WO2002101004A2 - Extension cyclique a basse temperature d'adn a haute specificite d'amorcage - Google Patents

Extension cyclique a basse temperature d'adn a haute specificite d'amorcage Download PDF

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WO2002101004A2
WO2002101004A2 PCT/IB2002/003341 IB0203341W WO02101004A2 WO 2002101004 A2 WO2002101004 A2 WO 2002101004A2 IB 0203341 W IB0203341 W IB 0203341W WO 02101004 A2 WO02101004 A2 WO 02101004A2
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dna
primer
dna polymerase
bacillus
cycle
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PCT/IB2002/003341
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WO2002101004A3 (fr
Inventor
Guo Fan Hong
Yongjie Yang
Jia Zhu
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Shanghai Mendel Dna Center Co., Ltd
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Priority claimed from US09/878,131 external-priority patent/US20030087237A1/en
Application filed by Shanghai Mendel Dna Center Co., Ltd filed Critical Shanghai Mendel Dna Center Co., Ltd
Priority to JP2003503756A priority Critical patent/JP2005514003A/ja
Priority to EP02758704A priority patent/EP1436391A4/fr
Priority to CA002449560A priority patent/CA2449560A1/fr
Publication of WO2002101004A2 publication Critical patent/WO2002101004A2/fr
Publication of WO2002101004A3 publication Critical patent/WO2002101004A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/113—Cycle sequencing

Definitions

  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • DNA consists of a chain of individual deoxynucleotides chemically linked in specific sequences. Each deoxynucleotide contains one of the four nitrogenous bases which may be adenine (A), cytosine (C), guanine (G) or thymine (T), and a deoxyribose, which is a pentose, with a hydroxyl group attached to its 3' position and a phosphate group attached to its 5' position.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • deoxyribose which is a pentose, with a hydroxyl group attached to its 3' position and a phosphate group attached to its 5' position.
  • the contiguous deoxynucleotides that form the DNA chain are connected to each other by a phosphodiester bond linking the 5' position of one pentose ring to the 3' position of the next pentose ring in such a manner that the beginning of the DNA molecule always has a phosphate group attached to the 5' carbon of a deoxyribose.
  • the end of the DNA molecule always has an OH (hydroxyl) group on the 3' carbon of a deoxyribose.
  • DNA usually exists as a double-stranded molecule in which two antiparallel DNA strands are held together by hydrogen bonds between the bases of the individual nucleotides of the two DNA strands in a strictly matched "A-T " and "C-G” pairing manner. It is the order or sequence of the bases in a strand of DNA that determines a gene which in turn determines the type of protein to be synthesized. Therefore, the accurate determination of the sequence of the bases in a DNA strand which also constitutes the genetic code for a protein is of fundamental importance in understanding the characteristics of the protein concerned.
  • DNA sequencing The process used to determine the sequence of the bases in a DNA molecule is referred to as DNA sequencing.
  • the enzymatic method developed by Sanger et al. (1) is most popular. It is based on the ability of a DNA polymerase to extend a primer annealed to the DNA template to be sequenced in the presence of four normal deoxynucleotide triphosphates (dNTPs), namely, dATP, dCTP, dGTP and dTTP, and on the ability of the nucleotide analogs, the dideoxynucleotide triphosphates (ddNTPs), namely, ddATP, ddCTP, ddGTP and ddTTP, to terminate the extension of the elongating deoxynucleotide polymers at various lengths.
  • dNTPs normal deoxynucleotide triphosphates
  • ddNTPs dideoxynucleotide triphosphates
  • the single-stranded template is again available for annealing with an oligonucleotide primer upon cooling, ready for another cycle of enzymatic DNA synthesis in the presence of a functioning DNA polymerase and dNTPs.
  • a heat-resistant DNA polymerase which can survive the heating to 95°C and is active at temperature between 55 and 72°C, is employed in the system so that no fresh enzyme needs to be added to initiate each cycle of DNA synthesis after denaturing at high temperature.
  • a primer is mixed in excess with a template and the temperature cycles repeat for a plurality of times, the number of the extended single- stranded target fragments increases one fold per cycle.
  • heat-resistant polymerases are usually associated with low processivity, and may lose their sequence-specific polymerase activity under certain unpredictable conditions, especially when GC-rich DNA segments (that is, segments containing a significantly higher content of guanine and cytosine, relative to the content of thymine and adenine) in a template are to be amplified or to be sequenced. Therefore, attempts have been made to develop conditions suitable for low temperature cycle sequencing and for low temperature cycle PCR using thermolabile DNA polymerases, which, in general, have higher fidelity and higher processivity than the heat-resistant DNA polymerases. For example, in U.S. Patent No.
  • the inventors have developed methods for extending a primer or a pair of primers in cycle DNA amplification for automated cycle sequencing and PCR.
  • the methods contemplate moderately thermostable DNA polymerases in the presence of a low concentration of glycerol or ethylene glycol, or the mixtures thereof, as an agent to reduce the melting temperature of DNA (that is, the temperature at which the double-strands of DNA are denatured).
  • the inventors observed that at a certain concentration range, glycerol and/or ethylene glycol not only reduced the melting temperature of the DNA template, but also increased the polymerization activity of the moderately thermostable DNA polymerases.
  • the temperature range of cycling is between 70°C and 37°C — much lower than what is usually required for denaturing DNA.
  • the methods use highly processive, moderately thermostable DNA polymerases preferably derived from Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus. These polymerases have an optimum reaction temperature at 65°C, but are rapidly inactivated above 70°C; thus, they are quite useful as the polymerizing enzymes for the cycle primer extension to overcome some of the shortcomings of the heat-resistant DNA polymerases, such as Taq and its corresponding mutants, and of the heat-labile DNA polymerases, such as the Klenow fragment.
  • the moderately thermostable DNA polymerases may be in their natural state (e.g., purified from the organisms), or modified.
