应用中十分关心的参数。
Phi29 DNA聚合酶是一种中温聚合酶，在65℃条件下加热10min即可失去活性。市面上一些商业化的Phi29 DNA聚合酶因为稳定性等原因，在诸如测序文库扩增、单细胞全基因组扩增等特殊应用中因为芯片上有效信号位点、扩增偏好性、覆盖度、变异检测评估等方面问题而难以满足试剂盒产品开发的要求。
发明公开
本发明提供了一种蛋白。
本发明提供的蛋白，为phi29 DNA聚合酶突变体，是如下A)或B)：
A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、217、224、 515和474这6位中至少一位的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白；
B)所示的蛋白为将A)所示蛋白的氨基酸序列末端添加标签序列且具有DNA聚合酶活性的由A)衍生的蛋白质。
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、217、224、515和474这6位中至少2位或至少3位或至少4位或至少5位或全部的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
上述修饰为仅对phi29 DNA聚合酶氨基酸序列中第97、123、217、224、515和474这6位按照上述需要修饰的位点进行修饰，除了这6位的其余氨基酸残基不变。
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、217、224和515这5位中全部的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白，具体为实施例中的phi29-337。
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第224、515和474这3位中全部的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白，具体为实施例中的phi29-464。
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123和515这3位中全部的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白，具体为实施例中的phi29-414；
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、217、515这4位中全部的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白，具体为实施例中的phi29-458；
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、224、515这4位的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白，具体为实施例中的phi29-459；
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第224、474这2位的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白，具体为实施例中的phi29-463；
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第217、224这2位的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白，具体为实施例中的phi29-335。
上述蛋白中，所述修饰为氨基酸置换。
上述蛋白中，所述第97、123、217、224、515和474这6位点的氨基酸置换方式分别为如下：
第97位的甲硫氨酸置换为丙氨酸或组氨酸或赖氨酸或苏氨酸；
第123位的亮氨酸置换为赖氨酸或苯丙氨酸或异亮氨酸或组氨酸；
第217位的甘氨酸置换为谷氨酸；
第224位的酪氨酸置换为赖氨酸；
第474位的异亮氨酸置换为赖氨酸；
第515位的谷氨酸置换为脯氨酸或甘氨酸。
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97位氨基酸残基由M突变为了K，且第123位由L突变成H，第515位由E突变成P，第224位由Y突变成K，第217位由G突变为E，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第224位氨基酸残基由Y突变为了K，且第474位由I突变成K，第515位由E突变成P。
上述蛋白中，A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97位氨基酸残基由M突变为了T，且第123位由L突变成H，第515位由E突变成P。
上述蛋白中，所述phi29 DNA聚合酶的氨基酸序列为如下(Ⅰ)或(Ⅱ)或(Ⅲ)：
(Ⅰ)序列表的序列2；
(Ⅱ)与序列表的序列2所示的蛋白质具有90％以上同一性且来源于枯草芽孢杆菌的蛋白质的氨基酸残基序列；
(Ⅲ)与序列表的序列2所示的蛋白质具有95％以上同一性且来源于枯草芽孢杆菌的蛋白质氨基酸残基序列。
上述蛋白中，所述蛋白质的稳定性高于所述phi29 DNA聚合酶。
上述蛋白中，所述稳定性为热稳定性。
编码上述蛋白的核酸分子也是本发明保护的范围。
含有上述核酸分子的表达盒、重组载体、重组菌或转基因细胞系也是本发明保护的范围。
上述蛋白在如下1)-11)中至少一种中的应用也是本发明保护的范围：
1)作为DNA聚合酶；
2)催化DNA复制和/或催化DNA扩增；
3)催化滚换扩增和/或催化多重链置换扩增；
4)进行DNA测序或RNA测序或全基因组测序；
5)RCA建库(DNA纳米球装载)；
6)基因组扩增覆盖度检测；
7)制备用于催化DNA复制和/或催化DNA扩增的试剂盒产品；
8)制备用于催化滚换扩增和/或催化多重链置换扩增的产品；
9)制备用于进行DNA测序或RNA测序或全基因组测序的产品；
10)制备用于RCA建库的产品；
11)制备用于基因组扩增覆盖度检测的产品。
编码上述蛋白的核酸分子、含有所述核酸分子的表达盒、含有所述核酸分 子的重组载体、含有所述核酸分子的重组菌或含有所述核酸分子的转基因细胞系在如下1)-10)中至少一种中的应用也是本发明保护的范围：
1)催化DNA复制和/或催化DNA扩增；
2)催化滚换扩增和/或催化多重链置换扩增；
3)进行DNA测序或RNA测序或全基因组测序；
4)RCA建库；
5)基因组扩增覆盖度检测；
6)制备用于催化DNA复制和/或催化DNA扩增的产品；
