EP0455430A2
Expired / Lapsed
EP
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Purified thermostable DNA polymerase obtainable from Thermococcus litoralis
Assignee: New England Biolabs Inc
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Filing: 1991-04-26
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Granted: —
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Expires: 2011-04-26
Abstract
Abstract There is provided an extremely thermostable enzyme obtainable from Thermococcus litoralis . The thermostable enzyme has a molecular weight of about 90,000 - 95,000 daltons, a half-life of about 60 minutes at 100°C in the absence of stabilizer, and a half-life of about 95 minutes at 100°C in the presence of stabilizer, such as octoxynol (TRITON X-100) or bovine serum albumin. The thermostable enzyme possesses a 3′-5′ proofreading exonuclease activity. The thermostable enzyme may be native or recombinant and may be used for second-strand cDNA synthesis in cDNA cloning, DNA sequencing, and DNA amplification.
Inventors
- 👤 Donald G. Comb
- 👤 Francine Perler
- 👤 Rebecca Kucera
- 👤 William E. Jack
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-04 19:53
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252
Claims Analysis
Total Claims: 54
Claim 1
Independent Claim
14 words
A purified thermostable enzyme obtainable from Thermococcus litoralis which catalyzes the polymerization of DNA.
Claim 2
Dependent Claim
16 words
The thermostable enzyme of claim 1, having a molecular weight of about 90,000 to 95,000 daltons.
Claim 3
Dependent Claim
11 words
The thermostable enzyme of claim 1, having a 3′-5′ exonuclease activity.
Claim 4
Dependent Claim
13 words
The thermostable enzyme of claim 3, wherein the 3′-5′ exonuclease activity is inactivated.
Claim 5
Dependent Claim
17 words
The thermostable enzyme of claim 4, wherein said enzyme is obtainable from E . coli NEB 681.
Claim 6
Dependent Claim
22 words
The thermostable enzyme of claim 1, having a half life of about 60 minutes at 100°C in the absence of a stabilizer.
Claim 7
Dependent Claim
22 words
The thermostable enzyme of claim 1, having a half life of about 95 minutes at 100°C in the presence of a stabilizer.
Claim 8
Dependent Claim
12 words
The thermostable enzyme of claim 7, wherein said stabilizer is a nonionic-detergent.
Claim 9
Dependent Claim
24 words
The thermostable enzyme of claim 8, wherein said nonionic-detergent is selected from the group consisting of octoxynol, polyoxyethylated sorbitan monolaurate and ethoxylated nonyl phenol.
Claim 10
Dependent Claim
12 words
The thermostable enzyme of claim 7, wherein said stabilizer is a protein.
Claim 11
Dependent Claim
21 words
The thermostable enzyme of claim 10, wherein said protein is selected from the group consisting of Bovine Serum Albumin and gelatin.
Claim 12
Dependent Claim
18 words
An isolated DNA sequence coding for the amino-terminal portion of a thermostable enzyme obtainable from Thermococcus litoralis .
Claim 13
Dependent Claim
18 words
The isolated DNA sequence of claim 12, wherein the isolated DNA sequence is about 1.3 kb in length.
Claim 14
Dependent Claim
20 words
The isolated DNA sequence of claim 13, wherein the isolated DNA comprises nucleotides 1 to 1274 of Figure No. 6.
Claim 15
Dependent Claim
10 words
A vector containing the isolated DNA sequence of claim 13.
Claim 16
Dependent Claim
12 words
The vector of claim 15, wherein such vector is bacteriophage NEB 618.
Claim 17
Dependent Claim
18 words
An isolated DNA sequence coding for the intermediate portion of a thermostable enzyme obtainable from Thermococcus litoralis .
Claim 18
Dependent Claim
18 words
The isolated DNA sequence of claim 17, wherein the isolated DNA sequence is about 1.6 kb in length.
Claim 19
Dependent Claim
20 words
The isolated DNA sequence of claim 18, wherein the isolated DNA comprises nucleotides 1269 to 2856 of Figure No. 6.
Claim 20
Dependent Claim
10 words
A vector containing the isolated DNA sequence of claim 19.
Claim 21
Dependent Claim
12 words
The vector of claim 20, wherein such vector is bacteriophage NEB 620.
Claim 22
Dependent Claim
17 words
An isolated DNA sequence coding for the carboxyl-terminal of a thermostable enzyme obtainable from Thermococcus litoralis .
Claim 23
Dependent Claim
17 words
The isolated DNA sequence of claim 22, wherein the isolated DNA is about 1.9 kb in length.
Claim 24
Dependent Claim
20 words
The isolated DNA sequence of claim 23, wherein the isolated DNA comprises nucleotides 2851 to 4771 of Figure No. 6.
Claim 25
Dependent Claim
10 words
A vector containing the isolated DNA sequence of claim 23.
