Jørn Koch

Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ

It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.

Assembly and structural analysis of a covalently closed nano-scale DNA cage

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson–Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helices that assembles from eight oligonucleotides with a yield of ∼30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures.

Construction of a panel of chromosome-specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ

We present the sequences of a set of oligonucleotides that, when used as primers for PRimed IN Situ (PRINS) labeling, are diagnostic for repetitive sequences in specific human chromosomes. Combined, they enable identification of all human chromosomes except 6, 19, and 20. However, as is also the case with cloned centromeric hybridization probes, chromosomes 14 and 22 are stained together. Along with the sequences of these oligonucleotides we offer a simple, universal procedures for their use. We also present an oligonucleotide that binds to both strands of alpha-satellite DNA, making it possible to specifically amplify alpha-satellite DNA by PCR with this one oligonucleotide alone as primer. The origin of the alpha-satellite DNA in the starting material (whether somatic cell hybrids, flow-sorted chromosomes, or microdissected material) can then be determined on test metaphase spreads by in situ hybridization or PRINS with the PCR product.

Staining of human telomeres with primed in situ labeling (PRINS).

As described, the PRINS method is a very rapid and reliable way of staining human telomeres. To obtain the maximum frequency of stained telomeres, the primer (CCCTAA)7 should be used, although the average frequency never quite reaches 100%. The frequency is strongly dependent on the age of the individual, being significantly higher in children and newborns than in adults. A difference between the (CCCTAA)7 primer and the complementary primer is demonstrated and a possible explanation is proposed, namely, that gaps in the C-rich strand cause chain elongation termination after the addition of only one dTTP molecule.

Droplet Microfluidics Platform for Highly Sensitive and Quantitative Detection of Malaria-Causing Plasmodium Parasites Based on Enzyme Activity Measurement

We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/μL. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings, may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water or food quality control, or other purposes within applied or basic science.

An improved method for chromosome-specific labeling of α satellite DNA in situ by using denatured double-stranded DNA probes as primers in a primed in situ labeling (PRINS) procedure

An improved primed in situ labeling (PRINS) procedure that provides fast, highly sensitive, and nonradioactive cytogenetic localization of chromosome-specific tandem repeat sequences is presented. The PRINS technique is based on the sequence-specific annealing in situ of unlabeled DNA. This DNA then serves as primer for chain elongation in situ catalyzed by a DNA polymerase. If biotin-labeled nucleotides are used as substrate for the chain elongation, the hybridization site becomes labeled with biotin. The biotin is subsequently made visible through the binding of FITC-labeled avidin. Tandem repeat sequences may be detected in a few hours with synthetic oligonucleotides as primers, but specific labeling of single chromosomes is not easily obtained. This may be achieved, however, if denatured double-stranded DNA fragments from polymerase-chain-reaction products or cloned probes are used as primers. In the latter case, single chromosome pairs are stained with a speed and ease (1 h reaction and no probe labeling) that are superior to traditional in situ hybridization. Subsequent high-quality Q banding of the chromosomes is also possible. The developments described here extends the range of applications of the PRINS technique, so that it now can operate with any type of probe that is available for traditional in situ hybridization.

Improved detection and comparative sizing of human chromosomal telomeres in situ

Abstract. Telomeric length dynamics are thought to play an important role both in the processes of cellular aging and cancer progression. We have revised the primed in situ (PRINS) labeling technique to allow an estimation of the relative length of individual telomeres. We illustrate the applicability of the approach by demonstrating different telomeric sizes not only between blood lymphocytes from a young and an old donor, but also among bone marrow cells from hematological cancer patients. In the latter case we found general variations in telomeric sizes as well as individual telomeric variations that would have escaped detection by other methods. An interesting finding was the selective expansion of a single telomere within a specific subset of cells.

Detection of Single Enzymatic Events in Rare or Single Cells Using Microfluidics

In the present study we demonstrate highly sensitive detection of rare, aberrant cells in a population of wild-type human cells by combining a rolling-circle-enhanced enzyme activity single-molecule detection assay with a custom-designed microfluidic device. Besides reliable detection of low concentrations of aberrant cells, the integrated system allowed multiplexed detection of individual enzymatic events at the single cell level. The single cell sensitivity of the presented setup relies on the combination of single-molecule rolling-circle-enhanced enzyme activity detection with the fast reaction kinetics provided by a picoliter droplet reaction volume and subsequent concentration of signals in a customized drop-trap device. This setup allows the fast reliable analyses of enzyme activities in a vast number of single cells, thereby offering a valuable tool for basic research as well as theranostics.

p16 as a diagnostic marker of cervical neoplasia: a tissue microarray study of 796 archival specimens

BackgroundTo evaluate the usefulness of this biomarker in the diagnosis of cases of cervical neoplasia we studied the immunohistochemical expression of p16INK4a in a large series of archival cervical biopsies arranged into tissue microarray format.MethodsTMAs were constructed with tissue cores from archival formalin fixed, paraffin-embedded donor tissues from 796 patients, and included cases of cervical intraepithelial neoplasia (CIN)1 (n = 249), CIN2 (n = 233), CIN3 (n = 181), and invasive cervical carcinoma (n = 133). p16INK4a expression was scored using two different protocols: 1) positive vs negative p16INK4a staining; 2) a semi-quantitative immunohistochemical score (0 to 8 points) according to the intensity of staining and the proportion of stained cellsResultsp16INK4A expression was not seen in normal cervix tissue, but was found with increasing frequency in the sequence: CIN1 (180/249; 72.3%) – CIN2 (212/233; 91.0%) – CIN3 (178/181; 98.3%) – invasive carcinoma (131/133; 98.5%). Using semi-quantitative scoring, all normal cervical samples had low scores (from 0 to 2 points), whilst the number of specimens with high scores was proportional to the degree of cervical dysplasia or the presence of invasive carcinoma.ConclusionImmunohistochemical analysis of p16INK4a expression is a useful diagnostic tool. Expression is related to the degree of histological dysplasia, suggesting that it may have prognostic and predicative value in the management of cervical neoplasia.

Single-Molecule Detection of Human Topoisomerase I Cleavage−Ligation Activity

In the present study, we demonstrate the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion is achieved by topoisomerase I mediated closure of a nicked DNA dumbbell structure, followed by rolling circle amplification. The resulting product consists of multiple tandem repeats of the DNA dumbbell and can subsequently be visualized by annealing to fluorescently labeled probes. Since amplification involves no thermal cycling, each fluorescent rolling circle product, which gives rise to an individual signal upon microscopic analysis, will correspond to a single human topoisomerase I mediated cleavage-ligation event. Regarding sensitivity, speed, and ease of performance, the presented activity assay based on single-molecule product detection is superior to current state of the art assays using supercoiled plasmids or radiolabeled oligonucleotides as the substrate for topoisomerase I activity. Moreover, inherent in the experimental design is the easy adaptation to multiplexed and/or high-throughput systems. Human topoisomerase I is the cellular target of clinically important anticancer drugs, and the effect of such drugs corresponds directly to the intracellular topoisomerase I cleavage-ligation activity level. We therefore believe that the presented setup, measuring directly the number of cleavage-ligation events in a given sample, has great diagnostic potential, adding considerably to the possibilities of accurate prognosis before treatment with topoisomerase I directed chemotherapeutics.