7)制备用于催化滚换扩增和/或催化多重链置换扩增的产品；
8)制备用于进行DNA测序或RNA测序或全基因组测序的产品；
9)制备用于RCA建库的产品；
10)制备用于基因组扩增覆盖度检测的产品。
本发明另一个目的是提供一种提高phi29 DNA聚合酶稳定性的方法。
本发明提供的方法，包括如下步骤：将phi29 DNA聚合酶氨基酸序列中第97、123、217、224、515和474这6位中至少一位的氨基酸残基按照上述中的修饰方式进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
上述方法中，所述稳定性为热稳定性。
本发明所述Phi29 DNA聚合酶既可以独立包装的DNA聚合酶产品形式存在，也可包装进DNA扩增试剂盒或DNA测序试剂盒中。本发明通过定点突变和筛选技术，获得热稳定性得到较大提升的突变体。其中一些突变体在单细胞全基因组扩增中扩增覆盖度有了较大提高，变异检测的准确度和灵敏度有所提升，达到QIAGEN同类商品的同等水平；本发明中的另外一些突变体则在RCA文库扩增应用中对DNB装载效果有良好的改善效果。
所述重组载体为在表达载体中插入所述核酸分子得到的。
所述表达载体具体可为pET28a(+)载体。
所述重组菌为将所述重组载体导入出发菌得到的菌。
所述出发菌可为大肠杆菌。
所述大肠杆菌具体可为大肠杆菌BL21(DE3)。
所述转基因细胞系可为将所述重组载体转化受体细胞获得的。所述转基因细胞系为非植物繁殖材料。
所述稳定性具体可为热稳定性。所述热稳定性具体可为37℃下的热稳定性。
所述phi29 DNA聚合酶具体可为如下(Ⅰ)或(Ⅱ)或(Ⅲ)：
(Ⅰ)序列表的序列2所示的蛋白质；
(Ⅱ)与序列表的序列2所示的蛋白质具有90％以上同一性且来源于枯草芽孢杆菌的蛋白质；
(Ⅲ)与序列表的序列2所示的蛋白质具有95％以上同一性且来源于枯草芽孢杆菌的蛋白质。
附图说明
图1为Phi29基因表达载体构建示意图。
图2为多重PCR检测MDA产物中看家基因覆盖度。
图3为RCA建库上机测试数据分析。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中的各个溶液或缓冲液，如无特殊说明，溶剂均为水。
pET28a(+)载体：Novagen公司。
大肠杆菌BL21(DE3)：TIANGEN，CB105-02。
储存缓冲液：10mM Tris-HCl，100mM KCl，1mM DTT，0.1mM EDTA，0.5％(v/v)
Figure PCTCN2018109777-appb-000001
20，0.5％(v/v)NP-40，50％(v/v)Glycerol；pH7.4@25℃。
下述实施例中的141RCA Primer：TCCTAAGACCGCTTGGCCTCCGACT。
下述实施例中的141Ad ssDNA：BGI自制，为一定大小范围的单链环文库，无固定序列(由A/T/C/G四种核苷酸组成的随机文库，主带长度为200-300bp)。
下面实施例中，序列表的序列1所示的DNA分子为野生型Phi29 DNA聚合酶的编码基因，其表达序列表的序列2所示的蛋白质即野生型Phi29 DNA聚合酶(用WT表示)。
实施例1、Phi29 DNA聚合酶突变体的制备
Phi29 DNA聚合酶突变体为将野生型Phi29 DNA聚合酶的氨基酸序列第97、123、217、224、474和515中至少一位进行突变，具体单点突变形式如表1所示，多点组合突变形式如表2所示。
一、表达Phi29 DNA聚合酶突变体的重组载体的构建
1、表达Phi29 DNA聚合酶单点突变体的重组载体
将序列表的序列2所示的DNA分子插入pET28a(+)载体(图1)的NdeI和BamHI酶切位点之间，得到表达野生型Phi29 DNA聚合酶的重组载体WT。
以重组载体WT为出发载体，采用表1所示的各个引物对引入点突变，得到各个表达Phi29 DNA聚合酶突变体的重组载体。
上述采用表1所示的各个引物对引入点突变的方法如下：
测重组载体WT作为模板，在含有如下成分的定点突变PCR反应体系(25ul)：2.5ul 10x Pfu Reaction Buffer with Mg 2+,2ul dNTP Mix(2.5mM each),25ng pET28a-Phi29 plasmid,0.5ul Pfu DNA Polymerase，分别加入突变位点的突变引物(表1)，中进行PCR扩增。
PCR条件为：95℃ 3min,19 cycle for[95℃ 30s，53℃ 30s,68℃ 8min],68℃ 10min，4℃ forever。反应产物经过DpnI消化后，转化DH5a感受 态细胞，涂布卡纳霉素抗性LB平板，37℃温箱过夜培养后挑取单克隆提质粒测序，验证特定氨基酸位点是否突变成功。
表1 单点突变引物
Figure PCTCN2018109777-appb-000002
与重组载体WT相比，表达Phi29 DNA聚合酶突变体M97H的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第289-291位核苷酸由“ATG”突变为了“CAT”。突变后的DNA分子编码突变体M97H。与野生型Phi29 DNA聚合酶相比，突变体M97H的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第97位氨基酸残基由M突变为了H。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体M97A的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第289-291位核苷酸由“ATG”突变为了“GCG”。突变后的DNA分子编码突变体M97A。与野生型Phi29 DNA聚合酶相比，突变体M97A的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第97位氨基酸残基由M突变为了A。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体M97K的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第289-291位核苷酸由“ATG”突变为了“AAA”。突变后的DNA分子编码突变体M97K。与野生型Phi29 DNA聚合酶相比，突变体M97K的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第97位氨基酸残基由M突变为了K。