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Results for “Primed In Situ Labeling”

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PubMedDate not supplied

Heteromorphism 18ph+ : with or without reproductive consequences?

Heteromorphism or chromosomal variants are usually attributed to structural variations in constitutive heterochromatin. In the case of chromosome 18, 25 cases of 18ph+ have been reported to date. Using the Primed In Situ Labelling technique (PRINS) to study 2 new cases of 18ph+, we have been able to confirm their molecular nature and assuming a mechanism of formation. Although such chromosomal variants are usually thought to have no adverse clinical consequence, a review of the literature shows that many cases were diagnosed because of recurrent abortion, malformed or mentally retarded children suggesting the possible relationship between 18ph+ and such clinical outcomes.

Amniotic Fluid↗

PubMed1999

Recycling of a single human blastomere fixed on a microscopic slide for sexing and diagnosis of specific mutations by various types of polymerase chain reaction.

OBJECTIVE: To investigate the suitability of recycling single blastomeres to assess multiple genetic variables for preimplantation genetic diagnosis. DESIGN: Prospective randomized study. SETTING: An academic medical center. PATIENT(S): Patients undergoing IVF-ET. INTERVENTION(S): Blastomeres were disaggregated from donated embryos obtained from patients. MAIN OUTCOME MEASURE(S): Polymerase chain reaction (PCR) amplification products. RESULT(S): Fifty-eight blastomeres individually fixed on slides were separated into four groups. Sequential PCRs (group I, n = 30), primed in situ labeling (PRINS) before five sequential PCRs (group II, n = 10), staining with hematoxylin before performing five sequential PCRs (group III, n = 11) and preamplification of whole DNAs by degenerate oligonucleotide primer (DOP) before performing PCR were executed. The amplification efficiencies of five sequential PCRs were 100%, 100%, 96.6%, 83.3%, 56.7% for group I; 100% 100%, 100%, 80%, 40% for group II; 54.5%, 36.4%, 18.2%, 9.1% for group III; and 100%, 100%, 100%, 100%, 100% for group IV. CONCLUSION(S): Blastomeres fixed for PRINS can be recycled for PCR to obtain more genetic information. Hematoxylin staining appears to increase the incidence of failed amplification. Preamplification of whole genomic DNAs by DOP-PCR appears to facilitate diagnosis with high efficiency.

Blastomeres↗

PubMed1997

Cycling-PRINS. A method to improve the accuracy of telomeric sequence detection in mammalian chromosomes.

The presence of (TTAGGG)n telomeric sequence was investigated in a Chinese hamster tumor-derived cell line, using single primed in situ labelling (single-PRINS) and multiple cycles of amplification (cycling-PRINS). The telomeric sequence hybridized the centromere of most chromosomes, using both techniques. However, signals visible at the telomeric regions of some chromosomes and an enhancement of the frequency signals at the centromere of chromosome 1 were obtained using cycling-PRINS. These results indicate that cycling-PRINS represents a promising improvement for detection of telomeric sequence in cell lines where the conventional methods failed to demonstrate their presence.

Animals↗

PubMed2001

Tetrasomy 8 is associated with a major cellular proliferative advantage and a poor prognosis. two cases of myeloid hematologic disorders and review of the literature.

We report two cases of acute myeloid leukemia (AML) with tetrasomy 8 detected in patients' bone marrow samples using chromosome GTG-banding, fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) techniques. Case 1 was a myelodysplastic syndrome (MDS) in transition to AML-M4 and case 2 was an AML-M2. In case 1, the tetrasomy 8 was found in 40% of metaphase cells and constituted the only chromosome abnormality. In case 2, it was accompanied by a double Ph, trisomy 18 and disomy Y and was found in 68% of metaphase cells. However, FISH and PRINS techniques revealed the coexistence of tetrasomy 8 and trisomy 8 in interphase nuclei of both cases. When the proportion of cells with tetrasomy 8 was compared between metaphases and interphase nuclei, it showed a much higher percentage of cells with tetrasomy 8 in metaphases than in interphase nuclei. Moreover, in case 2, although multi-PRINS and FISH-PRINS techniques showed other populations of interphase nuclei with different combinations of chromosome anomalies with respect to the copy numbers for chromosomes 8, 18, Y and Ph, only cells that contained either a single Ph or tetrasomy 8 plus trisomy 18, disomy Y, and double Ph could be seen in metaphases. This strongly suggests that tetrasomy 8 confers a higher proliferative advantage to cells. Our cases also show that the tetrasomy 8 is associated with a poor prognosis.

