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Biomedical subjects

Jørn Koch

Publications and source records attributed to Jørn Koch.

12 recordsLinked to original sources
PubMed2006

A microRNA detection system based on padlock probes and rolling circle amplification.

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19-24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA.

Animals↗
PubMed2006

Single-copy and point mutation analysis (rolling-circle PRINS).

This protocol is a prototype for a series of upcoming procedures aimed at the detection of single molecules of nucleic acids in situ. It uses circular probes that, upon hybridization to their target molecule, can template rolling-circle DNA synthesis, if the target also provides a primer for initiation of the DNA synthesis. This primer may be endogenous or artificial, for example, obtained through cleavage with a restriction enzyme. In this format, the primed in situ labeling (PRINS) product takes the form of a long-tandem repeat copy of the probe, covalently attached to the site of synthesis, and tagged it with copies of the probe. The amplification is sufficient for the detection of single, oligonucleotide size, targets and, depending on the probe format, the probe may detect variations in the targets down to single base substitutions.

Cell Nucleus↗
PubMed2006

Detection and sizing of telomeric and other simple repeats by dideoxy-PRINS.

The protocol is suited for the quantitative and qualitative detection of simple repeat target DNA composed of three or fewer of the four bases A, C, G, and T. A consequence of the lacking base(s) is that such DNA can be synthesized from nucleotide mixtures containing the particular bases as dideoxynucleotides. Most genomic DNA contains all four bases and can therefore not be synthesized from such a nucleotide mixture. The combined effects of probe specificity and selective DNA synthesis from the nucleotide mixture improves the signal-to-noise ratio for such targets approximately an order of magnitude, enabling the detection of exceedingly small hybridization targets (e.g., variant telomeric repeat variants embedded in, or situated next to, the main repeat), provided that they present enough template for the DNA synthesis.

Biotin↗
PubMed2005

A different approach to telomere analysis with ddPRINS in chronic lymphocytic leukemia.

Telomeric sequences, located at the very end of the chromosomes, compensate for the chromosomal shortening as it happens after each round of cell division. Telomeric sequences influence the progress of cellular senescence and cancer progression. It has been reported that telomeres are shortened in acute leukemias where the cell turnover is high. B-cell chronic lymphocytic leukemia (CLL) is a particularly interesting haematological malignancy in regard to telomere dynamics because most of the malignant cells in CLL are mitotically inactive. In this study, we analysed the telomere length in patients with B-cell CLL in a comparison with the control group by using ddPRINS technique. Twenty patients with CLL and four healthy donors as a control group were included. We found short telomeres and no detectable telomeric repeats at the sites of chromosome fusion. We hypothesise that the telomeric erosion in CLL may reflect the dominance of malignant cells with an abnormally long life span. These cells may have encountered many antigenic stimulants in the past and hence underwent multiple clonal expansions. Our findings imply that shortened telomeres in CLL may be reflecting the "history" of the disease and serve as an independent prognostic factor.

Aged↗
PubMed2005

Recurrent genomic imbalances in B-cell splenic marginal-zone lymphoma revealed by comparative genomic hybridization.

The cytogenetics of splenic marginal zone lymphoma (SMZL) is less well characterized than the cytogenetics of other non-Hodgkin B-cell lymphomas. The aim of this study was to address this issue by identifying characteristic copy number imbalances in SMZL, for which purpose we analyzed 20 SMZL cases by comparative genomic hybridization (CGH), adding chromosome banding and fluorescence in situ hybridization (FISH) in some cases. CGH identified copy number imbalances in 70% of the cases. Imbalances were recurrently observed for chromosomes 3 (20%), 6 (20%), 7 (25%), 12 (20%), and 14 (10%). The minimally involved regions of these chromosomes were gains of 3q25 approximately qter and 12q13 approximately q15, and loss of 6q23, 7q31, and 14q22 approximately q24. A compilation of our data with data from 3 previous SMZL CGH studies revealed a significant heterogeneity between the studies. Eleven imbalances were recurrently observed in the compiled data set, as opposed to only 5 in our data set. The most frequently observed imbalances in the 73 SMZL cases of the compiled data set were gains of 3q (27%) and 12q (15%), and loss of 7q (18%). Our data suggest that SMZL constitute a genetically heterogeneous disease where gain of 3q25 and loss of 7q31 are the most likely imbalances to be involved in the pathogenesis of the disease.

Adult↗
PubMed2005

Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction.

Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.

DNA, Bacterial↗
PubMed2004

In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes.

Methods are needed to study single molecules to reveal variability, interactions and mechanisms that may go undetected at the level of populations of molecules. We describe here an integrated series of reaction steps that allow individual nucleic acid molecules to be detected with excellent specificity. Oligonucleotide probes are circularized after hybridization to target sequences that have been prepared so that localized amplification reactions can be initiated from the target molecules. The process results in strong, discrete detection signals anchored to the target molecules. We use the method to observe the distribution, within and among human cells, of individual normal and mutant mitochondrial genomes that differ at a single nucleotide position.

DNA Mutational Analysis↗
PubMed2004

A narrow deletion of 7q is common to HCL, and SMZL, but not CLL.