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体L123K的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第367-369位核苷酸由“CTG”突变为了“AAA”。突变后的DNA分子编码突变体L123K。与野生型Phi29 DNA聚合酶相比，突变体L123K的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第123位氨基酸残基由L突变为了K。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体L123F的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第367-369位核苷酸由“CTG”突变为了“TTT”。突变后的DNA分子编码突变体L123F。与野生型Phi29 DNA聚合酶相比，突变体L123F的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第123位氨基酸残基由L突变为了F。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体L123I的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第367-369位核苷酸由“CTG”突变为了“ATT”。突变后的DNA分子编码突变体L123I。与野生型Phi29 DNA聚合酶相比，突变体L123I的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第123位氨基酸残基由L突变为了I。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体L123H的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第367-369位核苷酸由“CTG”突变为了“CAT”。突变后的DNA分子编码突变体L123H。与野生型Phi29 DNA聚合酶相比，突变体L123H的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第123位氨基酸残基由L突变为了H。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体E515P的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第1543-1545位核苷酸由“GAA”突变 为了“CCG”。突变后的DNA分子编码突变体E515P。与野生型Phi29 DNA聚合酶相比，突变体E515P的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第515位氨基酸残基由E突变为了P。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体Y224K的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第670-672位核苷酸由“TAT”突变为了“AAA”。突变后的DNA分子编码突变体Y224K。与野生型Phi29 DNA聚合酶相比，突变体Y224K的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第224位氨基酸残基由E突变为了K。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体G217E的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第649-651位核苷酸由“GGC”突变为了“GAA”。突变后的DNA分子编码突变体G217E。与野生型Phi29 DNA聚合酶相比，突变体G217E的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第217位氨基酸残基由G突变为了E。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体I474K的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第1420-1422位核苷酸由“ATT”突变为了“TTT”。突变后的DNA分子编码突变体I474K。与野生型Phi29 DNA聚合酶相比，突变体I474K的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第474位氨基酸残基由I突变为了K。
2、表达Phi29 DNA聚合酶多点突变体的重组载体的构建
以重组载体WT为出发载体，采用表1所示的各个引物对依次引入点突变，得到各个表达Phi29 DNA聚合酶突变体的重组载体，具体突变形式如表2所示：
表2
Figure PCTCN2018109777-appb-000003
与重组载体WT相比，表达Phi29 DNA聚合酶突变体M97K-L123H-E515P-Y224K-G217E的重组载体的差别仅在于：将序列表的序列1所 示的DNA分子第289-291位核苷酸由“ATG”突变为了“AAA”、序列表的序列1所示的DNA分子第367-369位核苷酸由“CTG”突变为了“CAT”、序列表的序列1所示的DNA分子第1543-1545位核苷酸由“GAA”突变为了“CCG”、序列表的序列1所示的DNA分子第670-672位核苷酸由“TAT”突变为了“AAA”、序列表的序列1所示的DNA分子第649-651位核苷酸由“GGC”突变为了“GAA”。突变后的DNA分子编码突变体M97K-L123H-E515P-Y224K-G217E。与野生型Phi29 DNA聚合酶相比，突变体M97K-L123H-E515P-Y224K-G217E的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第97位氨基酸残基由M突变为了K，且第123位由L突变成H，第515位由E突变成P，第224位由Y突变成K，第217位由G突变为E。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体M97T-L123K-E515P的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第289-291位核苷酸由“ATG”突变为了“ACC”、序列表的序列1所示的DNA分子第367-369位核苷酸由“CTG”突变为了“AAA”、序列表的序列1所示的DNA分子第1543-1545位核苷酸由“GAA”突变为了“CCG”。