Aged↗

PubMed2003

A unique clone involving multiple structural chromosome rearrangements in a myelodysplastic syndrome case.

In a young female patient presenting with a myelodysplastic syndrome (MDS), a unique clone involving six structural chromosome rearrangements was identified using G-banding and molecular cytogenetic techniques. Fifty GTG-banded metaphases from bone marrow were initially analyzed and all metaphases contained all of the six structural chromosome rearrangements. To further define the GTG-banded karyotype, a series of fluorescence in situ hybridization and primed in situ labeling experiments were performed and the karyotype was then characterized as: 46,XX,r(5)(p13q13),der(20)t(5;20),dup(11)(p11.2p15), r(11)(p15q25),del(13)(q14),idic(22)(p11). The patient quickly progressed to acute nonlymphocytic leukemia three months after the diagnosis and died of a hemorrhage in the brain parenchyma two months later. In this case, the multiple structural chromosome rearrangements conferred an obvious cellular proliferative advantage and indicated a very poor prognosis. Considering that multiple chromosome abnormalities associated with MDS transformation are often polyclonal, this unique clone involving six structural chromosome rearrangements make our case highly unusual.

Adult↗

PubMed1997

Using PRINS for gene mapping in polytene chromosomes.

We have adapted the primed in situ labelling (PRINS) protocol for gene mapping in polytene chromosomes of two dipteran species. The method was used to localize the genes for the Balbiani ring (BR) 2.1 and the iron-regulatory protein 1A (IRP1A) in polytene salivary gland chromosomes of Chironomus tentans, and Drosophila melanogaster respectively. Two oligonucleotides, corresponding to the BR 2.1 and IRP1A genes, were used as primers and the whole procedure was performed within 3-4 h. The strong labelling with low background revealed the localization of the BR 2.1 gene in polytene chromosome IV of C. tentans and the IRP1A gene in polytene chromosome 3R83 of D. melanogaster. The results demonstrated that PRINS is a fast, sensitive and suitable approach for physical gene mapping in polytene chromosomes.

Animals↗

PubMed2001

Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques.

Different types of nucleated fetal cells (trophoblasts, erythroblasts, lymphocytes, and granulocytes) have been recovered in maternal peripheral blood. In spite of many attempts to estimate the number of fetal cells in maternal circulation, there is still much controversy concerning this aspect. The numbers obtained vary widely, ranging from 1 nucleated cell per 104 to 1 per 109 nucleated maternal cells. The purpose of our project was to determine the absolute number of all different types of male fetal nucleated cells per unit volume of peripheral maternal blood. Peripheral blood samples were obtained from 12 normal pregnant women known to carry a male fetus between 18 and 22 weeks of pregnancy. Three milliliters (3 ml) of maternal blood has been processed without any enrichment procedures. Fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) were performed, and fetal XY cells were identified (among maternal XX cells) and scored by fluorescent microscopy screening. The total number of male fetal nucleated cells per milliliter of maternal blood was consistent in each woman studied and varied from 2 to 6 cells per milliliter within the group of normal pregnancies. The number of fetal cells in maternal blood, at a given period, is reproducible and can therefore be assessed by cytogenetic methods. This confirms the possibility of developing a non-invasive prenatal diagnosis test for aneuploidies. Furthermore, we demonstrate that it is possible to repeatedly identify an extremely small number of fetal cells among millions of maternal cells.

