EP0547920A2
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Recombinant thermostable DNA polymerase from archaebacteria
Assignee: New England Biolabs Inc
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Filing: 1992-12-18
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Granted: —
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Expires: 2012-12-18
Abstract
Abstract Recombinant DNA polymerases from archaebacteria as well as isolated DNA coding for such polymerases are provided. The isolated DNA is obtained by use of DNA or antibody probes prepared from the DNA encoding T. litoralis DNA polymerase and the T. litoralis DNA polymerase respectively. Also provided are methods for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymerases by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.
Inventors
- 👤 Donald G. Comb
- 👤 Francine Perler
- 👤 Rebecca Kucera
- 👤 William E. Jack
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 02:18
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252; C07; C07K; C07K16/00; C07K16/12
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
57 words
Recombinant thermostable DNA polymerase from archaebacteria which is encoded by a DNA sequence which hybridizes to a nucleotide sequence selected from the group consisting of the nucleotide sequence of Figure 6 or a portion thereof, nucleotides 1 to 1274 of Figure 6, nucleotides 1269 to 2856 of Figures 6 and nucleotides 2851 to 4771 of Figure 6.
Claim 2
Dependent Claim
57 words
Recombinant thermostable DNA polymerase from archaebacteria which is encoded by a DNA sequence which hybridizes to a nucleotide sequence selected from the group consisting of the nucleotide sequence of Figure 6 or a portion thereof, nucleotides 1 to 1274 of Figure 6, nucleotides 1269 to 2856 of Figures 6 and nucleotides 2851 to 4771 of Figure 6.
Claim 3
Dependent Claim
25 words
The recombinant thermostable polymerase of claim 1, wherein the portion of the nucleotide sequence of Figure 6 is at least about 20 nucleotides in length.
Claim 4
Dependent Claim
25 words
The recombinant thermostable polymerase of claim 1, wherein the portion of the nucleotide sequence of Figure 6 is at least about 50 nucleotides in length.
Claim 5
Dependent Claim
25 words
The recombinant thermostable polymerase of claim 1, wherein the portion of the nucleotide sequence of Figure 6 is at least about 150 nucleotides in length.
Claim 6
Dependent Claim
21 words
Recombinant thermostable DNA polymerase from archaebacteria which hybridizes to an antibody probe which has antigenic specificity to T. litoralis DNA polymerase.
Claim 7
Dependent Claim
21 words
Recombinant thermostable DNA polymerase from archaebacteria which hybridizes to an antibody probe which has antigenic specificity to T. litoralis DNA polymerase.
Claim 8
Dependent Claim
13 words
Isolated DNA which codes for the recombinant thermostable DNA polymerase of claim 1.
Claim 9
Dependent Claim
10 words
A cloning vector comprising the isolated DNA of claim 6.
Claim 10
Dependent Claim
10 words
A host cell transformed by the vector of claim 7.
Claim 11
Dependent Claim
33 words
A method for producing a recombinant thermostable DNA polmerase from archaebacteria comprising culturing a host cell transformed with the vector of claim 7 under conditions suitable for the expression of the DNA polymerase.
Claim 12
Dependent Claim
56 words
ADNAprobe which hybridizes to the DNAsequence coding for the archaebacteria thermostable DNA polymerase of claim 1, wherein the DNA probe is selected from the group consisting of the nucleotide sequence of Figure 6 or a portion thereof, nucleotides 1 to 1274 of Figure 6, nucleotides 1269 to 2856 of Figure 6 and nucleotides 2851 to 4771.
Claim 13
Dependent Claim
24 words
The DNA probe of claim 10, wherein the portion of the nucleotide sequence of Figure 6 is at least about 20 nucleotides in length.
Claim 14
Dependent Claim
24 words
The DNA probe of claim 10, wherein the portion of the nucleotide sequence of Figure 6 is at least about 50 nucleotides in length.
Claim 15
Dependent Claim
24 words
The DNA probe of claim 10, wherein the portion of the nucleotide sequence of Figure 6 is at least about 150 nucleotides in length.