突变后的DNA分子编码突变体M97T-L123K-E515P。与野生型Phi29 DNA聚合酶相比，突变体M97T-L123K-E515P的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第97位氨基酸残基由M突变为了T，且第123位由L突变成K，第515位由E突变成P。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体M97K-L123H-E515P的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第289-291位核苷酸由“ATG”突变为了“AAA”、序列表的序列1所示的DNA分子第367-369位核苷酸由“CTG”突变为了“CAT”、序列表的序列1所示的DNA分子第1543-1545位核苷酸由“GAA”突变为了“CCG”。突变后的DNA分子编码突变体M97T-L123H-E515P。与野生型Phi29 DNA聚合酶相比，突变体M97K-L123H-E515P的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第97位氨基酸残基由M突变为了T，且第123位由L突变成H，第515位由E突变成P。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体Y224K-I474K-E515P的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第670-672位核苷酸由“TAT”突变为了“AAA”、序列表的序列1所示的DNA分子第1420-1422位核苷酸由“ATT”突变为了“TTT”、序列表的序列1所示的DNA分子第1543-1545位核苷酸由“GAA”突变为了“CCG”。突变后的DNA分子编码突变体Y224K-I474K-E515P。与野生型Phi29 DNA聚合酶相比，突变体Y224K-I474K-E515P的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第224位氨基酸残基由Y突变为了K，且第474位由I突变成K，第515位由E突变成P。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体M97K-L123H-E515P-G217E的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第289-291位核苷酸由“ATG”突变为了“AAA”、序列表的序列1所示的DNA分子第367-369位核苷酸由“CTG”突变为了“CAT”、序列表的序列1所示的DNA分子第1543-1545位核苷 酸由“GAA”突变为了“CCG”、序列表的序列1所示的DNA分子第649-651位核苷酸由“GGC”突变为了“GAA”。突变后的DNA分子编码突变体M97K-L123H-E515P-G217E。与野生型Phi29 DNA聚合酶相比，突变体M97K-L123H-E515P-G217E的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第97位氨基酸残基由M突变为了K，第123位氨基酸残基由L突变为了H，且第515位由E突变成P,第217位由G突变成E。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体M97K-L123H-E515P-Y224K的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第289-291位核苷酸由“ATG”突变为了“AAA”、序列表的序列1所示的DNA分子第367-369位核苷酸由“CTG”突变为了“CAT”、序列表的序列1所示的DNA分子第1543-1545位核苷酸由“GAA”突变为了“CCG”、序列表的序列1所示的DNA分子第670-672位核苷酸由“TAT”突变为了“AAA”。突变后的DNA分子编码突变体M97K-L123H-E515P-G217E。与野生型Phi29 DNA聚合酶相比，突变体M97K-L123H-E515P-G217E的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第97位氨基酸残基由M突变为了K，第123位氨基酸残基由L突变为了H，且第515位由E突变成P,第224位由Y突变成K。
与重组载体WT相比，表达Phi29 DNA聚合酶突变体Y224K-I474K的重组载体的差别仅在于：将序列表的序列1所示的DNA分子第670-672位核苷酸由“TAT”突变为了“AAA”、序列表的序列1所示的DNA分子第1420-1422位核苷酸由“ATT”突变为了“TTT”。突变后的DNA分子编码突变体Y224K-I474K。与野生型Phi29 DNA聚合酶相比，突变体Y224K-I474K的差别仅在于将野生型Phi29 DNA聚合酶氨基酸序列第224位氨基酸残基由Y突变为了K，第474位氨基酸残基由I突变为了K。
二、表达Phi29 DNA聚合酶突变体的重组菌的构建
分别将步骤一构建的重组载体WT和各个表达Phi29 DNA聚合酶突变体的重组载体导入大肠杆菌BL21(DE3)，得到表达野生型Phi29 DNA聚合酶的重组菌和各个表达Phi29 DNA聚合酶突变体重组菌。
三、重组菌的诱导表达
取步骤二获得的表达野生型Phi29 DNA聚合酶的重组菌和各个表达Phi29 DNA聚合酶突变体重组菌，依次进行诱导和纯化，得到N端融合有His 6标签的野生型Phi29 DNA聚合酶和各个N端融合有His 6标签的Phi29 DNA聚合酶突变体。
N端融合有His 6标签的野生型Phi29 DNA聚合酶和各个N端融合有His 6标签的Phi29 DNA聚合酶突变体依次命名为具有His 6标签的野生型Phi29 DNA聚合酶、具有His6标签的Phi29 DNA聚合酶突变体M97H、具有His6标签的Phi29 DNA聚合酶突变体M97A、具有His6标签的Phi29 DNA聚合酶突变体M97K、具有His6标签的Phi29 DNA聚合酶突变体L123K、具有His6标签的Phi29 DNA聚合酶突变体L123F、具有His6标签的Phi29 DNA聚合酶突变体L123I、具有His6标签的Phi29 DNA聚合酶突变体L123H、具有His6标签的Phi29 DNA聚合酶突变体E515P、具有His6 标签的Phi29 DNA聚合酶突变体E515G、具有His6标签的Phi29 DNA聚合酶突变体Y224K、具有His6标签的Phi29 DNA聚合酶突变体G217E、具有His6标签的Phi29DNA聚合酶突变体I474K、具有His6标签的Phi29 DNA聚合酶突变体M97K-L123H-E515P-Y224K-G217E、具有His6标签的Phi29 DNA聚合酶突变体M97T-L123K-E515P、具有His6标签的Phi29 DNA聚合酶突变体M97K-L123H-E515P、具有His6标签的Phi29 DNA聚合酶突变体Y224K-I474K-E515P、具有His6标签的Phi29 DNA聚合酶突变体M97K-L123H-E515P-G217E、具有His6标签的Phi29 DNA聚合酶突变体M97K-L123H-E515P-Y224K、具有His6标签的Phi29 DNA聚合酶突变体Y224K-I474K、具有His6标签的Phi29 DNA聚合酶突变体Y224K-G217E。
