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US5834253A
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Bacillus stearothermophilus DNA polymerase with proof-reading 3'-5' exonuclease activity
Assignee: GUANGDONG HYBRIBIO BIOTECH Co Ltd
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Filing: 1996-05-03
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Granted: 1998-11-10
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Expires: 2016-05-03
Abstract
Abstract The invention relates to DNA polymerases which are capable of proofreading 3'-5' exonuclease activity during DNA sequencing of a DNA strand, 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 the nucleotides of the template, and which DNA polymerase does not exhibit 5'-3' exonuclease activity. The invention also relates to isolated and cloned DNA sequences derived from the Bacillus stearothermophilus thermostable DNA polymerase, as well as the expressed polymerase itself.
Inventors
- 👤 Guo Fan Hong
- 👤 Wei-hua Huang
- 👤 Feng Zhai, deceased
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 16:01
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
14 words
An isolated strain of Bacillus stearothermophilus which produces a peptide having SEQ ID NO:2.
Claim 2
Dependent Claim
14 words
An isolated strain of Bacillus stearothermophilus which produces a peptide having SEQ ID NO:2.
Claim 3
Dependent Claim
73 words
The isolated strain of Bacillus stearothermophilus of claim 1, wherein the peptide is a thermostable DNA polymerase which proofreads 3'-5' exonuclease activity during DNA sequencing of a DNA strand from 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.
Claim 4
Dependent Claim
13 words
The isolated strain of Bacillus stearothermophilus of claim 1 which is Bst 320.
Claim 5
Dependent Claim
22 words
The isolated strain of Bacillus stearothermophilus of claim 1 wherein the peptide is encoded by a DNA segment having SEQ ID NO:1.
Claim 6
Dependent Claim
8 words
An isolated DNA segment having SEQ ID NO:1.
Claim 7
Dependent Claim
8 words
An isolated DNA segment having SEQ ID NO:1.
Claim 8
Dependent Claim
11 words
The purified peptide encoded by the DNA segment of claim 5.
Claim 9
Dependent Claim
12 words
A transformed host cell which produces a peptide having SEQ ID NO:4.
Claim 10
Dependent Claim
12 words
A transformed host cell which produces a peptide having SEQ ID NO:4.
Claim 11
Dependent Claim
71 words
The transformed host cell of claim 7 wherein the peptide is a thermostable DNA polymerase which proofreads 3'-5' exonuclease activity during DNA sequencing of a DNA strand from 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.
Claim 12
Dependent Claim
11 words
The transformed host cell of claim 7 which is E. coli.
Claim 13
Dependent Claim
12 words
The transformed host cell of claim 9 which is E. coli JF1125.
Claim 14
Dependent Claim
20 words
The transformed host cell of claim 7 wherein the peptide is encoded by a DNA segment having SEQ ID NO:3.
Claim 15
Dependent Claim
9 words
An isolated DNA segment having nucleotide SEQ ID NO:3.
Claim 16
Dependent Claim
9 words
An isolated DNA segment having nucleotide SEQ ID NO:3.
Claim 17
Dependent Claim
11 words
The purified peptide encoded by the DNA segment of claim 12.
Claim 18
Dependent Claim
26 words
A DNA construct comprising: (i) the DNA segment of claim 12; and (ii) a vector, for introducing the DNA construct into eucaryotic or procaryotic host cells.
Claim 19
Dependent Claim
17 words
The DNA construct of claim 14 wherein the vector is a cloning vector or an expression vector.
Claim 20
Dependent Claim
27 words
A host cell stably transformed with the DNA construct of claim 14 wherein expression of the peptide encoded by the DNA segment in the construct is achieved.
Claim 21
Dependent Claim
70 words
The host cell of claim 16, wherein the peptide is a thermostable DNA polymerase which proofreads 3'-5' exonuclease activity during DNA sequencing of a DNA strand from 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.
Claim 22
Dependent Claim
29 words
A DNA construct comprising: (i) the DNA segment encoding the peptide of claim 13; and (ii) a vector, for introducing the DNA construct into eucaryotic or procaryotic host cells.
Claim 23
Dependent Claim
17 words
The DNA construct of claim 18 wherein the vector is a cloning vector or an expression vector.
Claim 24
Dependent Claim
28 words
A host cell stably transformed with the DNA construct of claim 18 in a manner allowing expression of the peptide encoded by the DNA segment in the construct.
Claim 25
Dependent Claim
26 words
The purified thermostable DNA polymerase produced by the isolated strain of Bacillus stearothermophilus of claim 1, or produced by the transformed host cell of claim 7.
Claim 26
Independent Claim
66 words
A method of replicating a DNA strand comprising the step of replicating a DNA strand using a DNA polymerase which proofreads 3'-5' exonuclease activity, such that the DNA polymerase functions to excise mismatched nucleotides from the 3' terminus of the polymerase functions to remove nucleotides matched correctly with nucleotides of the template; wherein the DNA polymerase has amino acid SEQ ID NO:2 or SEQ ID NO:4.
Claim 27
Independent Claim
66 words
A method of replicating a DNA strand comprising the step of replicating a DNA strand using a DNA polymerase which proofreads 3'-5' exonuclease activity, such that the DNA polymerase functions to excise mismatched nucleotides from the 3' terminus of the polymerase functions to remove nucleotides matched correctly with nucleotides of the template; wherein the DNA polymerase has amino acid SEQ ID NO:2 or SEQ ID NO:4.
Claim 28
Independent Claim
99 words
A method of sequencing a DNA strand comprising the steps of: i) hybridizing a primer to a DNA template to be sequenced; ii) extending the primer using a DNA polymerase which proofreads 3'-5' exonuclease activity, 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, under such conditions that the DNA strand is sequenced; wherein the DNA polymerase has amino acid SEQ ID NO:2 or SEQ ID NO:4.
Claim 29
Independent Claim
99 words
A method of sequencing a DNA strand comprising the steps of: i) hybridizing a primer to a DNA template to be sequenced; ii) extending the primer using a DNA polymerase which proofreads 3'-5' exonuclease activity, 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, under such conditions that the DNA strand is sequenced; wherein the DNA polymerase has amino acid SEQ ID NO:2 or SEQ ID NO:4.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-11-09 | 2002-05-11 | 2002-11-10 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-11-09 | 2006-05-11 | 2006-11-10 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-11-09 | 2010-05-11 | 2010-11-10 | $7700.00 | Window Closed / Lapsed |