Claim 16
Independent Claim
120 words
A method for isolating DNA coding for thermostable DNA polymerase from archaebacterium comprising the steps of: (a) forming a genomic library from the archaebacterium; (b) transforming or transfecting an appropriate host cell with the library of step (a); (c) contacting DNA from the transformed or transfected host cell with a DNA probe selected from the group consisting of the nucleotide sequence of Figure 6 or a portion thereof, nucleotides 1 to 1274 of Figure 6, nucleotides 1269 to 2856 of Figure 6 and nucleotides 2851 to 4771; (d) assaying the transformed or transfected cell of step (c) which hybridizes to the DNA probe for DNA polymerase activity; and (e) isolating a DNA fragment which codes for the thermostable DNA polymerase.
Claim 17
Independent Claim
120 words
A method for isolating DNA coding for thermostable DNA polymerase from archaebacterium comprising the steps of: (a) forming a genomic library from the archaebacterium; (b) transforming or transfecting an appropriate host cell with the library of step (a); (c) contacting DNA from the transformed or transfected host cell with a DNA probe selected from the group consisting of the nucleotide sequence of Figure 6 or a portion thereof, nucleotides 1 to 1274 of Figure 6, nucleotides 1269 to 2856 of Figure 6 and nucleotides 2851 to 4771; (d) assaying the transformed or transfected cell of step (c) which hybridizes to the DNA probe for DNA polymerase activity; and (e) isolating a DNA fragment which codes for the thermostable DNA polymerase.
Claim 18
Independent Claim
95 words
A method for isolating DNA coding for thermostable DNA polymerase from archaebacterium comprising the steps of: (a) forming a genomic library from the archaebacterium; (b) transforming or transfecting an appropriate host cell with the library of step (a); (c) contacting extract from the transformed or transfected host cell with an antibody probe which has specific affinity for T. litoralis DNA polymerase; (d) assaying the transformed or transfected cell of step (c) which is cross-reactive to the antibody probe for DNA polymerase activity; and (e) isolating a DNA fragment which codes for the thermostable DNA polymerase.
Claim 19
Independent Claim
95 words
A method for isolating DNA coding for thermostable DNA polymerase from archaebacterium comprising the steps of: (a) forming a genomic library from the archaebacterium; (b) transforming or transfecting an appropriate host cell with the library of step (a); (c) contacting extract from the transformed or transfected host cell with an antibody probe which has specific affinity for T. litoralis DNA polymerase; (d) assaying the transformed or transfected cell of step (c) which is cross-reactive to the antibody probe for DNA polymerase activity; and (e) isolating a DNA fragment which codes for the thermostable DNA polymerase.
Claim 20
Dependent Claim
45 words
A method for increasing the expression of a thermostable DNA polymerase from archaebacteria comprising the steps of: (a) identifying and locating any intervening nucleotide sequence in the isolated DNA of claim 14 or 15; and (b) removing the intervening nucleotide sequence from the isolated DNA.
Claim 21
Dependent Claim
11 words
The method of claim 16, wherein the archaebacteria comprises T. litoralis.
Claim 22
Dependent Claim
22 words
The method of claim 17, wherein the intervening nucleotide sequence is selected from the group of IVS1, IVS2 or IVS1 and IVS2.
Claim 23
Dependent Claim
31 words
The method of claim 16, wherein the intervening nucleotide sequence is identified and located with a DNA probe coding for an intron from the DNA sequence encoding T. litoralis DNA polymerase.
Claim 24
Dependent Claim
51 words
The method of claim 19, wherein the DNA probe is selected from the group consisting of a 1614 bp nucleotide sequence of Figure 6 comprising nucleotides 1776 to 3389 or a portion therof and an 1170 bp nucleotide sequence of Figure 6 comprising nucleotides 3544 to 4703 or a portion thereof.
Claim 25
Dependent Claim
18 words
A thermostable endonuclease obtainable from T. litoralis which cleaves double-stranded deoxynucleotide acid pBR322 at position 164 and 2411.
Claim 26
Dependent Claim
18 words
A thermostable endonuclease obtainable from T. litoralis which cleaves double-stranded deoxynucleotide acid pBR322 at position 164 and 2411.
Claim 27
Dependent Claim
12 words
The endonuclease of claim 21, having a molecular weight of about 33,000-37,000.
References Cited (Backward Citations)
No citation records indexed.
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