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Results for “Primed In Situ Labeling”

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PubMed2001

Asymmetrical segregation of chromosomes with a normal metaphase/anaphase checkpoint in polyploid megakaryocytes.

During differentiation, megakaryocytes increase ploidy through a process called endomitosis, whose mechanisms remain unknown. As it corresponds to abortive mitosis at anaphase and is associated with a multipolar spindle, investigation of chromosome segregation may help to better understand this cell-cycle abnormality. To examine this variation, a new method was developed to combine primed in situ labeling to label centromeres of one chromosome category and immunostaining of tubulin. Human megakaryocytes were obtained from normal bone marrow culture. By confocal microscopy, this study demonstrates an asymmetrical distribution of chromosomes (1 or 7) either between the spindle poles at anaphase stage of endomitosis and between the different lobes of interphase megakaryocyte nuclei. The metaphase/anaphase checkpoint appears normal on the evidence that under nocodazole treatment megakaryocytes progressively accumulate in pseudo-metaphase, without spontaneous escape from this blockage. Immunostaining of p55CDC/hCDC20 with similar kinetochore localization and dynamics as during normal mitosis confirms this result. HCdh1 was also expressed in megakaryocytes, and its main target, cyclin B1, was normally degraded at anaphase, suggesting that the hCdh1-anaphase-promoting complex checkpoint was also functional. This study found the explanation for these unexpected results of an asymmetrical segregation coupled to normal checkpoints by careful analysis of multipolar endomitotic spindles: whereas each aster is connected to more than one other aster, one chromosome may segregate symmetrically between 2 spindle poles and still show asymmetrical segregation when the entire complex spindle is considered.

Anaphase↗

PubMed2000

Chromosomal localization of 18S rDNA and telomere sequence in the aye-aye, Daubentonia madagascariensis.

Chromosomal localization of 18S rDNA and telomere sequence was attempted on the chromosomes of the aye-aye (2n = 30) using fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS), respectively. The rDNA was localized at the tip or whole of the short arm of acrocentric chromosomes 13 and 14 in all spreads observed. However, post-FISH silver-nitrate (Ag) staining showed that transcriptional activity of the rRNA genes was variable, particularly in chromosome 14, which was most frequently negative in one homologue carrying the smaller copy number of rDNA. This observation supports, at the molecular cytogenetic level, previous data concerning the relationship between the copy number of rDNA and its trancriptional activity. On the other hand, telomere sequence was localized only at the telomeric region of all chromosomes, the so-called telomere-only pattern, a characteristic similar to that of the greater bushbaby. These data may provide information on the chromosomal evolution of the lemur, because locations of rDNA and telomere sequences frequently offer important clues in reconstruction of karyotype differentiation.

Animals↗

PubMed2001

Strategies for signal amplification in nucleic acid detection.

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.

DNA Probes↗

PubMed1997

[Study on platelet-derived growth factor mRNA and copper-zinc superoxide dismutase mRNA expression changes of aortic artery endothelium with restenosis after aortic artery dilatation treated with buyang huanwu decoction].

OBJECTIVE: To study on dynamic mechanism of restenosis after PTCA treated with Buyang Huanwu Decoction (BYHWD). METHODS: Twenty-eight male New Zealand rabbits with mimic restenosis after aortic artery dilatation were detected by prime in situ labeling in aortic endothelium. The items included gene expression of copper zinc superoxide dismutase (SOD-1) and platelet-derived growth factor receptor (PDGFR) mRNA to analyse dynamic mechanism of restenosis. RESULTS: Seven days after aortic artery dilatation, PDGFR mRNA of aortic endothelium decreased clearly. The comparison between experimental and the control group showed significant difference (P < 0.05). By the same condition, the gene expression of SOD-1 was increasing, which showed a significant difference with the control group (P < 0.05). CONCLUSIONS: BYHWD increased SOD-1 gene expression of artery endothelium, to improve oxygen feed for ischemia reperfusion in the same way, BYHWD suppressed PDGFR mRNA expression to control cellular pathological proliferation.

