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Biomedical subjects

M Samiotaki

Publications and source records attributed to M Samiotaki.

10 recordsLinked to original sources
PubMed2001

C-MYC and IGF-II mRNA-binding protein (CRD-BP/IMP-1) in benign and malignant mesenchymal tumors.

Mouse coding region determinant-binding (mCRD-BP) and human IGF-II mRNA-binding 1 (hIMP-1) proteins are orthologous mRNA-binding proteins that recognize c-myc and IGF-II mRNA, respectively, and regulate their expression posttranscriptionally. Here, we confirm that human CRD-BP/IMP-1 binds to c-myc mRNA and that it is predominantly expressed in fetal tissues. Moreover, hCRD-BP/IMP-1 expression was detected in cell lines of neoplastic origin and in selected primary tumors. In a series of 33 malignant and 10 benign mesenchymal tumors, 73% and 40%, respectively, were found to express hCRD-BP/IMP-1. In particular, expression was significant in 14 Ewing's sarcomas, all of which were positive. The data suggest that hCRD-BP/IMP-1 plays a role in abnormal cell proliferation in mesenchymal tumors.

Amino Acid Motifs↗
PubMed2000

Expression profiling across many samples via manifold-assisted mRNA processing.

Analysis of mRNA provides a condensed view of gene structure, and quantitative analyses can reveal induction of physiological or pathological gene expression programs. One of the main hurdles for routine mRNA analyses is the need to prepare large sets of samples in a rapid and standardized manner. We describe here a procedure for mRNA isolation and cDNA synthesis using manifold devices, consisting of a set of prongs that project into individual reaction wells. The prongs have a high binding capacity for the polyA-tails of mRNA and the captured mRNA is directly used to synthesize cDNA on the supports, followed by amplification. The convenience and reproducibility of the procedure allows profiling of gene expression over time, by comparing many different samples. Using the device mRNA was simultaneously isolated and accurately measured from up to 96 different samples of anywhere between 10 and 200 000 cells. The amounts of a leukemia-specific transcript could be measured when the malignant cells represented </=0.01% of the sampled cells. We illustrate the possibility of analyzing a number of tissues and monitoring expression of sets of cytokines, involved in rejection, at variable times after transplantation.

Animals↗
PubMed1997

Seven-color time-resolved fluorescence hybridization analysis of human papilloma virus types.

Identification of human papilloma virus (HPV) types is important in order to determine the risk of cervical carcinoma in women. This requires a technique to probe individual samples for multiple virus specificities. Here we describe simultaneous multicolor analysis of amplification products for any of seven amplified HPV types 16, 18, 31, 33, 35, 39, and 45, associated with cancer of the cervix. A seminested polymerase chain reaction was performed in a single tube using a biotinylated inner primer. Sets of amplification products, immobilized on a 96-pronged manifold solid support, were rendered single stranded and probed with a mix of seven type-specific, differentially labeled oligonucleotides. These probes contained 10 or 20 lanthanide chelates at the 5' ends with seven distinct combinations of europium, terbium, and samarium ions. The seven viral strains were correctly identified by time-resolved fluorescence measurement of the specifically hybridized probes. Using this assay format, simultaneous detection of any of seven or even more target variants is possible.

Female↗
PubMed1996

Detecting Genes with Ligases

The combination of synthetic oligonucleotide probes and DNA ligases is central to several recently developed genetic assays. Among the advantages of ligase-mediated gene detection is that ligation of probe pairs provides highly specific detection of unique DNA sequences in genomic samples. The technique also allows for convenient distinction between sequence variants, since mismatched bases at the junction of the probe pair prevent ligation. Moreover, the circumstance that two probes are joined into one molecule can be exploited for detection in several ways, for instance by observing the change in probe size upon ligation. Alternatively, a detectable function on one probe can be demonstrated to become linked to a retrievable function on another one through ligation. Ligation products can also be recruited as templates for subsequent ligation reactions in powerful amplification schemes. So-called padlock probes lock to their targets by encircling them, remaining in place even after denaturing washes. Here, we will describe two ligase-mediated assays: one that serves to monitor the presence of common sequence variants in amplified samples of genomic DNA and another that is suitable to detect localized gene sequences.

Journal Article↗
PubMed1994

Padlock probes: circularizing oligonucleotides for localized DNA detection.

Nucleotide sequence information derived from DNA segments of the human and other genomes is accumulating rapidly. However, it frequently proves difficult to use such short DNA segments to identify clones in genomic libraries or fragments in blots of the whole genome or for in situ analysis of chromosomes. Oligonucleotide probes, consisting of two target-complementary segments, connected by a linker sequence, were designed. Upon recognition of the specific nucleic acid molecule the ends of the probes were joined through the action of a ligase, creating circular DNA molecules catenated to the target sequence. These probes thus provide highly specific detection with minimal background.

Base Sequence↗
PubMed1994

Solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis.

Oligonucleotides labelled with detectable groups are essential tools in gene detection. We describe here the synthesis of pyrimidine deoxynucleotide-building blocks, modified at their C-5 position with a protected form of a strongly chelating agent. These reagents can be used to introduce multiple metal ions into oligodeoxynucleotides during standard oligonucleotide synthesis. The chelating functions form strongly fluorescent complexes with europium ions, characterized by a wide separation between the excitation and emission spectra. Moreover, the long decay time of the fluorescence permits sensitive time-resolved fluorescence measurements. The chelates also have the stability required to function in triple-color assays involving europium, samarium, and terbium ions. We demonstrate the application of these reagents for ligase-based gene analysis reactions.

Base Sequence↗
PubMed1994

Dual-color detection of DNA sequence variants by ligase-mediated analysis.

Genetic screening for sequence variants associated with disease is assuming increasing importance in clinical medicine as well as in research. We describe an efficient method for such analyses, comprising a combination of practical features: (1) Amplified DNA samples are analyzed for their ability to serve as templates in standardized allele-specific ligation reactions between oligonucleotide probes; (2) Two allele-specific probes, differentially labeled with either of two lanthanide labels, compete for ligation to a third oligonucleotide (the signal from the two labeled probes can thus be directly compared in a sensitive time-resolved fluorescence detection reaction); and (3) Large sets of analyses are processed in parallel using a 96-pin capture manifold, serving to reduce pipetting steps and the risk of contamination. We present here the basis of the technique and its application to the screening for two common mutations causing cystic fibrosis and alpha 1-antiytrypsin deficiency.

Base Sequence↗
PubMed1993

A manifold support for molecular genetic reactions.

Large numbers of samples can be efficiently processed through sequential reaction steps using a 96-pronged support that projects into individual microtiter wells. The support was constructed by creating a porous surface on a disposable polystyrene manifold, with avidin coupled to this greatly expanded surface. The shape and high binding capacity of the device allow the parallel transfer of large sets of reaction intermediates between different binding or enzymatic processing steps. We have applied the support to increase the efficiency of preparative and analytical molecular genetic reactions. The support also reduce the risks of sample mix-up and contamination in applications such as PCR and DNA sequencing.

Avidin↗