AU4575799A - Creation of Inventions and Patents with Artificial Intelligence

AU4575799A – Fy7 polymerase
– Google Patents

AU4575799A – Fy7 polymerase
– Google Patents
Fy7 polymerase

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Publication number
AU4575799A

AU4575799A
AU45757/99A
AU4575799A
AU4575799A
AU 4575799 A
AU4575799 A
AU 4575799A
AU 45757/99 A
AU45757/99 A
AU 45757/99A
AU 4575799 A
AU4575799 A
AU 4575799A
AU 4575799 A
AU4575799 A
AU 4575799A
Authority
AU
Australia
Prior art keywords
dna
dna polymerase
polymerase
recombinant
sequencing
Prior art date
1998-06-17
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Granted

Application number
AU45757/99A
Other versions

AU768993B2
(en

Inventor
Maria Davis
Carl W Fuller
Lin Huang
Joseph A. Mamone
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Global Life Sciences Solutions USA LLC

Original Assignee
GE Healthcare Bio Sciences Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
1998-06-17
Filing date
1999-06-17
Publication date
2000-01-05

1999-06-17
Application filed by GE Healthcare Bio Sciences Corp
filed
Critical
GE Healthcare Bio Sciences Corp

2000-01-05
Publication of AU4575799A
publication
Critical
patent/AU4575799A/en

2002-05-23
Assigned to AMERSHAM BIOSCIENCES CORP.
reassignment
AMERSHAM BIOSCIENCES CORP.
Amend patent request/document other than specification (104)
Assignors: AMERSHAM PHARMACIA BIOTECH INC.

2004-01-15
Application granted
granted
Critical

2004-01-15
Publication of AU768993B2
publication
Critical
patent/AU768993B2/en

2006-08-17
Assigned to GE HEALTHCARE BIO-SCIENCES CORP.
reassignment
GE HEALTHCARE BIO-SCIENCES CORP.
Request to Amend Deed and Register
Assignors: AMERSHAM BIOSCIENCES CORP.

2019-06-17
Anticipated expiration
legal-status
Critical

Status
Ceased
legal-status
Critical
Current

Links

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108010014303
DNA-directed DNA polymerase
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triphosphate
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triphosphate
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alpha amino acid group
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spleen exonuclease
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supernatant
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Classifications

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

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

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

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

Abstract

A purified recombinant thermostable DNA polymerase polymerase which exhibits at least about 80% activity at salt concentations of 50 mM and greater, at least about 70% activity at salt concentrations of 25 mM and greater, and having a processivity of about 30 nucleotides per binding event. An isolated nucleic acid that encodes the thermostable DNA polymerase, as well as a recombinant DNA vector comprising the nucleic acid and a recombinant host cell transformed with the vector, are also disclosed. A method of sequencing DNA using the DNA polymerase as well as a kit for sequencing DNA is also disclosed.