  • the invention contemplates a method for extending a primer (or a pair of primers) using an enzymatic cycle primer extension reaction at low cycling temperatures (that is, temperatures below about 80°C), in a reaction mixture composition comprising between about 10% and about 20% (and preferably about 15%) (v/v) glycerol, ethylene glycol, or a mixture thereof, in the presence of a moderately thermostable (also referred to as mesophilic) DNA polymerase.
  • low cycling temperatures that is, temperatures below about 80°C
  • a reaction mixture composition comprising between about 10% and about 20% (and preferably about 15%) (v/v) glycerol, ethylene glycol, or a mixture thereof, in the presence of a moderately thermostable (also referred to as mesophilic) DNA polymerase.
  • “enzymatic cycle primer extension reaction” it is meant that in excess of primer over template, the limited number of template molecules can be used repeatedly for DNA polymerization catalyzed by a functional DNA polymerase when the temperature of the reaction mixture fluctuates repeatedly between the levels required for denaturing, annealing and primer extension in cycles.
  • DNA template may be mixed with a primer (or a pair of primers) and a natural or a modified form of a moderately thermostable DNA polymerase from one of Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus, in a solution containing between about 10% and about 20% (v/v) (preferably about 15% (v/v)) glycerol, ethylene glycol, or a mixture thereof.
  • the reaction may be carried out under conditions that the cycle reaction temperature fluctuates between a melting temperature of about 70°C and a cooling (or annealing) temperature of about 37°C, so that the DNA polymerase repeatedly extends the primer or pair of primers at the tempterature between about 45°C and 50°C.
  • the method may include the further step of repeating the cycle primer extension reaction, as many times as is desired.
  • copies of a selected segment of a double-stranded DNA are amplified in the presence of a forward primer and a reverse primer (where both may be of various lengths) to the template by repeated heating and cooling (or annealing) cycles (such as, for instance, in a PCR).
  • the reaction is run at low temperatures (that is, temperatures below about 80°C), in a reaction mixture composition comprising between about 10% and about 20% (and preferably about 15%) (v/v) glycerol, ethylene glycol, or a mixture thereof, in the presence of one of the moderately thermostable DNA polymerases described above.
  • the reaction may be carried out under conditions that the reaction temperature fluctuates between a melting temperature of about 70°C and a cooling (or annealing) temperature of about 37°C, so that the DNA polymerase repeatedly extends the forward and reverse primers at the temperature of between about 45°C and 50°C.
  • the method may include the further step of repeating the reaction, as many times (e.g., cycles) as is desired.
  • the DNA polymerase is one of those described in U.S. Patent No. 5,747,298, U.S. Patent No. 5,834,253 or U.S. Patent No. 6,165,765.
  • the DNA polymerase has an amino acid sequence that shares not less than 95% homology of a DNA polymerase isolated from Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus.
  • molecules of a single primer of various lengths are extended with specific nucleotide terminations in the presence of ddNTPs or their analogs for cycle sequencing.
  • the invention also contemplates a method for extending the molecules of a single primer annealed to a single-stranded copy of the doubled-stranded DNA product amplified in vitro without prior isolation or purification for direct cycle sequencing.
  • a diluted crude amplified reaction product preferably generated with a low- temperature PCR reaction catalyzed by a moderately thermostable DNA polymerase as described herein
  • a moderately thermostable DNA polymerase preferably one with a reduced innate selective discrimination against incorporation of a subset of dye-labeled ddNTPs
  • a suitable concentration of dNTPs dATP, dGTP, dTTP and dCTP
  • a composition comprising
  • a standard cycle primer extension reaction(s) may then be run at a temperature below 80°C for a sufficient number of times to extend the sequencing primer molecules to desired varying lengths, which extended molecules will be terminated specifically by fluorescently labeled ddNTPs or their corresponding analogs.
  • the cycle reaction temperature fluctuates between a melting temperature of about 70°C and a cooling/annealing temperature of about 37°C.
  • the method of sequencing a DNA strand may comprise the steps of: i) hybridizing a primer to a DNA template to be sequenced; and ii) extending the primer using one of the above-described DNA polymerases, in the presence of a solution containing between about 10% and about 20% (v/v) (preferably about 15% (v/v)) glycerol, ethylene glycol, or a mixture thereof, adequate amounts of the deoxynucleotide bases dATP, dGTP, dCTP and dTTP, and the four dideoxynucleotide terminators or their analogs, whereby the cycle reaction temperature fluctuates between a melting temperature of about 70°C and a cooling or annealing temperature of about 37°C, and under such conditions that the DNA strand is sequenced.
  • one of the deoxynucleotides is radioisotope-labeled, or the primer molecules are fluorescent dye- labeled, and more preferably all dideoxynucleo
  • the invention entails a dry or liquid ready-to-use reaction mixture or kit suitable for use in a low-temperature cycle primer extension reaction at temperatures below about 80°C.
  • This reaction mixture or kit comprises a moderately thermostable DNA polymerase (such as one of those described above) that is pre-mixed with at least one enzymatic DNA primer extension reaction component suitable for use in DNA amplification or for specific extension terminations with dideoxyribonucleotide analogs.
  • the reaction mixture is preferably pre-distributed into microcentrifuge tubes or in multiple-well plates, such as, for instance, those that are suitable for large-scale automated PCR or for large-scale automated DNA sequencing.
  • This ready-to-use reaction mixture or kit can be stored at room temperature between about 22°C and about 25°C for at least eight weeks without losing its specific polymerization activity for DNA primer amplification or extension terminations.
  • Figure 1 is a graph illustrating the effect of glycerol on 5 '-3' polymerization activity on Bst-II DNA polymerase.
  • Figure 2 is a picture of an electrophoresis gel (1% agarose), showing the results of low-temperature amplification with a moderately thermostable DNA polymerase in 40% glycerol.
  • Figure 3 A is a picture of an electrophoresis gel (1% agarose), showing the results of low-temperature cycle primer extension in 35% glycerol (lane 1) and in 15% glycerol (lane 2) with amplified products having a length of 250 base pairs.