1、诱导的具体步骤如下：
(1)活菌
将重组菌接种至3ml含Kana抗性的液体LB培养基中，过夜培养。
(2)转接
完成步骤(1)后，按1/100体积将以上菌液转接至2ml含Kana抗性的液体LB培养基中，37℃、220rpm振荡培养至OD 600nm＝0.6(实际应用中，OD 600nm＝0.4-0.8均可)。
(3)诱导
完成步骤(2)后，向体系中加入加入终浓度0.5mM的IPTG，16℃、220rpm振荡培养12h。
(4)收集菌体
完成步骤(3)后，4℃、8000rpm离心5min，收集菌体。
2、使用GE公司的
Figure PCTCN2018109777-appb-000004
Pure纯化系统进行纯化，具体步骤如下：
(1)取步骤1得到的菌体，用重悬缓冲液(20mM Tris-HCl，500mM NaCl，20mM Imidazole，5％Glycerol；pH 7.9@25℃)震荡混匀后，置冰上超声破碎，然后4℃、12000rpm离心30min，收集上清液。
(2)取步骤(1)得到的上清液，采用镍柱亲和层析(HisTrap FF 5ml预装柱)进行纯化。具体步骤：先用10个柱体积的Buffer A平衡；然后上样；然后用20个柱体积的Buffer A冲洗；然后用15个柱体积的洗脱液进行洗脱并收集具有目的蛋白的过柱后溶液(洗脱液由Buffer A和Buffer B组成，洗脱过程中，Buffer B的体积份数由0％线性上升至100％，相应的Buffer A的体积份数由100％线性下降至0％)。
Buffer A：20mM Tris-HCl，500mM NaCl，20mM Imidazole，5％(v/v)Glycerol；pH7.9@25℃。
Buffer B：20mM Tris-HCl，500mM NaCl，500mM Imidazole，5％(v/v)Glycerol；pH7.9@25℃。
(3)取步骤(2)得到的过柱后溶液，采用强阴离子柱层析(HiTrap Q HP 5ml预装柱)进行纯化。具体步骤：先用10个柱体积的由体积份数59％的Buffer A和体积份数41％的Buffer B组成的混合缓冲液平衡；然后上样；待蛋白峰出现后收集流 穿液(UV检测值升至20mAu后即收集，UV检测值降至50mAu后停止收集)。
Buffer A：20mM Tris-HCl,150mM NaCl,5％(v/v)Glycerol,pH7.5@25℃。
Buffer B：20mM Tris-HCl,1M NaCl,5％(v/v)Glycerol,pH7.5@25℃。
(4)取步骤(3)得到的流穿液，采用阳离子交换层析(HiTrap SP HP预装柱)进行纯化，获得纯度大于95％的蛋白样本溶液。具体步骤：先用10个柱体积的Buffer A平衡；然后上样；然后用15个柱体积的Buffer A冲洗；然后用10个柱体积的洗脱液进行洗脱(洗脱液由Buffer A和Buffer B组成，洗脱过程中，Buffer B的体积份数由0％线性上升至50％，相应的Buffer A的体积份数由100％线性下降至50％)并收集具有目的蛋白的过柱后溶液(UV检测值升至50mAu后即收集，UV检测值降至100mAu后停止收集)。
Buffer A：20mM Tris-HCl,150mM NaCl,5％(v/v)Glycerol,pH7.5@25℃。
Buffer B：20mM Tris-HCl,1M NaCl,5％(v/v)Glycerol,pH7.5@25℃。
(5)取步骤(4)收集到的目的蛋白转移到透析袋中，在透析缓冲液透析过夜后，收集透析袋中的蛋白溶液，加入其它试剂，得到蛋白浓度为1mg/ml的目的蛋白溶液。目的蛋白溶液中的其它组分的浓度如下：10mM Tris-HCl(pH7.4@25℃)，100mM KCl，1mM DTT，0.1mM EDTA，0.5％(v/v)NP-40，0.5％(v/v)Tween20，50％(v/v)Glycerol。
透析缓冲液：23.75mM Tris-HCl(pH7.4@25℃)，237.5mM KCl，2.375mM DTT，0.2375mM EDTA，5％(v/v)Glycerol。
实施例2、Phi29 DNA聚合酶野生型以及突变体的聚合活性测试
取实施例1制备的具有His 6标签的野生型Phi29 DNA聚合酶溶液和具有His 6标签的Phi29 DNA聚合酶突变体溶液作为待测酶液。
1、将各个待测酶液使用储存缓冲液梯度稀释至5000倍，涡旋振荡器充分混匀，冰上静置5min，得到各个待测溶液。
将预反应体系在PCR管中混匀，置于PCR仪，进行如下程序：95℃1min，65℃1min，40℃1min,热盖温度设置为102℃。
预反应体系(80.8μl)：50mM Tris-HCl(pH7.5)，4mM DTT，10mM(NH 4) 2SO 4，10mM MgCl 2,50nM dNTP Mixture，2pM 141RCA Primer和18ng单链环状DNA模板141Ad ssDNA。
2、完成步骤1后，当温度达到4℃时取出PCR管置于冰上。试验组分别加入1μl各个待测溶液，阴性对照组加入1μl储存缓冲液。然后使用漩涡振荡器震荡混匀，短暂离心机离心5s后置于PCR仪，进行如下程序：30℃60min，
热盖温度设置为65℃。
3、完成步骤2后，加入5μL 0.5M EDTA溶液终止反应，震荡混匀。
4、采用Qubit ssDNA Assay Kit(Q10212，INVITROGEN)并按说明书操作，使用Qubit fluorometer3.0检测反应产物中DNB(DNA Nano ball)浓度。
待测酶液的酶活＝ΔDNB×5000÷37.38。
说明：ΔDNB为试验组与阴性对照组终止反应后体系中的反应产物的浓度平均值的差值，5000是稀释倍数，37.38是酶活和ΔDNB之间函数关系的斜率。
部分待测酶液的酶活结果见表3。
表3
Figure PCTCN2018109777-appb-000005
