Universitetet I TromsØ—norges Arktiske Universitet

Arcticzymes As

Biotec Pharmacon Asa

Morten Elde

Olav Lanes

Marit Sjo Lorentzen

Terese Solstad

Atle Noralf Larsen

Yvonne Piotrowski

Nils Peder Willassen

Elisabeth Lill Andreassen

Dag Rune Gjellesvik

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.

The invention provides methods of removing nucleic acid contamination from reverse transcription reactions and hot-start PCR, wherein said hot-start PCR is a barrier hot-start PCR set up and/or involves a hot-start DNA polymerase, which methods comprise use of a DNase that is substantially irreversibly inactivated by heating at a temperature of about 50&#xb0; C. for 5 minutes, and that is substantially specific for double stranded DNA. The invention further provides a DNase that is substantially irreversibly inactivated by heating at a temperature of about 50&#xb0; C. for 5 minutes, and that is substantially specific for double stranded DNA, nucleic acids encoding said DNase and kits or compositions comprising said DNase or said nucleic acid.

Method of removing nucleic acid contamination in reverse transcription and amplification reactions

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Tromsø, Norway

Morten Elde