  • Figure 3B is a picture of an electrophoresis gel (1% agarose), showing the results of low-temperature cycle primer extension in 35%> glycerol (lane 1) and in 15% glycerol (lane 2) with amplified products having a length of 400 base pairs.
  • Figure 3C is a picture of an electrophoresis gel (1% agarose), showing the results of low-temperature cycle primer extension in 35% glycerol (lane 1) and in 15% glycerol (lane 2) with amplified products having a length of 1 kilobase.
  • Figure 3D is a picture of an electrophoresis gel (1% agarose), showing the results of low-temperature cycle primer extension in 35% glycerol (lane 1) and in 15% glycerol (lane 2) with amplified products having a length of 2 kilobases.
  • Figure 4 is a picture of an electrophoresis gel (1% agarose), showing the results of low-temperature cycle extension reaction of 17mer and 30mer primers with moderately thermostable DNA polymerases and Klenow fragment.
  • the reaction products with 17mer primers are Al (Klenow fragment using the lakobashvili and Lapidot system), A2 (Klenow fragment with the Bst system), A3 (Bst-I polymerase with the Bst system), A4 (Bst-II polymerase with the Bst system), and A5 (Bca polymerase with the Bst system).
  • the reaction products with 30mer primers are Bl (Klenow fragment using the lakobashvili and Lapidot system), B2 (Klenow fragment with the Bst system), B3 (Bst-I polymerase with the Bst system), B4 (Bst-II polymerase with the Bst system), and B5 (Bca polymerase with the Bst system).
  • Figures 5 A and 5B represent two automated fluorescent DNA sequencing tracings of a GC-rich segment, comparing the performance of AmpliTaqTM in the ABI PrismTM BigDyeTM Terminator cycle sequencing kit (5 A) with that of the Bst-II cycle sequencing system (5B).
  • Figure 6 is a picture of an electrophoresis gel (1% agarose), showing the results of cycle primer extension reactions conducted at various temperature steps, using a moderately thermostable DNA polymerase (Bst-II), with no glycerol and with 15% glycerol.
  • Bst-II moderately thermostable DNA polymerase
  • this invention entails a unique combination of a moderately thermostable DNA polymerase (such as Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus ) in the presence of a low concentration of an agent selected from the group consisting of glycerol, ethylene glycol and mixtures of these, to provide a way to extend a primer (or pair of primers) in cycle DNA amplification for automated cycle sequencing and PCR at temperatures below about 80°C.
  • glycerol and ethylene glycol at low concentrations increase the sequence-specific DNA polymerization activity of the moderately thermostable DNA polymerases in vitro.
  • both glycerol and ethylene glycol exhibit a detrimental inhibitory effect on the DNA polymerization activity of these enzymes.
  • the inventors achieved a reaction mixture with an optimum concentration of glycerol or ethylene glycol, in which double-stranded DNA templates are denatured at 70°C while the polymerization activity of the moderately thermostable DNA polymerases can be preserved during low temperature sequence-specific cycle primer extension.
  • the inventors first observed that at the optimum enzymatic reaction temperature of 65 °C, a final concentration of glycerol of up to about 20% increased the 5 '-3' polymerization activity of the moderately thermostable DNA polymerases (for instance, see Figure 1). However, when the concentrations of glycerol was increased to greater than about 35% it invariably suppressed this enzymatic activity. When the glycerol concentration increases to 40% (v/v), this group of DNA polymerases usually lost more than two thirds (2/3) of the original polymerization activity. It was found that low-temperature cycle extensions with moderately thermostable DNA polymerases in a solution containing 40% of glycerol generates poorly defined non-specific amplified products of varying fragment sizes (for instance, see Figure 2).
  • moderately thermostable DNA polymerases such as a Bst mutant or Bca
  • the reaction products are non-specific. (For instance, see Figure 3).
  • a low concentration of glycerol or ethylene glycol for example between about 10% and about 20% v/v, preferably 15%, can be used to lower the DNA melting temperature for cycle primer extension in conjunction with a moderately thermostable DNA polymerase to generate sequence-specific amplification products.
  • DNA fragments of a wide range in length including those having less than 30 base pairs, even shorter than 20 base pairs in length can be used as the primers for sequence-specific extensions (for instance, see Figure 4).
  • the thermolabile DNA polymerases such as the Klenow fragment, fail to generate any significant amount of amplification products useful for further analysis (see, for instance, Figure 4).
  • ThermoSequenaseTM and AmpliTaqTM both being modified forms of the heat-resistant Taq DNA polymerase, cannot generate sequence-specific products useful for further analysis in the low-temperature cycle extension system.
  • the heat-resistant DNA polymerases generate no sequence-specific ddNTP terminations down-stream to the GC-rich segment of the template whereas a moderately thermostable enzyme, for example the mutated Bst-II, can successfully overcome the GC-rich obstacle during DNA polymerase cycle extensions (see, for instance, Figure 5).
  • moderately thermostable DNA polymerases are quite critical to the methods of this invention.
  • Moderately thermostable it is meant that these polymerases have an optimum reaction temperature at 65°C, but are rapidly inactivated above 70°C.
  • the invention contemplates DNA polymerases obtained or derived from one or more of Bacillus stearothermophilus (Bst), Bacillus caldotenax (Bca) or Bacillus caldolyticus (Bey). All three of these organisms are classified as mesophilic microbes because, although their DNA polymerases are referred to as thermostable (most active at 65°C), they are inactivated at 70°C or above.
  • Taq enzymes
  • Other enzymes such as Taq, which are truly thermophilic — that is, the Taq DNA polymerase tolerates and remains active at temperatures higher than 95°C.
  • a moderately thermostable (also sometimes referred to as mesophilic) DNA polymerase may have proofreading 3'-5' exonuclease activity during DNA primer extension over a template, such that the DNA polymerase functions to excise mismatched nucleotides from the 3' terminus of the DNA strand at a faster rate than the rate at which the DNA polymerase functions to remove nucleotides matched correctly with nucleotides of the template.