Claim 26
Dependent Claim
11 words
The vector of claim 25, wherein such vector is plasmid V153-2.
Claim 27
Dependent Claim
35 words
A vector comprising the isolated DNA sequence of claim 13 operably linked in the proper reading frame with the isolated DNA sequence of claim 17 to produce Thermococcus litoralis DNA polymerase or a portion thereof.
Claim 28
Dependent Claim
31 words
The vector of claim 27, further comprising the isolated DNA sequence of claim 23 operably linked in the proper reading frame to produce Thermococcus litoralis DNA polymerase or a portion thereof.
Claim 29
Dependent Claim
13 words
An isolated DNA sequence which codes for the thermostable enzyme of claim 1.
Claim 30
Dependent Claim
9 words
A vector containing the DNA sequence of claim 29.
Claim 31
Dependent Claim
10 words
A microbial host transformed by the vector of claim 30.
Claim 32
Dependent Claim
21 words
An isolated DNA sequence according to claim 29 contained within an approximately 14 kb BamHI restriction fragment of bacteriophage NEB 619.
Claim 33
Dependent Claim
15 words
The isolated DNA sequence of claim 29, comprising the DNA sequence of Figure No. 6.
Claim 34
Dependent Claim
15 words
The isolated DNA sequence of claim 33, wherein nucleotides 1776 to 3389 have been deleted.
Claim 35
Dependent Claim
19 words
An isolated DNA sequence according to claim 29, comprising an approximately 4 kb BamHI/NdeI restriction fragment of plasmid pPR969.
Claim 36
Dependent Claim
8 words
A vector containing the DNA of claim 33.
Claim 37
Dependent Claim
8 words
A vector containing the DNA of claim 34.
Claim 38
Dependent Claim
11 words
The vector of claim 37, wherein said vector is plasmid V174-1B1.
Claim 39
Dependent Claim
11 words
The vector of claim 37, wherein said vector is plasmid pPR969.
Claim 40
Dependent Claim
13 words
A microbial host transformed with the vector of claim 38 or claim 39.
Claim 41
Dependent Claim
16 words
The transformant of claim 40, wherein said transformant is E . coli NEB 671 (ATCC 68447).
Claim 42
Dependent Claim
15 words
The transformant of claim 40, wherein said transformant is E . coli NEB687 (ATCC 68487).
Claim 43
Dependent Claim
42 words
A process for the preparation of Thermococcus litoralis DNA polymerase comprising culturing the transformed microbial host of any of the claims 40, 41, or 42 under conditions suitable for the expression of Thermococcus litoralis DNA polymerase and recovering Thermococcus litoralis DNA polymerase.
Claim 44
Dependent Claim
11 words
Thermococcus litoralis DNA polymerase produced by the process of claim 43.
Claim 45
Independent Claim
96 words
A method for producing Thermococcus litoralis DNA polymerase comprising the steps of (a) purifying total DNA from Thermococcus litoralis ; (b) isolating DNA from the total DNA of step (a) which codes for the DNA polymerase; (c) removing an intervening DNA from said isolated DNA of step (b); (d) ligating the DNA of step (c) into an appropriate vector; (e) transforming a host with the vector of step (d); (f) cultivating the transformed host of step (e) under conditions suitable for expression of the T . litoralis DNA polymerase; (g) recovering the Thermococcus litoralis DNA polymerase.
Claim 46
Dependent Claim
16 words
The method of claim 45, wherein the isolated DNA comprises the DNA of Figure No. 6.
Claim 47
Dependent Claim
44 words
The method of claim 44, wherein the intervening DNA is removed via a splice junction, said splice junction spanning from about nucleotides 1761-1775 and 3384-3392 of Figure No. 6, whereby a concensus region corresponding to concensus region III of Figure No. 8 is formed.
Claim 48
Dependent Claim
18 words
The method of claim 47, wherein the intervening DNA comprises nucleotides 1776 to 3389 of Figure No. 6.
Claim 49
Dependent Claim
19 words
A thermostable endonuclease obtainable from Thermococcus litoralis which cleaves double-stranded deoxynucleotide acid pBR322 at position 164 and position 2411.
Claim 50
Dependent Claim
14 words
The thermostable endonuclease of claim 49, having a molecular weight of about 33,000-37,000 daltons.
Claim 51
Dependent Claim
16 words
The isolated DNA of claim 33, which further codes for the thermostable endonuclease of claim 49.
Claim 52
Dependent Claim
17 words
The isolated DNA of claim 51, wherein the coding sequence for the endonuclease starts at nucleotide 3534.
Claim 53
Dependent Claim
13 words
The vector of claim 30, wherein such vector is bacteriophage NEB619 (ATCC 40795).
Claim 54
Dependent Claim
18 words
A microbial host transformed with the vector pCAS4, wherein said transformant is E . Coli NEB681 (ATCC 68473).
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