Description

WO 99/65938 PCT/US99/13741 FY7 POLYMERASE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to United States Provisional Application Serial No. 60/089,556, filed on June 17, 1998, the entire disclosure of which is incorporated in its herein. BACKGROUND OF THE INVENTION Field of the Invention The instant disclosure pertains to thermostable DNA polymerases which exhibit improved robustness and efficiency. Background DNA polymerases are enzymes which are useful in many recombinant DNA techniques such as nucleic acid amplification by the polymerase chain reaction (“PCR”), self sustained sequence replication (“3SR”), and high temperature DNA sequencing. Thermostable polymerases are particularly useful. Because heat does not destroy the polymerase activity, there is no need to add additional polymerase after every denaturation step. However, many thermostable polymerases have been found to display a 5′ to 3′ exonuclease or structure-dependent single-stranded endonuclease (“SDSSE”) activity which may limit the amount of product produced or contribute to the plateau phenomenon in the normally exponential accumulation of product. Such 5′ to 3′ nuclease activity may contribute to an impaired ability to efficiently generate long PCR products greater than or equal to 10kb, particularly for G+C rich targets. In DNA sequencing applications and cycle sequencing applications, the presence of 5′ to 3′ nuclease activity may contribute to a reduction in desired band intensities and/or generation of spurious or background bands. Additionally, many of the enzymes presently available are sensitive to high salt environments, a condition commonly Presently available enzymes have so-so processing ability (are more distributive – fall off more often – explain in more detail) dITP added to address compression problems – usually kills activity of enzyme Thus, a need continues to exist for an improved DNA polymerase having increased tolerance to high salt conditions, efficient utilization of dITP, high productivity, and improved performance on GC-rich templates.
WO 99/65938 2 PCT/US99/13741 BRIEF SUMMARY OF THE INVENTION The instant disclosure teaches a purified recombinant thermostable DNA polymerase comprising the amino acid sequence set forth in Figure 1, as well as a purified recombinant thermostable DNA polymerase which exhibits at least about 80% activity at salt concentations of 50 mM and greater. The instant disclosure further teaches a purified recombinant thermostable DNA polymerase which exhibits at least about 70% activity at salt concentrations of 25 mM and greater, and a purified recombinant thermostable DNA polymerase having a processivity of about 30 nucleotides per binding event. The instant disclosure also teaches an isolated nucleic acid that encodes a thermostable DNA polymerase, wherein said nucleic acid consists of the nucleotide sequence set forth in Figure 1, as well as a recombinant DNA vector that comprises the nucleic acid, and a recombinant host cell transformed with the vector. The instant disclosure also teaches a method of sequencing DNA comprising the step of generating chain terminated fragments from the DNA template to be sequenced with the DNA polymerase in the presence of at least one chain terminating agent and one or more nucleotide triphosphates, and determining the sequence of said DNA from the sizes of said fragments. The instant disclosure also teaches a kit for sequencing DNA comprising the DNA polymerase. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS FIGURE 1 depicts the amino acid sequence (and DNA sequence encoding therefor) for the FY7 polymerase. FIGURE 2 depicts the DNA sequence of M 13mp 18 DNA sequenced using the FY7 polymerase formulated in Mn conditions, as shown by a print out from an ABI model 377 automated fluorescent DNA sequencing apparatus. FIGURE 3 depicts the DNA sequence of M1 3mp 18 DNA sequenced using the FY7 polymerase formulated in Mg conditions, as shown by a print out from an ABI model 377 automated fluorescent DNA sequencing apparatus. FIGURE 4 depicts the percent of maximum polymerase activity for Thermo Sequenasem enzyme DNA polymerase versus FY7 DNA polymerase under varyingKCl concentrations.
WO 99/65938 3 PCTIUS99/13741 FIGURE 5 depicts the effect of high salt concentrations on DNA sequencing ability in radioactively labeled DNA sequencing reactions using Thermo SequenaseTM enzyme DNA polymerase versus FY7 DNA polymerase. FIGURES 6-10 depict the effect of increasing salt concentration on the performance of Thermo Sequenase. At concentrations as low as 25mM data quality is affected with the read length being decreased from at least 600 bases to about 450 bases. At 50mM salt the read length is further decreased to about 350 bases, 75mM to about 250 bases and at 100mM the read length is negligible. FIGURES 11-15 depict the effect of increasing salt concentration on the performance of FY7 DNA polymerase. There is no detrimental effect on performance to at least 75mM KCl and only a slight decrease in data quality at 100mM KCl. FIGURE 16 depicts the processivity measured for Thermo Sequenase DNA polymerase, AmpliTaq FS DNA polymerase, compared with the processivity measured for FY7 DNA polymerase. FIGURE 17 depicts the improved read length obtained when using FY7 polymerase versus Thermo Sequenase DNA polymerase in radioactively labeled sequencing reactions incorporating the dGTP (Guanosine triphosphate) analog dITP (Inosine triphosphate) at 72 “C. FIGURES 18-22 show the effect of increasing extension step time on the read length and data quality produced by Thermo Sequenase DNA polymerase in fluorescently labeled terminator DNA sequencing reactions FIGURES 23-27 show the effect of increasing extension step time on the read length and data quality produced by FY7 DNA polymerase in fluorescently labeled terminator DNA sequencing reactions. DETAILED DESCRIPTION OF THE INVENTION A series of polymerase mutants were constructed with the aim of obtaining an improved polymerase for DNA sequencing, by reducing the exonuclease activity found in full length Thermus thermophilus and Thermus aquaticus DNA polymerase I enzymes. Six conserved motifs (Gutman and Minton (1993) Nucleic Acids Research 21, 4406 – 4407) can be identified in the amino-terminal domain of pol I type polymerases, in which the 5′ to 3′ exonuclease activity has been shown to reside. Further, six carboxylate residues in these conserved regions have been shown in a crystal structure to be located at the active site of the exonuclease domain of Thermus aquaticus DNA pol I (Kim et al., (1995) Nature 376, 612 616). Point mutations were made by site-directed mutagenesis to carboxylates and other residues in three of six conserved motifs in Tth and Taq polymerases as follows: WO 99/65938 4 PCTIUS99/13741 Taq D18A, Taq T140V, Taq D142N/D144N. All of these have the mutation F667Y outside of the exonuclease domain. Tth D18A, Tth T141V, Tth D143N/D145N. All of these have the mutation F669Y outside of the exonuclease domain. All polymerases were evaluated for exonuclease activity, processivity, strand displacement, salt tolerance, thermostability, and sequencing quality. One FY7 polymerase, Tth D1 8A, F669Y, is described in further detail below. EXAMPLES Methods In vitro mutagenesis PCR was employed to introduce an aspartic acid to alanine amino acid change at codon 18 (D18A) of cloned full length F669Y Tth (plasmid pMR10). Mutagenic Primer 1 (CTGTTCGAACCCAAAGGCCGTGTCCTCCTGGTGGCCGGCCACCAC) spans nucleotides 19-60 of pMR10 including codon 18 and a BstBI restriction site. Oligonucleotide Primer 2 (GAGGCTGCCGAATTCCAGCCTCTC) spans an EcoRl site of pMR10. pMR10 was used as template DNA. The PCR product was digested with BstBI and EcoRI and ligated to two fragments of pMR10: a 5000 bp Kpnl/ BstBI and a 2057 bp EcoRI / KpnI, creating plasmid pMR12. Cells of E. coli strain DHlX* were used for primary transformation, and strain M5248 (X c1857) was used for protein expression, although any comparable pair of E. coli strains carrying the cI* and c1857 alleles could be utilized. Alternatively, any rec’ cI* strain could be induced by chemical agents such as nalidixic acid to produce the polymerase. Purification of Polvmerase M5248 containing plasmid pMR12 was grown in one liter of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCi), preferably 2X LB medium, containing 100 mg/ml ampicillin at 30’C. When the OD 600 reached 1.0, the culture was induced at 42*C for 1.5 hours. The cultures were then cooled to <20 0 C and the cells harvested by centrifugation in a Sorvall RC-3B centrifuge at 5000 rpm 4'C for 15 to 30 minutes. Harvested were stored -80*C. The cell pellet was resuspended 25 ml pre-warmed lysis buffer (50 mM Tris-HCI pH 8.0, 10 MgCl 2 , 16 (NH 4 ) 2 SO 4 1 EDTA, 0.1%, preferably 0.2% Tween 20, NP40). Preferably, contains 300 NaCl. Resuspended incubated 75 - 85 10-20 minutes, sonicated minute, WO 99>50-100 more nucleotides under standard 3 P[a-dATP sequencing conditions than Thermo Sequenase. Example 9 Effect of Extension Step Time on Length of Read These series of experiments examined the effect of increasing extension step time of the read length and data quality of Thermo Sequenase and FY7 DNA polymerases in fluorescently labeled terminator DNA sequencing reactions. The results are presented in Figures 18-27. Figures 18-22 show the effect of increasing extension step time on the read length and data quality produced by Thermo Sequenase DNA polymerase. This data shows that a minimum of a two WO 99/65938 9 PCT/US99/13741 minutes extension step is required by Thermo Sequenase in order to achieve a quality read of at least 600 bases. Signal strength generally increases to a maximum at a four minute extension (the time specified in the commercial product utilizing this enzyme and method). Figures 23-27 show the effect of increasing extension step time on the read length and data quality produced by FY7 DNA polymerase. This data shows that a minimum of a 30 second extension step is required by FY7 in order to achieve a quality read of at least 600 bases. Signal strengths plateau at about one minute extension time. The FY7 DNA polymerase can produce data of equivalent quality to Thermo Sequenase in one-quarter to one-half the time of extension reaction. Although the above examples describe various embodiments of the invention in detail, many variations will be apparent to those of ordinary skill in the art. Accordingly, the above examples are intended for illustration purposes and should not be used in any way to restrict the scope of the appended claims.