  • DNA polymerases are also described by the inventors in U.S. Patent No. 5,834,253, U.S. Patent No. 5,747,298, and U.S. Patent No. 6,165,765 (the contents of all of which are incorporated herein by reference in their entirety).
  • strain No. 320 for identification purposes; described in U.S. Patent 5,747,298
  • Bst 320 a DNA polymerase
  • Bst 320 a proof-reading 3'-5' exonuclease activity which is absent in DNA polymerases isolated from other strains of Bacillus stearothermophilus.
  • the term "proof-reading" is intended to denote that the DNA polymerase is capable of removing mismatched nucleotides from the 3' terminus of a newly formed DNA strand at a faster rate than the rate at which nucleotides correctly matched with the nucleotides of the template are removed during DNA sequencing.)
  • the strain Bst 320 was deposited on October 30, 1995 in the American Type Culture Collection, located at 12301 Parklawn Drive, Rockville, Maryland 20852, and has been given ATCC Designation No. 55719.
  • the DNA polymerase isolated from Bst 320 is composed of 587 amino acids as are the DNA polymerases of other known strains of Bacillus stearothermophilus, such as, for instance, the strains deposited by Riggs et al (Genbank Accession No. L42111) and by Phang et al. (Genbank Accession No. U23149).
  • the Bst 320 shares only 89.1% sequence identity at protein level with the Bacillus stearothermophilus DNA polymerase deposited by Riggs et al., and shares only 87.4% sequence identity at protein level with the Bacillus stearothermophilus DNA polymerase deposited by Phang et al.
  • the above-referenced enzyme deposited by Riggs et al. and the enzyme deposited by Phang et al. share 96.9% of their amino acid sequence identity.
  • Bst 320 DNA polymerase shares 88.4% of the amino acid sequence identity with Bca DNA polymerase (Uemori et al. J. Biochem. 113: 401-410, 1993). Based on homology of the amino acid sequences, Bst 320 DNA polymerase is as close to DNA polymerases isolated from Bacillus stearothermophilus as to the DNA polymerase isolated from Bacillus caldotenax, i.e. another species of bacillus.
  • Bst 320 DNA polymerase and Bca DNA polymerase functionally exhibit 3'-5' exonuclease activity, which is not associated with known amino acid sequence exonuclease motifs I, II and III as in the E. coli DNA polymerase I model, or other known Bacillus stearothermophilus polymerases.
  • One preferred Bst DNA polymerase is isolated from strain 320 with an amino acid sequence as follows:
  • D aspartic acid (Asp) P: proline (Pro)
  • E glutamic acid (Glu)
  • Q glutamine
  • Gin Gin
  • R arginine (Arg)
  • G glycine
  • S serine (Ser)
  • H histidine (His) T: threonine (Thr) I: isoleucine (He) V: valine (Val) K: lysine (Lys) W: tryptophan (Trp) L: leucine (Leu) Y: tyrosine (Tyr)
  • This Bst 320 DNA polymerase is characterized by possessing a proofreading 3'-5' exonuclease activity.
  • the nucleotide sequence encoding the Bst 320 DNA polymerase is indicated in SEQ ID NO: 1, below.
  • DNA sequence isolated/purified: GCCGAAGGGG AGAAACCGCT TGAGGAGATG GAGTTTGCCA TCGTTGACGT CATTACCGAA GAGATGCTTG CCGACAAGGC AGCGCTTGTC GTTGAGGTGA TGGAAGAAAA CTACCACGAT GCCCCGATTG TCGGAATCGC ACTAGTGAAC GAGCATGGGC GATTTTTTAT GCGCCCGGAG ACCGCGCTGG CTGATTCGCA ATTTTTAGCA TGGCTTGCCG ATGAAACGAA GAAAAAGC ATGTTTGACG CCAAGCGGGC AGTCGTTGCC TTAAAGTGGA AAGGAATTGA GCTTCGCGGC GTCGCCTTTG ATTTATTGCT CGCTAT TTGCTCAATC TGCCGGCGAT ATCGCTGCGG TGGCGAAAAT GAAACAATA
  • a disadvantage of the DNA polymerases of the mesophilic strains Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus is that during DNA sequencing they all exhibit a high degree of selective discrimination against incorporation of certain particular members of fluorescent dye-labeled ddNTPs, namely the fluorescent dye-labeled ddCTP and fluorescent dye-labeled ddATP, as terminators onto the 3' end of the extending DNA fragments during enzymatic reaction.
  • the DNA polymerase used is a mesophilic bacillus DNA polymerase (such as Bacillus stearothermophilus, Bacillus caldotenax and Bacillus caldolyticus) which, during dye-labeled terminator automated DNA cycle sequencing, reduces the innate selective discrimination against the incorporation of fluorescent dye-labeled ddCTP and fluorescent dye-labeled ddATP, without increasing the rate of incorporation of the other two dye-labeled ddNTP terminators (ddTTP and ddGTP) excessively.
  • ddTTP and ddGTP dye-labeledNTP terminators
  • polymerases having this ability to reduce selective discrimination may be obtained or otherwise derived from a strain of Bacillus stearothermophilus, Bacillus caldotenax and Bacillus caldolyticus, or made synthetically, where the amino acid sequences of the naturally-occurring DNA polymerase have leucine-glutamate- glutamate at positions corresponding respectively to positions 342-344 of Bst 320 DNA polymerase and phenylalanine at a position corresponding to position 422 of Bst 320 DNA polymerase.
  • DNA polymerases derived from other strains of Bacillus stearothermophilus, Bacillus caldotenax and Bacillus caldolyticus may be easily modified using conventional DNA modification techniques to include the amino acid or nucleotide substitutions identified above.