Angioplasty, Balloon, Coronary↗

PubMed2004

Comparison of three methods for the gene analysis of fetal cells from maternal peripheral blood.

BACKGROUND: Although great advances in techniques for noninvasive prenatal diagnosis using fetal cells from maternal peripheral blood have achieved, current technology does not meet the demands required for clinical use. In this study, we aimed to establish reliable methods for the gene analysis of fetal cells from maternal peripheral blood. METHODS: Primed extension preamplification (PEP)-polymerase chain reaction (PCR), multiple primed in situ labeling (PRINS), and nested PCR were individually applied to detect the sex determining region Y (SRY) gene in single fetal cells collected from maternal peripheral blood. RESULTS: The sensitivity and specificity of the detection of the SRY gene by PEP-PCR were 97.39% (149/153) and 99.17% (119/120), respectively. The sensitivity and specificity of PRINS were 97.56% (40/41) and 100% (35/35), respectively. The sensitivity and specificity of nested-PCR were 80.00% (24/30) and 87.50% (14/16), respectively. CONCLUSIONS: PEP-PCR and PRINS are reliable techniques for the gene analysis of single fetal cells from maternal peripheral blood because of their high sensitivity and specificity. PEP-PCR and PRINS can be used as standard methods of noninvasive prenatal diagnosis using single fetal cells from maternal peripheral blood.

Adult↗

PubMedDate not supplied

[Molecular cytogenetic techniques and their application in clinical diagnosis].

Cytogenetics analysis is at present the basic element of the diagnostic process of genetic disorders which are caused by chromosomal abnormalities. Since the chromosome banding technique has been introduced in the 1970s, it has been available as a diagnostic tool of a number of clinical syndromes. It enabled to prove the causal association between specific chromosomal abnormalities and clinical features observed in patients. However, since banding resolution is not always sufficient for the identification of chromosomal abnormalities, additional techniques for solving diagnostic dilemma of classical cytogenetics are needed. A new field of cytogenetics -- molecular cytogenetics, the product of a combination of cytogenetics and molecular biology, has increased the resolution and diagnostic utility of cytogenetic analysis. The basic method of molecular cytogenetics is fluorescence in situ hybridization (FISH). It enables a specific detection of unique sequences, chromosomal regions or entire chromosomes in metaphase, interphase cells or in tissue sections. In this article FISH technique and its modifications such as multicolor FISH (M-FISH, SKY, CCK), Primed In Situ Labelling (PRINS) and Comparative Genomic Hybridization (CGH) are presented. The range of applications and the use of these techniques for the identification of chromosomal abnormalities in relation to diagnostic possibilities of the classical methods of karyotyping is also discussed.

Chromosome Aberrations↗

PubMed1994

Fluorescence in situ hybridization in cancer and radiation biology.

This review describes molecular cytogenetic approaches to genetic analysis including fluorescence in situ hybridization, primed in situ labeling and comparative genomic hybridization. It also summarizes the applications of this technology to physical mapping, cancer diagnosis and prognostication and in radiation biology.

Animals↗

PubMed1997

Detection of rare RNA sequences by single-enzyme in situ reverse transcription-polymerase chain reaction. High-resolution analyses of interleukin-6 mRNA in paraffin sections of lymph nodes.

To study the distribution pattern of interleukin-6 (IL-6)-producing cells in normal human lymph nodes, we applied the in situ reverse transcription-polymerase chain reaction technique. We describe a new modification of this technique for monitoring small amounts of specific nucleotide sequences in conventional paraffin sections. This technique differs in at least two respects from those described earlier. The two decisive steps are: 1) the reverse transcription of mRNA and the subsequent amplification of cDNA by polymerase chain reaction are performed by a new single enzyme capable of both reaction types in one and the same medium without buffer exchange; and 2) for the specific detection of the amplified cDNA, a modified version of the primed in situ labeling technique was used. The technique, carried out on normal human lymph nodes, traces a low load of IL-6 mRNA in fibroblasts, endothelial cells, and a minor population of T lymphocytes in the pulp region. High levels of expression were encountered in about 20% of perisinusoidal pulp macrophages. In addition, moderate activity was detectable in sinus lining cells. Because no major activity was found in the germinal centers of the lymphoid B follicles and in the T zone, it is suggested that the plasma cell differentiation ensuing from primary and secondary B-cell immunization is mainly effected by the sinus lining cells as well as perifollicular and perisinusoidal pulp macrophages capable of producing high amounts of IL-6.

