Universitetet I Tromsø—norges Arktiske Universitet

Arcticzymes As

Nils Peder Willassen

Olav Lanes

Marit Sjo Lorentzen

Terese Solstad

Morten Elde

Atle Noralf Larsen

Yvonne Piotrowski

Elisabeth Lill Andreassen

Marcin Pierechod

Ulli Rothweiler

The present invention relates to the use of a single-strand DNA binding protein (SSB) which exhibits at least 50% of its maximum ssDNA binding capability in the presence of 500 mM of sodium ions, to dehybridize a DNA molecule or to prevent hybridisation of a complementary ssDNA, wherein the SSB comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which is at least 75% identical to SEQ ID NO:1, or a functional fragment thereof, and wherein the DNA molecule or ssDNA is present in or exposed to a solution containing one or more of the following (i) at least 350 mM of sodium ions; (ii) at least 50 mM of potassium ions; (iii) at least 150 mM of magnesium ions; or (iv) at least 200 mM of calcium ions.

Single-strand binding protein

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

Nils Peder Willassen