  • the following amino acid sequence represents the modified Bst 320 DNA polymerase (also referred to herein as "Bst II” or "HiFi Bst II”) as another preferred embodiment of this invention, modified from the naturally-occurring Bst 320 DNA polymerase at positions 342-344 to substitute threonine, proline and leucine, respectively, for leucine, glutamate and glutamate, and at position 422 to substitute tyrosine for phenylalanine.
  • Bst II also referred to herein as "Bst II” or "HiFi Bst II”
  • underlined amino acids are substituted amino acids produced by site-directed mutation of the naturally-occurring Bst 320 DNA polymerase.
  • the modified Bst 320 DNA polymerase is encoded by a DNA sequence such as the following (SEQ ID NO:3):
  • the underlined nucleotides TAC are substituted nucleotides produced by site- directed mutation of the naturally-occurring Bst 320 polymerase. (As would be apparent to someone skilled in this art, this DNA sequence does not indicate the starting codon.)
  • the DNA polymerase may also be one that has a DNA sequence that is complementary to Bst 320 or the modified Bst 320 DNA sequence, for instance, DNA sequences that would hybridize to one of the above DNA sequences of under stringent conditions. As would be understood by someone skilled in the art, the DNA sequence also contemplates those that encode a peptide having these characteristics and properties (including degenerate DNA code).
  • DNA sequences and amino acid sequences contemplated include allelic variations and mutations (for instance, adding or deleting nucleotide or amino acids, sequence recombination or replacement or alteration) which result in no substantive change in the function of the DNA polymerase or its characteristics.
  • the DNA polymerases encompass non-critical substitutions of nucleotides or amino acids that would not change functionality (i.e., such as those changes caused by a transformant host cell).
  • the invention is intended to include fusion proteins and muteins of the DNA polymerases.
  • DNA sequences and amino acid sequences for the modified and ummodified DNA polymerases are also obtainable by, for instance, isolating and purifying DNA polymerase from a Bacillus stearothermophilus, or a bacterial strain otherwise derived from Bacillus stearothermophilus, or other mesophilic bacillus strains such as Bacillus caldotenax or Bacillus caldolyticus.
  • the DNA polymerases obtained from these organisms may be easily modified using conventional DNA modification techniques to achieve the properties of high fidelity, high processivity, thermostability and reduction in fluorescent dye-labeled ddCTP and ddATP selective discrimination, as long as the unmodified amino acid sequences have leucine-glutamate-glutamate at positions corresponding respectively to positions 342-344 of Bst 320 DNA polymerase and phenylalanine at a position corresponding to position 422 of Bst 320 DNA polymerase.
  • a DNA polymerase having the same properties and function from other strains.
  • a DNA polymerase which has highly stable enzymatic activity — for instance, stable enough to withstand drying-down processes yet remain viable for DNA sequencing.
  • DNA polymerases are described by the inventors in U.S. Patent Application No. 09/735,677 (the contents of which is incorporated herein by reference in its entirety).
  • modified Bst DNA polymerases have increased stability properties, such that they can be freeze-dried or dried-down in cold temperatures, or stored in ready-to-use liquid reaction mixtures, for extended lengths of time (e.g., at least eight weeks) at room temperature without significant loss of its quality as a DNA polymerase for accurate incorporation of dNTPs and ddNTPs, or their analogs, onto the 3' end of an extending primer upon reconstitution in solution. That is, upon reconstitution in solution and use in standard DNA sequencing there is no significant variability in the quality of sequences produced, when compared to control (e.g., non-freeze-dried or non-dried-down) DNA polymerase.
  • these polymerases can be used in known DNA sequencing protocols to generate excellent quality DNA sequences. These DNA polymerases also demonstrate higher thermostability than the wild-type Bst DNA polymerases. For instance, these polymerases typically have a half-life of polymerase activity at 65°C for about 16 minutes, which is roughly twice as long as the wild-type Bst DNA polymerase.
  • HiFi Bst or "Bst 320" DNA polymerase refers to the unmodified naturally occurring DNA polymerase having proofreading 3'-5' exonuclease activity, either isolated from the cells of a strain designated no. 320 of Bacillus stearothermophilus or produced by overexpression of the gene encoding this naturally occurring DNA polymerase.
  • this Bst strain no. 320 and DNA polymerase are described in U.S. Patent 5,747,298 and U.S.
  • HiFi Bst- II refers to the modified form of "HiFi Bst” DNA polymerase which has the ability to reduce selective discrimination against fluorescent dye-labeled ddCTP and ddATP.
  • HiFi Bst-II is an example of one preferred embodiment of this invention. (This Bst strain and DNA polymerase are described in U.S.
  • Bst-II also has sufficient stability to be dried-down or freeze-dried or stored in ready-to-use liquid reaction mixtures, at room temperature for an extended period of time (such as at least eight weeks), without significant loss of its quality as a DNA polymerase for accurate incorporation of dNTPs and ddNTPs, or their analogs, onto the 3' end of an extending primer upon reconstitution in appropriate solution.
  • This Bst strain and DNA polymerase are described in copending U.S. Patent application 09/735,677.
  • the invention contemplates a method for extending a primer (or a pair of primers) using an enzymatic cycle primer extension reaction at low cycling temperatures (that is, temperatures below about 80°C).
  • the reaction mixture composition that comprises between about 10% and about 20%o (and preferably about 15%>) (v/v) glycerol, ethylene glycol, or a mixture thereof.
  • the reaction is run in the presence of a moderately thermostable DNA polymerase such as one of those described above.
  • the reaction is carried out under conditions that the cycle reaction temperature fluctuates between a melting temperature of about 70°C and a cooling (or annealing) temperature of about 37°C, so that the DNA polymerase repeatedly extends the primer or pair of primers.
  • the method may include the further step of repeating the cycle primer extension reaction, as many times as is desired.
  • a PCR or PCR-like reaction may be run at low temperatures below 80°C.
  • copies of a selected segment of a double- stranded DNA are amplified in the presence of a forward primer and a reverse primer (where both may be of various lengths) to the template by repeated heating and cooling (or annealing) cycles.