Humans↗

PubMed1996

[Fetal erythroblasts from maternal blood for prenatal genetic diagnosis].

OBJECTIVE: To study fetal erythroblasts (FE) from maternal peripheral blood for the diagnosis of fetal aneuploidies. METHODS: FE expressing the glycophorin A(GPA) were isolated from 13 pregnant women with male fetus (8-14 w) by fluorescence-activated cell sorting(FACS), FE were identified by oligonucleotide primed in situ labelling (PRINS) with Y centromeric satellite DNA primer. The concentration of pregnancy-associated plasma protein A (PAPP-A) was measured by enzyme-labelled immunosorbent assay (ELISA) in serum samples of 41 normal pregnant women (8-14 w). In 5 pregnant women suspicious of fetal Down's syndrome (10-13 w) the serum and FE were examined by PAPP-A, GPA/FACS and PRINS with 21 chromosome centrometric primer. RESULTS: Detection of flow sorted FE from 13 pregnant women by Y primer showed 14.5% of GPA positive signal. There was no difference in serum level of PAPP-A between 5 pregnant women and 41 normal controls, and all GPA positive cell nuclei of the 5 cases displayed two signals with 21 chromosome. CONCLUSION: Measurement of fetal erythroblasts from maternal blood for the diagnosis of genetic fetal aneuploidies is a promising non-invasive, rapid and reliable technique.

Adult↗

PubMed2003

Sequence subfamilies of satellite repeats related to rDNA intergenic spacer are differentially amplified on Vicia sativa chromosomes.

We cloned and sequenced the Vicia sativa 25S-18S rDNA intergenic spacer (IGS) and the satellite repeat S12, thought to be related to the spacer sequence. The spacer was shown to contain three types of subrepeats (A, B, and C) with monomers of 173 bp (A), 10 bp (B), and 66 bp (C), separated by unique or partially duplicated sequences. Two spacer variants were detected in V. sativa that differed in length (2990 and 3168 bp) owing to an extra copy of the subrepeat A. The A subrepeats were also shown to be highly homologous to the satellite repeat S12, which is located in large clusters on chromosomes 4, 5, and 6, and is not associated with the rDNA loci. Sequencing of additional S12 clones retrieved from a shotgun genomic library allowed definition of three subfamilies of this repeat based on minor differences in their nucleotide sequences. Two of these subfamilies could be discriminated from the rest of the S12 sequences as well as from the IGS A subrepeats using specific oligonucleotide primers that labeled only a subset of the S12 loci when used in the primed in situ DNA labeling (PRINS) reaction on mitotic chromosomes. These experiments showed that, in spite of the high overall similarity of the IGS A subrepeats and the S12 satellite repeats, there are S12 subfamilies that are divergent from the common consensus and are present at only some of the chromosomes containing the S12 loci. Thus, the subfamilies may have evolved at these loci following the spreading of the A subrepeats from the IGS to genomic regions outside the rDNA clusters.

Base Sequence↗

PubMed1998

[Application of the PRINS technique for chromosome examination in fetal cells present in maternal blood].

Both detection and chromosomal analysis of fetal cells present in the maternal circulation can be performed using Histopaque double density gradient centrifugation followed by primed in situ (PRINS) labeling technique. This approach has been tested on blood samples from 15 pregnant women and 6 control donors with primers specific for chromosomes 9, X and Y. The cell separation technique allows recovery of both mononuclear cells and polynuclear cells with a 97% efficiency. PRINS labeling was successful in 100% cells from control blood samples. Among patient samples, 2 "false-negative" results were observed. These preliminary results suggest that the present protocol might be efficient for non-invasive prenatal chromosome analysis.