实施例3、Phi29 DNA聚合酶野生型以及突变体的热稳定性测试
37℃10min实验：
取实施例1制备的目的蛋白溶液(具有His 6标签的野生型Phi29 DNA聚合酶溶液和具有His 6标签的Phi29 DNA聚合酶突变体溶液)，分成两份，分别处理如下：
第一份：置于预热至37℃的金属浴中10min，4℃、13000rpm离心1min，收集上清液；然后，取所述上清液，用储存缓冲液稀释至1000倍体积，用涡旋振荡器充分混匀，然后冰上静置5min，得到待测溶液1。
第二份：用储存缓冲液稀释至5000倍体积，用漩涡震荡仪充分混匀，然后冰上静置5min，得到待测溶液2。
按照实施例2的步骤1至4进行。
未热处理的酶活(U1)＝ΔDNB×5000÷37.38；ΔDNB为试验组(第二份)与阴性对照组终止反应后体系中的反应产物的浓度平均值的差值；
热处理后的酶活(U2)＝ΔDNB×1000÷37.38；ΔDNB为试验组(第一份)与阴性对照组终止反应后体系中的反应产物的浓度平均值的差值；
5000和1000分别是稀释倍数，37.38是酶活和ΔDNB之间函数关系的斜率；
根据未经热处理和经过热处理的酶液的活性计算酶活损失比，
酶活损失比(％)＝(U1-U2)÷U1×100％；
U1为未经热处理酶液活性，U2为经热处理酶液活性。
-20℃实验：与37℃10min实验不同的仅为将37℃的金属浴中10min替换为-20℃的冰箱中10min。
各个突变体的比酶活和酶活损失比如表4所示。相比于野生型(WT)，示例突变体的酶活损失比均有一定程度的提升；酶活损失比数值越大，酶越不稳定，热稳定性越差。
表4
Figure PCTCN2018109777-appb-000006
Figure PCTCN2018109777-appb-000007
上述表中，NC是背景值，空白处是因为没有数据的计算处理。
实施例4、基因组扩增覆盖度检测
为在上机测序前检测样本扩展到覆盖度，使用多重PCR方式扩增MDA产物中多重看家基因。
具体操作步骤如下：往200ul PCR管中加入NA12878参考基因组样本(Coriell Institute)，总体积为4ul，不足的话用加入PBS补足至4ul。向PCR管中加入3ul Buffer D2(QIAGEN REPLI-g Single Cell Kit)，枪头要贴管壁，不要插入液面下，离心，不要振荡。离心后放到PCR仪上，65℃10min。结束后，置于冰上，向PCR管中加入3ul中和buffer(QIAGEN REPLI-g Single Cell Kit)，枪头要贴管壁，不要插入液面下，离心，不要振荡。向PCR管中加入MDA reaction buffer(10x phi29 Reaction Buffer 5ul，25mM dNTP 2ul，1mM N8Primer 2ul,100x BSA 0.5ul，10％F68 0.05ul，H2O 29.75ul)39ul后，分别加入1ul实施例1制备的phi29-337、phi29-456、phi2,9-458、phi29-36、phi29-335、phi29-414、phi29-464和1ul QIAGEN REPLI-g Single Cell Kit附带的Phi29 DNA聚合酶。枪头要贴管壁，不要插入液面下，离心，不要振荡。离心后放到PCR仪上30℃ 8h，65℃ 3min。反应完后振荡离心，用Qubit BR测浓度。测完浓度后进行多重看家基因PCR检测。DNA量取50～200ng，要设置阴性(N)和阳性对照(P)，阴性为水，阳性为没有扩增的NA12878参考基因组，实验组设置3个不同的样品。多重PCR引物及程序见如下表5：
表5 多重PCR引物及PCR反应体系
Figure PCTCN2018109777-appb-000008
Figure PCTCN2018109777-appb-000009
PCR程序：95℃ 5min，35cycles x[94℃ 30s,60℃ 50s,72℃ 1min],72℃ 5min,12℃ holder。
结果如图2所示，phi29-337、phi29-456、phi29-458、phi29-36、phi29-414相比野生型WT，扩增覆盖度提高了很多，8条看家基因产物至少能扩增出6条以上。在扩增产物的浓度上，phi29-337、phi29-456、phi29-458、phi29-36、phi29-414比野生型WT和商品Enzymatics公司的phi29产品要高。
实施例5、Phi29 DNA聚合酶野生型和Phi29 DNA聚合酶突变体在单细胞全基因组测序中的应用效果
为了测试Phi29 DNA聚合酶野生型和突变体在单细胞基因组测序中的效果，分别将实施例1制备的Phi29 DNA聚合酶野生型和Phi29 DNA聚合酶突变体扩增NA12878微量gDNA(200pg)的产物进行建库，后进行高深度测序；同时以商业化的Qiagen单细胞扩增试剂盒扩增的相同的NA12878微量gDNA(200pg)进行对照。
对测序数据进行分析及变异检测分析，用NA12878标准变异数据集进行评估，比较各个突变体及与Qiagen试剂盒的差异。具体数据分析结果见表6和表7。
可以看出，突变体Phi29-337、Phi29-456、Phi29-335、Phi29-414在mapping rate,coverage上与野生型WT和QIAGEN试剂盒表现相当，但duplication rate参数方面，野生型WT表现最差，表中所列突变体均比野生型稍好些，其中337突变体表现最好，与QIAGEN(REPLI-g Single Cell Kit)试剂盒相当。同时，变异检测评估参数snp_Precision，snp_Sensitivity，snp_F-measure和方面，337突变体表现最好，比QIAGEN试剂盒略好，野生型WT和456、335、414突变体均比QIAGEN试剂盒差；indel_Precision，indel_Sensitivity和indel_F-measure参数方面也是337突变体变现最好，数据比QIAGEN试剂盒要好些。
表6.Phi29突变体及野生型在单细胞测序中的测序数据分析
突变体编号 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 1.01E+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
表7.Phi29突变体及野生型在单细胞测序中的测序数据分析
突变体编号 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
(注：QIAGEN是商品REPLI-g Single Cell Kit对照)
实施例6、RCA建库上机测试效果检测