  • the reaction mixture composition comprises between about 10% and about 20% (and preferably about 15%) (v/v) glycerol, ethylene glycol, or a mixture thereof, in the presence of one of the moderately thermostable DNA polymerases described above.
  • the reaction is preferably carried out under conditions that the reaction temperature fluctuates between a melting temperature of about 70°C and a cooling (or annealing) temperature of about 37°C, so that the DNA polymerase repeatedly extends the forward and reverse primers.
  • the method may include the further step of repeating the reaction, as many times as is desired.
  • molecules of a single primer of various lengths are extended by a moderately thermostable DNA polymerase with specific nucleotide terminations in the presence of ddNTPs or their analogs for low-temperature cycle sequencing below about 80°C.
  • the ddNTP analogs may be fluorescent dye-labeled so that each members of the ddNTPs may emit different wavelengths, as those used in automated dye-labeled terminator DNA cycle sequencing.
  • the sequencing primer will be labeled with four different dyes- to be used in pairing with the corresponding unlabeled member of the ddNTPs for a modified Sanger reaction as in fluorescent dye-labeled primer DNA cycle sequencing technology.
  • the low-temperature cycle primer extension termination reaction can be used in the classic Sanger protocol with radioactive isotope-labeled dATP for manual direct sequencing of a small amount of DNA template without prior PCR amplification.
  • Another embodiment contemplates a method for extending the molecules of a single primer annealed to a single-stranded copy of the double-stranded DNA product amplified in vitro without prior isolation or purification for direct cycle sequencing.
  • a diluted crude amplified reaction product preferably generated with a low- temperature PCR reaction catalyzed by a moderately thermostable DNA polymerase as described herein
  • a sequencing primer preferably generated with a low- temperature PCR reaction catalyzed by a moderately thermostable DNA polymerase as described herein
  • the four standard ddNTP terminators ddATP, ddGTP, ddTTP and ddCTP
  • a moderately thermostable DNA polymerase preferably one with a reduced innate selective discrimination against incorporation of a subset of dye-labeled ddNTPs
  • a suitable concentration of dNTPs dATP, dGTP, dTTP and dCTP
  • a standard cycle primer extension reaction(s) may then be run at a temperature below 80°C for a sufficient number of times to extend the sequencing primer molecules to desired varying lengths, which extended molecules will be terminated specifically by fluorescently labeled ddNTPs or their corresponding analogs.
  • the cycle reaction temperature fluctuates between a melting temperature of about 70°C and a cooling/annealing temperature of about 37°C.
  • the method of sequencing a DNA strand may comprise the steps of: i) hybridizing a primer to a DNA template to be sequenced; and ii) extending the primer using one of the above-described DNA polymerases, in the presence of a solution containing between about 10% and about 20% (v/v) (preferably about 15% (v/v)) glycerol, ethylene glycol, or a mixture thereof, adequate amounts of the deoxynucleotide bases dATP, dGTP, dCTP and dTTP, and the four dideoxynucleotide terminators, or their analogs, whereby the cycle reaction temperature fluctuates between a melting temperature of about 70°C arid a cooling or annealing temperature of about 37°C, and under such conditions that the DNA strand is sequenced.
  • one of the deoxynucleotides is radioisotope-labeled, or the primer molecules are fluorescent dye-labeled, and more preferably all are fluorescent dye-
  • the invention entails a dry or liquid ready-to-use reaction mixture or kit suitable for use in a low-temperature cycle primer extension reaction at temperatures below about 80°C.
  • This reaction mixture or kit comprises a moderately thermostable DNA polymerase (such as one of those described above) that is pre-mixed with at least one enzymatic DNA primer extension reaction component suitable for use in DNA amplification or for specific extension terminations with dideoxyribonucleotide analogs.
  • the reaction mixture is preferably pre-distributed into microcentrifuge tubes or in multiple-well plates, such as, for instance, those that are suitable for large-scale automated PCR or for large-scale automated DNA sequencing.
  • This ready-to-use reaction mixture or kit can be stored at room temperature between about 22°C and about 25°C for at least eight weeks without losing its specific polymerization activity for DNA primer amplification or extension terminations.
  • both annealing and primer extension can take place simultaneously at 45°C.
  • 37°C can be used as the annealing temperature and 45-50°C the primer extension temperature. (See Figure 6). Therefore, both the following protocol A and protocol B can be used for the low- temperature cycling steps with effective specific amplification:
  • protocol B is preferred when the enzymatic primer extension is to generate reaction products with fluorescent dye-labeled ddNTP terminations for automated cycle sequencing. It is noted that the methods of this invention are not limited to either protocols A or B, but that these two protocols are exemplary of temperatures and cycles that work effectively with these methods. For instance, under certain circumstances, the extension time may be desired to be prolonged to about 11 minutes for long target segment amplification.
  • a single primer in excess can be added to a reaction mixture containing 15% glycerol and a moderately thermostable DNA polymerase.
  • the single-stranded primer oligonucleotides can then be extended to various lengths with specific nucleotide terminations in the presence of ddNTPs or their analogs, which may be fluorescently labeled.
  • the template used for the cycle sequencing can be any purified double-stranded or single-stranded DNA fragments containing the target sequence, or an aliquot of the diluted amplification products derived from the low- temperature cycle-extended primer strands of the double-stranded DNA template described in this invention, without prior isolation and purification. Since the amplification products derived from the low-temperature cycle primer extension using a moderately thermostable DNA polymerase with high fidelity and high processivity as described in this invention are highly sequence-specific, prior isolation of the PCR product from the reaction mixture before being used as the template for DNA cycle sequencing is generally unnecessary.
  • Example 1 The effect of Glycerol on 5'- 3' polymerization activity of moderately thermostable DNA polymerases
  • Reaction Buffer lOOmM Tris-Cl, pH 8.5 containing lOOmM MgCl 2 .