Case-Control Studies↗

PubMed2002

Rapid chromosome detection by PRINS in human sperm.

Primed in situ (PRINS) labeling may be used for direct estimation of disomy rate in human sperm. We combined the PRINS procedure with a rapid and efficient NaOH pretreatment allowing simultaneous decondensation and denaturation of sperm nuclei. This enabled double labeling of human sperm within a 2-hr time span. Chromosome-specific primers have been defined for most human chromosomes, including those most frequently involved in aneuploidy. Using the dual-color PRINS method, we estimated incidences of disomy for 15 autosomes and the sex chromosomes in sperm samples from 6 normal fertile men. The frequency of disomy ranged from 0.28% to 0.36%. There were no significant interchromosomal differences in disomy rate, but chromosome 21 displayed a higher incidence of nondisjunction than did the other chromosomes.

Base Sequence↗

PubMed2006

PRINS and C-PRINS: promising tools for the physical mapping of the lupin genome.

Two molecular cytogenetics methods, PRINS (primed in situ DNA labeling) and C-PRINS (cycling PRINS), were optimized for the physical mapping of several types of DNA sequences on the mitotic chromosomes of the narrow-leafed lupin (Lupinus angustifolius L.). The fragment of the FokI element from Vicia faba was localised by indirect PRINS reaction. Two other sequences, fragments of the coding sequences of L. luteus and of L. angustifolius, were localised by indirect C-PRINS. These techniques are faster and more sensitive than FISH, and they allowed the mapping of short DNA fragments. The data obtained shows that both types of PRINS are valuable tools for chromosome identification in lupin.

Chromosomes, Plant↗

PubMed1998

Assessment of chromosomal heterogeneity in tumoral cell lines using PRINS technique.

The primed in situ (PRINS) labeling technique has been used for the interphase cytogenetic investigation of 3 colon cancer cell lines (Caco-2, TC7 and PF1 1) derived from a same primary tumor. A panel of 10 chromosome-specific primers (for chromosomes 1, 2, 3, 7, 8, 9, 10, 13, 16 and 18) has been utilized in simple and double color PRINS reactions. Each cell line displayed a heterogeneous distribution of copy number for several chromosomes. The karyotypic heterogeneity was also significant between the 3 cell lines. These data indicate the common occurrence of chromosome heterogeneity in tumoral cell lines and demonstrate the feasibility of interphase PRINS procedure for analysis of numerical changes in tumoral cells.

Chromosomes, Human↗

PubMed2000

Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS.

BACKGROUND: Flow-sorted plant chromosomes are being increasingly used in plant genome analysis and mapping. Consequently, there is a need for a rapid method for identification of sorted chromosomes and for determination of their purity. We report on optimization of procedures for primed in situ DNA labeling (PRINS) and cycling-PRINS (C-PRINS) for fluorescent labeling of repetitive DNA sequences on sorted plant chromosomes suitable for their identification. METHODS: Chromosomes of barley, wheat, and field bean were sorted onto microscope slides, dried, and subjected to PRINS or C-PRINS with primers for GAA microsatellites (barley and wheat) or FokI repeat (field bean). The following parameters were optimized to achieve the highest specificity and intensity of fluorescent labeling: ratio of labeled versus unlabeled nucleotides, nucleotide concentration, and the number and concentration of primers. RESULTS: Under optimal conditions, C-PRINS resulted in strong and specific labeling of GAA microsatellites on sorted barley and wheat chromosomes and FokI repeats on sorted field bean chromosomes. The labeling patterns were characteristic for each chromosome and permitted their unequivocal identification as well as determination of purity after sorting, which ranged from 96% to 99%. A standard polymerase chain reaction (PCR) with chromosome-specific primers was not sensitive enough to detect low-frequency contamination. CONCLUSIONS: The results indicate that a single C-PRINS assay with primers that give chromosome-specific labeling pattern is sufficient not only to determine chromosome content of peaks on flow karyotype but also to determine the purity of sorted chromosome fractions. The whole procedure can be performed in less than 3 h on the next day after sorting. Numerous applications are expected in the area of plant flow cytogenetics.

