Søren Møller

Single nucleotide polymorphism genotyping using locked nucleic acid (LNA

Locked nucleic acid (LNA™) is a new class of bicyclic high affinity DNA analogs. LNA-containing oligonucleotides confer significantly increased affinity against their complementary DNA targets, increased mismatch discrimination (ΔTm) and allow full control of the melting point of the hybridization reaction. LNA chemistry is completely compatible with the traditional DNA phosphoramidite chemistry and therefore LNA–DNA mixmer oligonucleotides can be designed with complete freedom for optimal performance. These properties render LNA oligonucleotides very well suited for SNP genotyping and have enabled several approaches for enzyme-independent SNP genotyping based on allele-specific hybridization. In addition, allele-specific PCR assays relying on enzymatically-enhanced discrimination can be improved using LNA-modified oligonucleotides. The use of LNA transforms enzyme-independent genotyping approaches into experimentally simple, robust and cost-effective assays, which are highly suited for genotyping in clinical and industrial settings.

Using inactivated microbial biomass as fertilizer: the fate of antibiotic resistance genes in the environment.

The waste product produced by Novo Nordisk A/S from microbial fermentations is used as agricultural fertilizer in Denmark (NovoGro) after being treated by heat and chemicals to destroy the microorganisms. The fertilizer contains DNA fragments from the genetically modified microorganisms used in industrial production. This DNA contains genes coding for the desired industrial products as well as genes used as genetic selection markers during production strain development. The antibiotic resistance markers used as genetic selection markers are chloramphenicol (Cm), kanamycin (Km) and ampicillin (Ap). The aim of the present study was to examine whether DNA and intact genes were present in NovoGro and whether horizontal transfer of DNA isolated from inactivated production strains occurred either in the laboratory or in the fields treated with NovoGro. DNA isolated from NovoGro was analysed by PCR and intact genes coding for a protease and chloramphenicol resistance were amplified. This isolated DNA was used for in vitro experiments including electroporation and transformation but no transfer of DNA to Escherichia coli or Bacillus subtilis was observed. The antibiotic resistance profile of the indigenous bacterial population in the fields treated with NovoGro compared with fields treated with inorganic fertilizers showed no differences. In addition, DNA isolated directly from the fields treated with NovoGro for up to 7 years was analysed by PCR and no specific production gene constructs could be detected.