  • Calf Thymus DNA DNase I activated, 1.5ug/ul.
  • Example 2 The effect of 40% glycerol (v/v) on low-temperature cycle primer extension with moderately thermostable DNA polymerases
  • reaction products were run on a 1% agarose gel for electrophoresis and stained by ethidium bromide.
  • Example 3 The effect of reduced concentrations of glycerol on low-temperature cycle primer extension with moderately thermostable DNA polymerases
  • the low temperature cycle extension was carried out as follows:
  • Reverse primer A, B, C or D (10 pmol/ul) 2.5 ul dNTP(2.5mM each) 4 ul
  • reaction products were run on a 1% agarose gel for electrophoresis and stained by ethidium bromide.
  • the reaction products from the mixt e containing 35% glycerol were loaded in lane 1, and the reaction products from the mixture containing 15 % glycerol were loaded in lane 2.
  • the experiments were designed to demonstrate that the low- temperature cycle extension system with moderately thermostable DNA polymerases of this invention can be used for sequence-specific extension of primers of up to 30 base pairs in length.
  • the polymerases used were Bst-I (wild type produced according to US patent 5,834,254), Bst-II, and Bca (TaKaRa Co.).
  • the Klenow fragment (Sigma Chemical Co.) was used as a thermolabile DNA polymerase for comparison (lakobashvili and Lapidot).
  • the template used was rice genome BAC B414f7.
  • the two pairs of primers used were:
  • the following reaction system (referred to hereafter as the Bst system) was used.
  • reaction products were run on a 1% agarose gel for electrophoresis and stained by ethidium bromide.
  • Al Klenow fragment using the lakobashvili and Lapidot system.
  • A2 Klenow fragment with the Bst system.
  • A3 Bst-I polymerase with the Bst system.
  • A4 Bst-II polymerase with the Bst system.
  • A5 Bca polymerase with the Bst system.
  • Bl Klenow fragment using the lakobashvili and Lapidot system.
  • B2 Klenow fragment with the Bst system.
  • B3 Bst-I polymerase with the Bst system.
  • B4 Bst-II polymerase with the Bst system.
  • B5 Bca polymerase with the Bst system.
  • M2 DL 2,000 (from TaKaRa Co., with the DNA fragment of 2000, 1000, 750, 500, 250 and 100 bp respectively).
  • Figure 4 shows that the moderately thermostable DNA polymerases, namely the natural form of Bst-I, the mutated Bst-II and Bca, all generated specific amplification products as a result of 17mer primer extension (A3-A5) and of 30mer primer extension (B3-B5) in the Bst system containing 15 % glycerol in the reaction mixture as recommended for low temperature cycling.
  • the thermolabile DNA polymerase, Klenow fragment failed to produce a specific amplification product from 17mer or 30mer primer extension either in the lakobashvili and Lapidot system (Al and Bl) or in the Bst system (A2 and B2).
  • Example 5 High fidelity low-temperature linear cycle sequencing with Bst-II DNA polymerase in stored ready-to-use reaction pre-mixture.
  • the current invention can be used to perform DNA sequencing with a genetically modified moderately thermostable DNA polymerase, Bst-II, to extend the primer over the GC-rich segments of the template which the commonly used heat-stable DNA polymerases with low processivity, such as ThermoSequenaseTM or AmpliTaqTM, are unable to overcome.
  • all pre-measured ingredients of the reaction mixture with or without the primer pre-added can be pre-mixed and stored in individual microcentrifuge tubes or 96-well plates for at least eight (8) weeks at temperatures between 23°C and 25°C.
  • Template bg08. This was a GC-rich segment of a subclone of rice genome BAG 129.
  • Pre-mixed dye-ddNTPs Optimized R6G-ddATP, ROX-ddCTP, TAMRA-ddUTP, and Bodipy Fl-14-ddGTP, purchased from NENTM Life Sciences Products.
  • reaction pre-mixture in microcentrifuge tubes was stored at temperatures between 23°C and 25°C until use within eight (8) weeks.
  • Figure 5 shows the ABI PrismTM BigDyeTM Terminator cycle sequencing kit with AmpliTaqTM
  • Figure 5 A failed to accomplish efficient specific fluorescent dye-labeled ddNTP terminations during cycle primer extension over the GC-rich segment of the DNA template.
  • the Bst-II Cycle Sequencing system even after the Bst-II DNA polymerase had been stored in a pre-mixed form for eight (8) weeks at 23-25°C, successfully overcame the GC-rich barrier in the template and generated adequate specific dye-labeled ddNTP terminations for DNA sequencing analyses (Figure 5 B).
  • Thermo SequenaseTM used with the Amersham DYEnamicTM ET terminator cycle sequencing kit also failed to overcome the GC-rich segment of the template during the cycle primer extension reaction for automated fluorescent DNA sequencing (tracing not shown here).
  • the Bst-II Cycle Sequencing system which remains stable in ready- to-use pre-mixture at room temperature for at least eight (8) weeks is most suitable for large-scale high fidelity automated fluorescent DNA sequencing, especially when the templates contain GC-rich segments.
  • Figure 5 shows DNA sequencing over a GC-rich segment, including a comparison of the performance of AmpliTaqTM in the ABI PrismTM BigDyeTM Terminator cycle sequencing kit (A) with that of the Bst-II Cycle Sequencing System (B).
  • Figure 5 A and B represent two automated fluorescent DNA sequencing tracings of a GC-rich segment of the same template using the same prime for cycle extension. Both sequences were run in an ABI 377 sequencer.
  • the shadowed zone illustrated in A represents the region out of quality control evaluated and reported by the computer.
  • Example 6 Optimum temperature steps for cycle primer extension with moderately thermostable DNA polymerases This experiment was designed to determine the optimum temperature steps for cycle primer extension with moderately thermostable DNA polymerases in a reaction mixture containing 15% glycerol as the agent to lower the DNA melting temperature.
  • Bst-II was the DNA polymerase used.