综合了表4的热稳定性结果，本实施例挑选若干个热稳定性提升的突变体，在BGISEQ-500测序仪上机测试，检测不同突变体在DNA测序中的应用效果，参照BGISEQ-500测序仪标准。整个测试所用试剂为BGI生产的全套PE50V2.0试剂盒和BGI生产的的E.coil Ad153标准文库及Invitrogen生产的Qubit ssDNA Assay试剂。以下所用试剂除文库以及QubitssDNA Assay外均包括在PE50V2.0试剂盒中，而下述所写PE50V2.0试剂槽仅指上机测试所用试剂。
1、DNB制备
从-20℃冰箱取出DNB制备缓冲液、标准文库、DNB聚合酶混合液和DNB聚合酶混合液II于冰盒上融化，待完全溶解后，置于漩涡振荡器振荡持续混匀5s，并于掌上离心机短暂离心3s，置于冰盒上备用。从4℃冰箱里取出分子级水和DNB终止缓冲液，置于冰盒上备用。在标记好的八连管中，依次加入20ul DNB制备缓冲液和6ng ssDNA(E.coli标准品文库)，用Nuclease Free Water补足至40ul。将八连管置于漩涡振荡持续混匀5s，并于掌上离心机短暂离心3s。将上述八连管置于PCR仪中，设置或检查反应条件：95℃1min，65℃1min，40℃1min，4℃保持(热盖温度设置为103℃)。反应完成后，取出PCR八连管，置于冰盒上，等八连管盖温度降至室温后，将其置于掌上离心机短暂离心3s，立即放置于冰盒上。向八连管中依次加入如下各组份:40ul变性后八连管内反应液，40ul DNB聚合酶混合液，4ul DNB聚合酶混合液II。将八连管置于漩涡振荡持续混匀5s，并于掌上离心机短暂离心3s。立即置于PCR仪中或水浴锅中开始反应。设置或检查反应条件：30℃20min，4℃保持(不需设置热盖)。当上述RCA反应结束后，将八连管取出放置于冰盒上，立即加入20μL DNB终止缓冲液。用带100μL阔口枪头的移液器缓慢地吹打混匀5-8次(一吸一吹记为1次)，混匀后置于4℃保存备用。使用
Figure PCTCN2018109777-appb-000010
ssDNA Assay Kit和
Figure PCTCN2018109777-appb-000011
Fluorometer仪器对上述DNB制备产物进行浓度测定，当浓度为8ng/ul～40ng/ul时判定合格，备用后续实验。
2、DNB加载
DNB制备好后，需把DNB加载至芯片上，即DNB加载。
取出样品加载试剂板V2.1置于室温融化，震荡混匀，短暂离心后放于冰盒上备用。取出DNB加载缓冲液II，震荡均匀，短暂离心后放于冰盒上备用。首先，将芯片和样品加载试剂板V2.1置于BGIDL-50上。其次，取35μL DNB加载缓冲液II加到装有含100μL DNB的PCR管中，用阔口吸头温和混匀15次。然后，将上述混合液放于加载系统指定DNB放置区，选择DNB加载程序(Sample load 2.0)，开始加载。最后，加载完成后室温孵育30min后，放在2～8℃备用。
3、上机测试
对待测的phi29聚合酶突变体或野生型，在BGISEQ-500测序仪上进行上机测序检验，使用一张芯片和一个BGISEQ-500RS高通量测序试剂槽(PE50V2.0)。上机测试前，首先对测序试剂槽II，dNTPs混合液(V3.0)、dNTPs混合液II(V2.0)解冻融化后置于4℃冰箱或者冰盒上备用；并对测序酶振荡混匀，置于冰盒上备用。首先，配置5号孔试剂，即用1ml移液器移取1150μL DNA聚合酶混合液加入到5号孔以及移取1150μL dNTPs混合液(V3.0)加入到5号孔，并用移液器吹打混匀10-15次。其次，配置6号孔试剂，即用1ml移液器移取890μL DNA聚合酶混合液加入到6号孔以及移取890μL dNTPs混合液II(V2.0)加入到6号孔，并用移液器吹打混匀10-15次。然后，配置14号孔试剂，即用5ml移液器移取全部14号孔试剂，取其中2.8ml14号孔试剂与400μL phi29聚合酶突变体混匀后加入到14号孔。并把配置好的试剂槽组装。最后，进行上机测试，即启动测序仪并进行清洗，把试剂槽放入测序仪指定位置，按操作顺序进行实际预载，预载结束后，安装上述(2)步骤制备好的芯片，并填写相关测序信息，即开始测序，结束后取出芯片和试剂槽及清洗仪器。本实施例中测试一个循环。
4、数据分析
测序结束后，下载分析报告，比照先前指定的标准，判断phi29 DNA聚合酶突变体的性能。
比较的各个参数说明如下：ESR(Effective Spots Rates)有效spot数的比例，Bic(basecall information content)，可用于作basecalling的DNB比率；fit(crosstalk fit score)，反映crosstalk的情况，校正后各base的intensity分布越集中，其fit值越高；SNR(Signal to Noise Ratio)，信噪比,以单个DNB SNR的计算为例，base A(max intensity)作为信号，CGT则为背景，CGT intensity的方差则为噪声；RHO(Rho Intensity)，为corrected intensity去除normalization的intensity值，直观上解释为dye的原始强度经过校正后的intensity值。
结果如图3所示(A图为各个突变体的BIC、FIT、ESR参数的比较；B图为各个突变体SNR参数的比较；C图为各个突变体RHO参数的比较)，可以看出，综合上述各参数指标分析，测试的突变体中最好的为464，其次是463。
工业应用
在本发明基础上，可通过对本发明所述的突变位点的氨基酸进行饱和突变，进 一步筛选出效果良好的突变体；或者在本发明所述突变体的基础上，再对本发明所包含氨基酸位点之外的其他氨基酸进行突变，获得类似的效果。本法明还可能应用的技术领域包括食品检测、病毒检测、RNA检测、单细胞测序等，也有可能用于开发第三、四代测序仪技术。

Claims (22)

  1. 一种蛋白，是如下A)或B)：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、217、224、515和474这6位中至少一位的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白；
    B)所示的蛋白为将A)所示蛋白的氨基酸序列末端添加标签序列且具有DNA聚合酶活性的由A)衍生的蛋白质。
  2. 根据权利要求1所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、217、224、515和474这6位中至少2位或至少3位或至少4位或至少5位或全部的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
