Christoph Kessler

The digoxigenin:anti-digoxigenin (DIG) technology--a survey on the concept and realization of a novel bioanalytical indicator system.

A review is given on the novel non-radioactive digoxigenin:anti-digoxigenin (DIG) bioanalytical indicator system. After a general introduction on direct and indirect indicator systems based on previous non-radioactive indicator reactions as well as in vitro and in vivo amplification procedures the principle of the new digoxigenin:anti-digoxigenin technology is demonstrated. The novel system is based on the specific high-affinity interaction between the cardenolide digoxigenin from Digitalis plants and a digoxigenin-specific antibody coupled with a reporter group. A variety of methods for digoxigenin modification of nucleic acids, proteins and glycans are presented. In addition, various applications of the novel non-radioactive indicator system in a variety of direct or indirect detection approaches with either insoluble or soluble substrates are described. It is also shown that with these applications alternative reaction formats are used which are partly characterized by additional amplification steps.

Recognition sequences of restriction endonucleases and methylases — a review

The properties and sources of all known endonucleases and methylases acting site-specifically on DNA are listed. The enzymes are crossindexed (Table I), classified according to homologies within their recognition sequences (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328 and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (Table III), the structure of the restriction fragment ends (Table IV), and the sensitivity to different kinds of DNA methylation (Table V). Table VI classifies the methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises those restriction endonucleases, which are known to be inhibited by the modified nucleotides. Furthermore, this review includes a restriction map of bacteriophage lambda DNA based on sequence data. Table VII lists the exact nucleotide positions of the cleavage sites, the length of the generated fragments ordered according to size, and the effects of the Escherichia coli dam- and dcmI-coded methylases M X Eco dam and M X Eco dcmI on the particular recognition sites.

Non-radioactive analysis of biomolecules

An overview on non-radioactive bioanalytical indicator systems is presented. The nature of labels being important for direct as well as indirect systems is discussed. This is followed by the description of enzymatic, photochemical and chemical methods for labeling nucleic acids, proteins and glycans. These methods can be applied either for direct labeling of these biomolecules or for labeling of respective probes (DNA, RNA, oligonucleotides, antibodies, lectins). In the second part, various optical, luminescent and fluorescent detection approaches are described. The possibility to enhance the sensitivity by coupled amplification reactions (signal amplification, target-specific signal amplification, target amplification) is shown in a separate section. Finally, the wide variety of qualitative and quantitative reaction formats related to different applications is collected.

Nonradioactive labeling and high-sensitive detection of PCR products

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 nonradioactivc labeled nucleotides during PCR byTaq 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, sensilivities in the 0.1-pg range are obtained in Southern blot procedures.

The Digoxigenin: Anti-Digoxigenin (DIG) System

The digoxigenin:anti-dioxigenin (DIG) indicator system is based on the specific interaction between the cardenolide steroid DIG, a chemically derived aglycon of digoxin and lanatoside C (see figure), and a high-affinity, DIG-specific antibody (Kessler, 1991).

Labeling of Specific DNA Sequences with Digoxigenin during Polymerase Chain Reaction

A broad spectrum of methods for the detection of specific nucleic acids is applied in molecular biology, genetics and medicine.

SphI restriction map of bacteriophage lambda DNA.

Abstract Upon reinvestigation, the number of cleavage sites for site-specific endonuclease Sph I on λ DNA was found to be six. The Sph I restriction map of the λ c I857 S am7 genome was determined.

Nonradioactive Labeling of Polymerase Chain Reaction Products

Polymerase chain reaction (PCR) was originally introduced to amplify in vitro particular DNA sequences by the application of temperature cycles (1). In a modification, RNA molecules also may serve as templates by an additional reverse transcription step converting RNA in complementary DNA sequences (2).

RleAI: a novel class-IIS restriction endonuclease from Rhizobium leguminosarum recognizing 5′-CCCACA(N)12-3′3′-GGGTGT(N)9-5′

Abstract The novel class-IIS restriction endonuclease, Rle AI, has the 5′-CCCACA(N) 12 -3′ 3′-GGGTGT(N) 9 -5′ , specificity.

Novel specific endonuclease activity recognizing a 10-bp sequence.

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.