  • Reverse primer (10 pmol/ul) 2.5 ul dNTPs (2.5 mM each) 4 ul
  • Steps 3 and Steps 5 can be used for specific primer extension in DNA amplification
  • the cycling protocol of Steps 3 with 70°C 30s, 37°C 20s, 50°C 3min, 35 cycles is preferred (Lane 7) when the enzymatic primer extension is used to generate reaction products with fluorescent dye- labeled ddNTP terminators for automated cycle DNA sequencing.
  • Example 7 Direct low temperature cycle sequencing of amplified products generated by moderately thermostable DNA polymerases in stored ready-to-use reaction pre- mixture.
  • Bst-II was used as the DNA polymerase.
  • Template H525d9, a BAC of rice genome
  • Forward primer 5' TTT CAG GGT CCC TTA TAT CTC 3'
  • Reverse primer 5'TCG CTT CTC CTC ATA ATC GAT 3'.
  • Pre-mixed dye-ddNTPs Optimized R6G-ddATP, ROX-ddCTP, TAMRA-ddUTP, and Bodipy Fl-14-ddGTP, purchased fro NENTM Life Sciences Products.
  • Reverse primer (10 pmol/ul) 2 ul dNTPs (2.5 mM each) 2 ul
  • reaction pre-mixture in the microcentrifuge tube was stored at temperature between 23°C and 25°C until use within eight (8) weeks.
  • reaction products were loaded onto a 1% low melting point agarose gel for electrophoresis.
  • NENTM Pre-mixed dye-ddNTPs
  • reaction pre-mixture in microcentrifuge tubes was stored at temperatures between 23 °C and 24°C until use within eight (8) weeks.

Abstract

L'invention concerne des méthodes d'extension d'une amorce ou d'une paire d'amorces dans l'amplification d'ADN cyclique à basse température de séquençage de cycle et PCR. Ces méthodes consistent en particulier à utiliser de façon combinée des ADN polymérases modérément thermostables en présence d'une faible concentration de glycérol ou d'éthylène glycol, ou de mélanges de ceux-ci, comme agents destinés à réduire la température de fusion de l'ADN (c'est-à-dire la température à laquelle les deux brins d'ADN sont dénaturés). L'invention concerne également des mélanges de réaction prédistribués d'ADN polymérase haute fidélité à grande capacité de traitement, stables à température ambiante pendant plusieurs semaines dans des kits prêts à l'emploi.
PCT/IB2002/003341 2001-06-08 2002-06-05 Extension cyclique a basse temperature d'adn a haute specificite d'amorcage WO2002101004A2 (fr)

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US9968902B2 (en) 2009-11-25 2018-05-15 Gen9, Inc. Microfluidic devices and methods for gene synthesis
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US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
US10457935B2 (en) 2010-11-12 2019-10-29 Gen9, Inc. Protein arrays and methods of using and making the same
US11072789B2 (en) 2012-06-25 2021-07-27 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
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WO2023093061A1 (fr) * 2021-11-23 2023-06-01 广州达安基因股份有限公司 Procédé de préparation d'un système enzymatique mixte d'amplification à température constante
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US10640764B2 (en) 2002-09-12 2020-05-05 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US9051666B2 (en) 2002-09-12 2015-06-09 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US10450560B2 (en) 2002-09-12 2019-10-22 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US20130095479A1 (en) * 2004-10-18 2013-04-18 Brandeis University Primers, probes and methods for nucleic acid amplification
US9745624B2 (en) * 2004-10-18 2017-08-29 Brandeis University Methods for sequential DNA amplification and sequencing
WO2007123742A3 (fr) * 2006-03-31 2008-02-28 Codon Devices Inc Méthodes et compositions améliorant la fidélité d'assemblage de plusieurs acides nucléiques
WO2007123742A2 (fr) * 2006-03-31 2007-11-01 Codon Devices, Inc. Méthodes et compositions améliorant la fidélité d'assemblage de plusieurs acides nucléiques
US10202608B2 (en) 2006-08-31 2019-02-12 Gen9, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
US10207240B2 (en) 2009-11-03 2019-02-19 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
US9968902B2 (en) 2009-11-25 2018-05-15 Gen9, Inc. Microfluidic devices and methods for gene synthesis
US9925510B2 (en) 2010-01-07 2018-03-27 Gen9, Inc. Assembly of high fidelity polynucleotides
US11071963B2 (en) 2010-01-07 2021-07-27 Gen9, Inc. Assembly of high fidelity polynucleotides
US11084014B2 (en) 2010-11-12 2021-08-10 Gen9, Inc. Methods and devices for nucleic acids synthesis
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US10457935B2 (en) 2010-11-12 2019-10-29 Gen9, Inc. Protein arrays and methods of using and making the same
US11845054B2 (en) 2010-11-12 2023-12-19 Gen9, Inc. Methods and devices for nucleic acids synthesis
US11702662B2 (en) 2011-08-26 2023-07-18 Gen9, Inc. Compositions and methods for high fidelity assembly of nucleic acids
EP2751264A4 (fr) * 2011-09-01 2015-07-22 New England Biolabs Inc Compositions et procédés associés à des variants d'adn polymérases et d'adn polymérases synthétiques
EP2751264A1 (fr) * 2011-09-01 2014-07-09 New England Biolabs, Inc. Compositions et procédés associés à des variants d'adn polymérases et d'adn polymérases synthétiques
WO2013033528A1 (fr) 2011-09-01 2013-03-07 Jennifer Ong Compositions et procédés associés à des variants d'adn polymérases et d'adn polymérases synthétiques
US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
US10927369B2 (en) 2012-04-24 2021-02-23 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US10081807B2 (en) 2012-04-24 2018-09-25 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US11072789B2 (en) 2012-06-25 2021-07-27 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
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CA2449560A1 (fr) 2002-12-19
EP1436391A2 (fr) 2004-07-14
JP2005514003A (ja) 2005-05-19

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