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° C. for 10 minutes in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mM MgCl 2 ; (ii) is substantially specific for single stranded DNA; and (iii) has a 3′-5′ 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.
1. A method of nucleic acid amplification, said method comprising:
(a) amplifying nucleic acid by a nucleic acid amplification reaction selected from the group consisting of a polymerase chain reaction (PCR), a ligase amplification reaction (LAR), a strand displacement amplification (SDA), a nucleic acid sequence based amplification (NASBA) and a loop-mediated isothermal amplification (LAMP) to produce a nucleic acid amplification product after a final amplification step of the nucleic acid amplification reaction and
(b) subsequently contacting the nucleic acid amplification product with an exonuclease under conditions which permit the digestion of at least a portion of any single stranded DNA present in the nucleic acid amplification product and optionally then heating the mixture to inactivate said exonuclease,
wherein said exonuclease has the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which is at least about 85% identical thereto, wherein said exonuclease
(i) is at least 90% irreversibly inactivated by heating at a temperature of about 55° C. for 10 mins in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mM MgCl 2 ;
(ii) has activity against double stranded DNA that is equal to or less than 15% of its activity against an equivalent amount of single stranded DNA under the same assay conditions; and
(iii) has a 3′-5′ exonuclease activity.
2. The method of claim 1 , wherein said nucleic acid amplification product is the direct product of the final amplification step.
3. The method as claimed in claim 1 , wherein said exonuclease has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11.
4. The method as claimed in claim 2 , wherein said exonuclease has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11.