Chromosomes↗

PubMed2000

PRINS analysis of the telomeric sequence in seven lemurs.

We examined the chromosomal localization of the telomeric sequence, (TTAGGG)n, in seven species of the lemurs and one greater galago, as an outgroup, using the primed in-situ labeling (PRINS) technique. As expected, the telomeric sequence was identified at both ends of all chromosomes of the eight prosimians. However, six species showed a signal at some pericentromeric regions involving constitutive heterochromatin as well. The pericentromeric region of chromosome 1 of Verreaux's sifaka (Propithecus verreauxi verreauxi) was labeled with a large and intense signal. The range of the signal considerably exceeded the area of DAPI positive heterochromatin. On the other hand, in the five lemurs, a large signal was detected also in the short arm of acrocentric chromosomes. Acquisition of the large block of the telomeric sequence into the acrocentric short arm might be interpretable in terms of the tandem growth of the heterochromatic short arm and the reciprocal translocation between heterochromatic short arms involving the telomeric sequence. Subsequently, it was postulated that meta- or submetacentric chromosomes possessing the telomeric sequence at the pericentromeric region could be formed by centric fusion between such acrocentric chromosomes.

Animals↗

PubMed1999

Chromosomal localization of 5S rDNA genes in Leporinus fish (Anostomidae, Characiformes).

The large 45S rDNA chromosome sites have often been analyzed in fish. In contrast, little is known about the 5S genes in this animal group. In the genus Leporinus, the NOR chromosomal location has been shown to be very diverse. In the present work, chromosome mapping of 5S rDNA in three anostomids, Leporinus elongatus, L. obtusidens and L. friderici, is investigated using fluorescence in-situ hybridization (FISH) with PCR-obtained 5S probes and primed in-situ labeling (PRINS). Major 5S rDNA chromosomal sites were found to be subterminally located in a small metacentric pair, while minor ones were detected near the centromeric region of a medium-sized submetacentric pair in all studied species. The 5S rDNA genes were not associated with the NORs or sex chromosomes. A highly conserved chromosomal location of these genes appears to characterize the karyotype evolution of this fish group.

Animals↗

PubMed2003

Characterization of the atypical karyotype of the black-winged kite Elanus caeruleus (Falconiformes: Accipitridae) by means of classical and molecular cytogenetic techniques.

The karyotype of the black-winged kite (Elanus caeruleus), a small diurnal raptor living in Africa, Asia and southern Europe, was studied with classical (G-, C-, R-banding, and Ag-NOR staining) and molecular cytogenetic methods, including primed in-situ labelling (PRINS) and fluorescence in-situ hybridization (FISH) with telomeric (TTAGGG) and centromeric DNA repeats. The study revealed that the genome size, measured by flow cytometry (3.1 pg), is in the normal avian range. However, the black-winged kite karyotype is particularly unusual among birds in having a moderate diploid number of 68 chromosomes, and containing only one pair of dot-shaped microchromosomes. Moreover, the macrochromosomes are medium-sized, with the Z and W gonosomes being clearly the largest in the set. C-banding shows that constitutive heterochromatin is located at the centromeric regions of all chromosomes, and that two pairs of small acrocentrics and the pair of microchromosomes are almost entirely heterochromatic and G-band negative. The distribution pattern of a centromeric repeated DNA sequence, as demonstrated by PRINS, follows that of C-heterochromatin. The localization of telomeric sequences by FISH and PRINS reveals many strong telomeric signals but no extratelomeric signal was observed. The atypical organization of the karyotype of the black-winged kite is considered in the context of the modes of karyotypic evolution in birds.

Animals↗