Claims (13)

1. A purified recombinant thermostable DNA polymerase comprising the amino acid sequence set forth in Figure 1.

2. A purified recombinant thermostable DNA polymerase which exhibits at least about 80% activity at salt concentations of 50 mM and greater.

3. A purified recombinant thermostable DNA polymerase which exhibits at least about 70% activity at salt concentrations of 25 mM and greater.

4. A purified recombinant thermostable DNA polymerase having a processivity of about 30 nucleotides per binding event.

5. An isolated nucleic acid that encodes a thermostable DNA polymerase, wherein said nucleic acid consists of the nucleotide sequence set forth in Figure 1

6. A recombinant DNA vector that comprises the nucleic acid of Claim 3.

7. The recombinant DNA sequence of Claim 4 comprising the plasmid pMR10.

8. A recombinant host cell transformed with the vector of Claim 5.

9. The recombinant host cell of Claim 6 that is E. coli.

10. The recombinant host cell of Claim 7 which is E. coli carrying the cI* and c1857 alleles.

11. The recombinant host cell of Claim 7 selected from the group consisting of DHIk’ [gyrA96, recAl, relAl, endAl, thi-1, hsdR17, supE44, k’] and M5248 [k (bio275. c1857, cIII+, N+, k (HI))].

12. Method of sequencing DNA comprising the step of generating chain terminated fragments from the DNA template to be sequenced with the DNA polymerase of Claim 1 in the presence of at least one chain terminating agent and one or more nucleotide triphosphates, and determining the sequence of said DNA from the sizes of said fragments. WO 99/65938 1 PCT/US99/13741

13. A kit for sequencing DNA comprising the DNA polymerase of Claim 1.

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