ENHANCED DETECTION OF GENETICALLY-ENGINEERED CORYNEBACTERIUM-GLUTAMICUM PUN1 IN DIRECTLY EXTRACTED DNA FROM SOIL, USING THE T4 GENE-32 PROTEIN IN THE POLYMERASE CHAIN-REACTION

The single stranded DNA stabilizing T4 gene 32 protein (T4gp32) was us ed to enhance the polymerase chain reaction (PCR) amplification of a r ecombinant gene (174 bp) in samples contaminated with humic acids or u nspecific DNA, as well as in direct soil extracted DNA samples seeded with Corynebacterium glutamicum pUN1. Compared to PCR amplification wi thout T4gp32 the inhibitory action of standard humic acids was reduced 7-fold, when T4gp32 was included in the PCR reaction mix. Unspecific soil DNA also inhibited the detection of 2000 target molecules by PCR amplifications at concentrations of 20.1 mu g unspecific DNA/ml or abo ve. With T4gp32 the minimal inhibitory concentration increased 4-fold. The threshold of detection of C. glutamicum pUN1 using directly extra cted DNA from non-sterile soil after PCR amplification was 10 cells pe r g.