DOMAIN Glycosylases have found use in a number of methods aimed at analyzing DNA for the presence of altered nucleobases that could result from ionizing radiation or chemical oxidants, identifying mismatched base pairs, studying repair of abasic sites in double-stranded DNA and as a means to prevent carry- over contamination of PCR reactions by products from previously generated amplicons. Several different glycosylases have been identified, most of which are specific for a certain type of altered base, andt also some that exhibit a more relaxed specificity.
DESCRIPTION NTNU researchers have cloned and expressed both the mitochondrial as well as the nuclear version of the UNG enzyme from E.coli and have generated various mutants that display either altered specificity or have increased sensitivity to heat inactivation. The researchers have also cloned and expressed the wild-type SMUG1 glycosylase (single-strand-selective monofunctional uracil DNA glycosylase 1).
The new enzymes exhibit:
• A novel, temperature sensitive Uracil N-Glycosylase with very favourable characteristics for use in PCR de-contamination procedures.
• A novel Thymine DNA-glycosylase which is the first enzyme ever capable of hydrolysing thymine-glycosidic bonds in ssDNA and in T:A base-pairs in dsDNA.
• A novel Cytosine DNA Glycosylase which is the first enzyme ever capable of hydrolysing cytosine-glycosidic bonds in DNA
• A novel, protease resistant Uracil-N-glycosylase
• A recombinant Single Strand Monofunctional Uracil DNA Glycosylase 1 (SMUG1).
The enzymes can be divided into the tsUNG enzymes that address the significant PCR sterilisation market and a likely smaller research market for glycosylases with novel specificities.
UNIQUE SELLING POINTS • Effective in removing uracil from both dsDNA and ssDNA in a suitable temperature interval (20 to 37oC) under PCR buffer conditions.
• The enzyme is effectively and irreversibly inactivated by short incubations at elevated temperatures
• The enzyme is cheap to manufacture, free of contaminating exo – and endonculease and RNAse activity and have long shelf–life.
• The portfolio of enzymes cover “rare” glycosylases
POTENTIAL APPLICATIONS • Reagents for biochemical analysis of DNA
• Core reagents in ready-to-use kits for PCR sterilization
STATE OF DEVELOPMENT NTNU has the capability to produce each of these enzymes in commercial quantities and at competitive prices for commercial partners for worldwide marketing and sale.
Hans E Krokan
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