Universitetet I TromsØ—norges Arktiske Universitet

Arcticzymes As

Universitetet I TromsØ—norges Artiske Universitet

Yvonne Piotrowski

Atle Noralf Larsen

Olav Lanes

Marit Sjo Lorentzen

Terese Solstad

Morten Elde

Nils Peder Willassen

Elisabeth Lill Andreassen

Netsanet Gizaw Assefa

The present invention provides a DNA polymerase including the sequence of SEQ ID NO. 1 or a sequence which is at least 70% identical thereto, but wherein the aspartic acid residue at position 18 of SEQ ID NO. 1, or the equivalent aspartic acid residue in other sequences, has been replaced by a non-negatively charged amino acid residue. It further provides DNA polymerases comprising the amino acid sequences of SEQ ID NO. 2, 11 and 12 and variants thereof. The present invention also provides nucleic acids encoding the DNA polymerases, a method of producing said DNA polymerases, and compositions, expression vectors and host cells or viruses comprising said DNA polymerases. The present invention also provides uses of said DNA polymerases in nucleotide polymerisation, amplification and sequencing reactions.

DNA polymerases

The present invention relates to DNA polymerases. In particular, the present invention relates to DNA polymerases based on a DNA polymerase from asp. The present invention provides an isolated DNA polymerase or an enzymatically active fragment thereof, said DNA polymerase comprising the amino acid sequence of SEQ ID NO:1 or comprising an amino acid sequence which is at least 70% identical to SEQ ID NO:1. The invention also provides nucleic acid molecules comprising a nucleotide sequence that encodes the DNA polymerase. The invention also provides a method of nucleotide polymerisation and a method of amplifying a nucleic acid in which the DNA polymerase or an enzymatically active fragment thereof is used.

Marine DNA polymerases

The invention provides an exonuclease or an enzymatically active fragment thereof, said exonuclease having the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which is at least about 50% identical thereto, wherein said exonuclease or enzymatically active fragment thereof (i) is substantially irreversibly inactivated by heating at a temperature of about 55&#xb0; C. for 10 minutes in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25&#xb0; C., 50 mM KCl and 5 mM MgCl; (ii) is substantially specific for single stranded DNA; and (iii) has a 3'-5&#x2032; exonuclease activity. The invention further provides a method of removing single stranded DNA from a sample, a method of nucleic acid amplification, a method of reverse transcription and a method of nucleic acid sequence analysis in which the exonuclease or enzymatically active fragment thereof is used. The invention still further provides nucleic acids encoding said exonuclease or an enzymatically active fragment thereof and kits or compositions comprising the same.

Thermolabile exonucleases

The invention provides an exonuclease or an enzymatically active fragment thereof, said exonuclease having the amino acid sequence of SEQ ID No. 1 or an amino acid sequence which is at least about 50% identical thereto, wherein said exonuclease or enzymatically active fragment thereof (i) is substantially irreversibly inactivated by heating at a temperature of about 55&#xb0; C. for 10 minutes in a buffer consisting of 10 mM Tris-HCI, pH 8.5 at 25&#xb0; C., 50 mM KCI and 5 mM MgCI; (ii) is substantially specific for single stranded DNA; and (iii) has a 3'-5&#x2032; exonuclease activity. The invention further provides a method of removing single stranded DNA from a sample, a method of nucleic acid amplification, a method of reverse transcription and a method of nucleic acid sequence analysis in which the exonuclease or enzymatically active fragment thereof is used. The invention still further provides nucleic acids encoding said exonuclease or an enzymatically active fragment thereof and kits or compositions comprising the same.

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Tomasjord, Norway

Yvonne Piotrowski