To further characterise the genetic background of the two closely related B-lymphocytic malignancies hairy cell leukaemia (HCL), and splenic marginal zone lymphoma (SMZL) we have identified characteristic copy number imbalances by comparative genomic hybridisation (CGH). Based on these findings, areas of special interest were fine mapped, and relevant probes constructed for use in interphase-fluorescence in situ hybridisation (FISH) investigations. Thus, using the CGH data from 52 HCL and 61 SMZL patients, we identified the characteristic profiles of copy number imbalances for both diseases. These were a gain of 5q13-31 (19%) and loss of 7q22-q35 (6%) for HCL, and gain of 3q25 (28%), loss of 7q31 (16%), and gain of 12q15 (16%) for SMZL. A partial loss of 7q unusual for low-malignant B-cell diseases was found to be common to the two diseases. This loss was therefore fine mapped with BAC/PAC clones. Fine mapping revealed that in SMZL the minimal lost region covers 11.4 Mb spanning from 7q31.33 to 7q33 located between sequence tagged site (STS)-markers SHGC-3275 and D7S725. This area was distinct from the commonly deleted 7q region of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML). A FISH probe specific for the 7q region was constructed. Using this probe in an interphase-FISH investigation we showed the minimal lost 7q-region of HCL and SMZL to be one and the same. In one HCL case, this investigation furthermore showed the extent of the deleted region to be below the detection limit of CGH, whereas interphase-FISH screening of 36 chronic lymphocytic leukaemia (CLL) cases showed no deletion of the 7q area. In conclusion, we have identified characteristic profiles of copy number imbalances in HCL and SMZL and fine mapped the minimal extent of a commonly lost 7q area of special interest. We hypothesise that this region may contain (a) gene(s) important for the pathology of HCL and SMZL.

Biopsy↗
PubMed2002

Multiple telomeric aberrations in a telomerase-positive leukemia patient.

Bone marrow samples from a pancytopenia/leukemia patient were routinely analyzed at first and second admission. At the first presentation, the karyotype was normal, whereas 17 months later several chromosome aberrations were recognized including presumed additions to the short arms of chromosomes 1 and 16 in all cells, and numerous other aberrations in subpopulations of cells. From the predominance of aberrations at chromosome ends, we suspected insufficient telomere maintenance as an underlying mechanism behind the karyotype changes, in particular as an interstitial deletion in the region harboring the gene for the RNA component (hTERC) of the telomerase enzyme was also noticed; however, while molecular cytogenetic investigation confirmed the terminal aberrations, we found the malignant cells positive for telomerase activity and the presence of an hTERC gene on both chromosomes 3. A presumed chromosome 1 addition turned out to reflect an amplification of a tandemly repeated sequence element. Labeling of multiple tandem repeat sequences in situ by a novel multicolor primed in situ hybridization showed no evidence of instability of other repeated DNA elements.

Adult↗
PubMed2002

Active, but not inactive, human centromeres display topoisomerase II activity in vivo.

Eukaryotic centromeres are composed of centromere DNA and the multiple proteins directly or indirectly associated with it. One important DNA-binding protein in the centromere is DNA topoisomerase II (topo II). In the genome in general, topo II has two functions, one structural and one enzymatic, the latter catalyzing DNA strand-passage reactions. It has been demonstrated that topo II accumulates at centromeres during the first part of mitosis, and disappears again at anaphase, but it has not been clear whether it serves a structural or an enzymatic function at the centromere. To investigate this issue, we developed the topo II-induced self-primed in situ assay (Topo-SPRINS). In this assay, DNA breaks created by topo II are stabilized with the topo II inhibitor VM-26 in vivo, and used as 'primers' for localized DNA synthesis in vitro. The assay revealed that topo II has enzymatic activity at mitotic centromeres and that the activity is relatively constant across centromeres. Furthermore, the activity was observed at a neocentromere, and, in multicentric chromosomes, the activity was restricted to the active centromere. The topo II activity is thus selectively present at functioning centromeres, indicating that it plays a role in mitotic centromere function.

Centromere↗
PubMed2002

Extensive cytogenetic analysis of a stable dicentric isochromosome 21, idic(21), formed by fusion of the terminal long arms.

The dicentric isochromosome 21 described in this paper was formed by fusion of the terminal parts of the long arms of two chromosomes 21. No interstitial telomeric AGGGTT repeats could be detected at the fusion point, but G-banding, comparative genomic hybridization, and fluorescence in situ hybridization with painting probes for 21qter revealed no loss of other terminal DNA sequences at the fusion point. Thus, only the telomeric repeats seem to have been lost prior to, or as a consequence of, isochromosome formation. Both short arms of the isochromosome were intact with complete NORs, and staining for alpha-satellite DNA showed that the DNA content of the two centromeres was the same. Antibody staining for the centromeric proteins CENP-C and CENP-E and for topoisomerase IIalpha and IIbeta demonstrated that these proteins were localized predominantly or exclusively at the centromere in the primary constriction. A novel functional in situ assay for topoisomerase activity in vivo similarly demonstrated enzyme activity exclusively at the primary constriction centromere.

Antigens, Neoplasm↗