Recombinant thermostable DNA polymerase from archaebacteria

Claim 1

An isolated DNA which codes for a recombinant DNA polymerase from thermophilic archaebacteria, consisting essentially of a DNA fragment which hybridizes in a Southern blot to an isolated DNA fragment selected from the group consisting of a DNA fragment consisting essentially of nucleotides 1-1274 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 291-1772 of SEQ ID NO: 1, a DNA fragment consisting essentially of nucleotides 3387-3533 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 4704-5396 of SEQ ID NO: 1, and a DNA fragment consisting essentially of nucleotides 4718-5437 of SEQ ID NO:1, wherein hybridization is conduction under the following conditions: a) hybridization: 0.75M NaCI, 0.15 Tris, 10mM EDTA, 0.1% Sodium Pyrophosphate, 0.1% SLS, 0.03% BSA, 0.03% Ficoll 400, 0.03% PVP and 100ug/ml boiled calf thymus DNA at 50° C. for about 12 hours and; b) wash: 3×30 minutes with 0.1X SET, 0.1% SDS, 0.1% sodium pryrophosphate and 0.1M phosphate buffer at 45° C.

Claim 7

A method for isolating a target DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase from thermophilic 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 which hybridizes to a DNA fragment selected from the group consisting of a DNA fragment consisting essentially of nucleotides 1-1274 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 291-1772 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 3387-3533 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 4704-5396 of SEQ ID NO: 1, and a DNA fragment consisting essentially of nucleotides 4718-5437 of SEQ ID NO:1, wherein hybridization is conducted under the following conditions: a) hybridization: 0.75M NaCI, 0.15 Tris, 10mM EDTA, 0.1% Sodium Pyrophosphate, 0.1% SLS, 0.03% BSA, 0.03% Ficoll 400, 0.03% PVP and 100ug/ml boiled calf thymus DNA at 50° C. for about 12 hours and; b) wash: 3×30 minutes with 0.1X SET, 0. 1% SDS, 0.1% sodium pryrophosphate and 0.1M phosphate buffer at 37°-55° C.; (d) assaying the transformed or transfected cell of step (c) which hybridizes to the DNA probe for DNA polymerase activity; (e) isolating a target DNA fragment which codes for the thermostable DNA polymerase.

Claim 8

A method for isolating a target DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase from thermophilic 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 which hybridizes to a DNA fragment selected from the group consisting of a DNA fragment consisting essentially of nucleotides 1-1274 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 291-1772 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 3387-3533 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 4704-5396 of SEQ ID NO: 1, and a DNA fragment consisting essentially of nucleotides 4718-5437 of SEQ ID NO:1, wherein hybridization is conducted under the following conditions: a) hybridization: 0.75M NaCI, 0.15 Tris, 10mM EDTA, 0.1% Sodium Pyrophosphate, 0.1% SLS, 0.03% BSA, 0.03% Ficoll 400, 0.03% PVP and 100ug/ml boiled calf thymus DNA at 50° C. for about 12 hours and; b) wash: 3×30 minutes with 0.1X SET, 0. 1% SDS, 0.1% sodium pryrophosphate and 0.1M phosphate buffer at 37°-55° C.; (d) assaying the transformed or transfected cell of step (c) which hybridizes to the DNA probe for DNA polymerase activity; (e) isolating a target DNA fragment which codes for the thermostable DNA polymerase.

Claim 9

A method for isolating a DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase from thermophilic 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 cellular polypeptide 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 10

A method for isolating a DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase from thermophilic 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 cellular polypeptide 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.