  3. 根据权利要求1或2所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、217、224和515这5位中全部的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
  4. 根据权利要求1或2所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第224、515和474这3位中全部的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
  5. 根据权利要求1或2所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、217、515这4位中全部的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
  6. 根据权利要求1或2所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97、123、224、515这4位的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
  7. 根据权利要求1或2所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第224、474这2位的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
  8. 根据权利要求1或2所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第217、224这2位的氨基酸残基进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
  9. 根据权利要求1-8中任一所述的蛋白，其特征在于：所述修饰为氨基酸置换。
  10. 根据权利要求9所述的蛋白，其特征在于：
    所述第97、123、217、224、515和474这6位点的氨基酸置换方式分别为 如下：
    第97位的甲硫氨酸置换为丙氨酸或组氨酸或赖氨酸或苏氨酸；
    第123位的亮氨酸置换为赖氨酸或苯丙氨酸或异亮氨酸或组氨酸；
    第217位的甘氨酸置换为谷氨酸；
    第224位的酪氨酸置换为赖氨酸；
    第474位的异亮氨酸置换为赖氨酸；
    第515位的谷氨酸置换为脯氨酸或甘氨酸。
  11. 根据权利要求10所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97位氨基酸残基由M突变为了K，且第123位由L突变成H，第515位由E突变成P，第224位由Y突变成K，第217位由G突变为E，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
  12. 根据权利要求10所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第224位氨基酸残基由Y突变为了K，且第474位由I突变成K，第515位由E突变成P。
  13. 根据权利要求10所述的蛋白，其特征在于：
    A)所示的蛋白为将phi29 DNA聚合酶氨基酸序列中第97位氨基酸残基由M突变为了T，且第123位由L突变成H，第515位由E突变成P。
  14. 根据权利要求1-13中任一所述蛋白，其特征在于：所述phi29 DNA聚合酶的氨基酸序列为如下任一种：
    (Ⅰ)序列表的序列2所示的蛋白；
    (Ⅱ)与序列表的序列2所示的蛋白具有90％以上同一性且来源于枯草芽孢杆菌的蛋白质；
    (Ⅲ)与序列表的序列2所示的蛋白具有95％以上同一性且来源于枯草芽孢杆菌的蛋白质。
  15. 根据权利要求1-14中任一所述的蛋白，其特征在于：所述蛋白质的稳定性高于所述phi29 DNA聚合酶。
  16. 根据权利要求15所述蛋白，其特征在于：所述稳定性为热稳定性。
  17. 编码权利要求1-16中任一所述蛋白的核酸分子。
  18. 含有权利要求17所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
  19. 权利要求1-16中任一所述蛋白在如下1)-11)中至少一种中的应用：
    1)作为DNA聚合酶；
    2)催化DNA复制和/或催化DNA扩增；
    3)催化滚换扩增和/或催化多重链置换扩增；
    4)进行DNA测序或RNA测序或全基因组测序；
    5)RCA建库；
    6)基因组扩增覆盖度检测；
    7)制备用于催化DNA复制和/或催化DNA扩增的试剂盒产品；
    8)制备用于催化滚换扩增和/或催化多重链置换扩增的产品；
    9)制备用于进行DNA测序或RNA测序或全基因组测序的产品；
    10)制备用于RCA建库的产品；
    11)制备用于基因组扩增覆盖度检测的产品。
  20. 编码权利要求1-16中任一所述蛋白的核酸分子、含有所述核酸分子的表达盒、含有所述核酸分子的重组载体、含有所述核酸分子的重组菌或含有所述核酸分子的转基因细胞系在如下1)-10)中至少一种中的应用：
    1)催化DNA复制和/或催化DNA扩增；
    2)催化滚换扩增和/或催化多重链置换扩增；
    3)进行DNA测序或RNA测序或全基因组测序；
    4)RCA建库；
    5)基因组扩增覆盖度检测；
    6)制备用于催化DNA复制和/或催化DNA扩增的产品；
    7)制备用于催化滚换扩增和/或催化多重链置换扩增的产品；
    8)制备用于进行DNA测序或RNA测序或全基因组测序的产品；
    9)制备用于RCA建库的产品；
    10)制备用于基因组扩增覆盖度检测的产品。
  21. 一种提高phi29 DNA聚合酶稳定性的方法，包括如下步骤：将phi29 DNA聚合酶氨基酸序列中第97、123、217、224、515和474这6位中至少一位的氨基酸残基按照权利要求1)-17)中任一中的修饰方式进行修饰，其余氨基酸残基不变，得到具有DNA聚合酶活性的蛋白。
  22. 根据权利要求22所述的方法，其特征在于：所述稳定性为热稳定性。
PCT/CN2018/109777 2018-10-11 2018-10-11 热稳定性提升的Phi29 DNA聚合酶突变体及其在测序中的应用 Ceased WO2020073266A1 (zh)

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