Christoph Kessler’s research while affiliated with Roche and other places

The highly sensitive non-radioactive DIG system allows specific detection Principle and of 0.01-0.03 pg DNA or RNA within 1-16 h with colorimetric BCIP/NBT applications detection (McGadey, 1970) or 15-30 min with chemiluminescent detection [Disodium 3- (4-methoxyspiro-{1,2 - dioxetane-3,2’-(5’-chloro) tricyclo [3.3.1.1 3’7]decan}-4-yl)phenyl phosphate (CSPD) or Disodium 2-chloro5-(4-methoxyspiro {1,2-dioxetane-3,2’-(5’-chloro)tricyclo [3.3.1.13’7] decan}-4-y1)-1-phenyl phosphate (CDP-Star) (Bronstein et al., 1989)] in dot-, slot-, Southern and Northern blots, avoiding any significant background on nylon membranes (Kessler et al., 1990; Höltke et al., 1990; Seibl et al., 1990; Mühlegger et al., 1990; Kessler, 1991; Höltke, 1995). Nitrocellulose membranes in combination with chemiluminescence substrates are less sensitive and require the addition of an enhancer [e.g., NitroBlock, from Tropix, Inc. (Höltke, 1995)].

The genes encoding a class-IIN restriction-modification (R-M) system (MamI, sequence specificity [symbol: see text] from Microbacterium ammoniaphilum have been cloned in Escherichia coli. The vector used for cloning was plasmid pUC18 modified by the inclusion of three MamI recognition sites. Recombinant clones containing the mamIM gene in its genomic context became fully methylated in vivo and remained completely resistant against digestion with the R.MamI restriction endonuclease (ENase). Determination of the nucleotide (nt) sequence revealed three open reading frames with lengths of 1089 bp (ORF1), 276 bp (ORFc) and 927 bp (ORF2). On the basis of expression and deletion experiments, the 1089-bp ORF1 was assigned to mamIM encoding the M.MamI DNA methyltransferase (MTase). By amino acid sequencing of the N terminus of R.MamI and comparison of the deduced nt sequence with ORF2, the 927-bp ORF2 was identified as the mamIR gene encoding R.MamI. The 276-bp ORFc, located between mamIR and mamIM, is part of the DNA sequence downstream from mamIM shown to be necessary for controlled mamIM expression.

We report here the generation of a novel restriction endonuclease (ENase) activity with the 10-bp recognition sequence, [symbol: see text]. This specificity could be achieved by first methylating a substrate DNA with M.MamI in vivo, followed by in vitro R.DpnI restriction.

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactive labeled nucleotides during PCR by Taq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin-(DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin-labeled amplification products as hybridization probes, sensitivities in the 0.1-pg range are obtained in Southern blot procedures.

Listeria monocytogenes is associated with meningoencephalitis, septicemia and abortion in humans (Gray et a11966). Pregnant women, newborns, and immunocompromised individuals are particularly susceptible to infection; however, in some instances, apparently healthy individuals develop clinical disease after ingestion of food contaminated with this pathogen (Schlech et al 1983; Schwartz et al 1989). There have been at least five serious foodborne outbreaks of listeriosis reported in North America and Europe in recent years (Schwartz et al 1989).