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
W. Vahjen et Cc. Tebbe, 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, European journal of soil biology, 30(2), 1994, pp. 93-98
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.