Adult↗

PubMedDate not supplied

Detection of a concomitant distal deletion in an inverted duplication of chromosome 3. Is there an overall mechanism for the origin of such duplications/deficiencies?

We describe the first inverted duplication of the p21.3p26 region of chromosome 3 in a child with phenotypic features of the trisomy 3p syndrome. This uncommon type of aberration was verified by multicolour fluorescence in situ hybridisation (FISH) using yeast artificial chromosome (YAC) clones from chromosome 3 (CEPH library). With a newly constructed YAC clone from the 3p26 region an unexpected subtelomeric deletion was diagnosed in the aberrant chromosome 3. Using the primed in situ labelling (PRINS) method, telomeres were found to be present on the recombinant chromosome 3. The repeated appearance of concomitant distal deletions in inverted duplications suggests that an overall mechanism exists for the origin of such duplications/deficiencies.

Chromosome Aberrations↗

PubMedDate not supplied

Soybean chromosome painting: a strategy for somatic cytogenetics.

Cytological identification of soybean mitotic metaphase chromosomes (2n = 40) has been severely limited by their small size and uniform karyomorphology. We have developed fluorescent in situ hybridization (FISH), PCR-primed in situ labelling (PCR-PRINS) procedures, and molecular probes for routine cytological identification and for the physical mapping of soybean somatic chromosomes. Chromosome preparation has been achieved by modifications of previous protocols and through the preparation of root-tip protoplasts prior to chromosome spreading. Initially our probe selection focused on highly repeated DNAs that provide very intense localized hybridization signals. Repetitive gene probes that have proven valuable include the rDNA loci (5S and 45S) which are chromosome specific. We have also developed satellite DNA probes for two different sequence families: the SB92 and the STR120 satellites. Both of these are tandemly arranged at multiple chromosomal loci. By using different cloned examples of each family, we have been able to selectively label unique subsets of soybean chromosomes. Double hybridization with biotin and digoxigenin labeled probes has allowed us to determine the chromosomal overlap between different probes. In addition, we have joined portions of the metaphase chromosome painting patterns with the genetic map by single-copy FISH and PCR-PRINS detection of the RFLP loci G8.15, G17.3, and A199a and A199b. Total genomic DNA in situ hybridization (GISH) patterns were also used to characterize the soybean chromosomes.

Chromosome Mapping↗

PubMed1997

Low frequency of microsatellites in the avian genome.

A better insight into the occurrence of microsatellites in a range of taxa may help to understand the evolution of simple repeats. Previous studies have found the relative abundance of several repeat motifs to differ among mammals, invertebrates, and plants. Absolute numbers of microsatellites also tend to correlate positively with genome size. We analyzed the occurrence, frequency, and distribution of microsatellites in birds, a taxon with one of the smallest known genome sizes among vertebrates. Dot-blot hybridization revealed that about half of 22 different di-, tri-, and tetranucleotide repeat motifs were clearly more common in human than in three species of birds: chicken, woodpecker, and swallow. For the remaining motifs no clear difference was found. From searching avian database sequences we estimated there to be 30,000-70,000 microsatellites longer than 20 bp in the avian genome. The number of (CA) > or = 10 would be around 7000-9000 and the number of (CA) > or = 14 about 3000. The calculated density of avian microsatellites (total, one every 20-39 kb; (CA) > or = 10, one every 136-150 kb) is much lower than that estimated for the human genome (one every 6 and 30 kb, respectively). This may be explained by the fact that the avian genome contains relatively less noncoding DNA than most mammals and that avian SINE/LINE elements do not terminate in poly(A) tails, which are known to provide a resource for the evolution of simple repeats in mammals. We found no association between microsatellites and SINEs in birds. Primed in situ labeling suggested fairly even distribution of (CA)n repeats over chicken macrochromosomes and intermediate chromosomes, whereas the microchromosomes, a large part of the Z and W chromosomes, and most telomeres and centromeres had very low concentrations of (CA)n microsatellites. The scarcity of microsatellites on the microchromosomes is compatible to these regions likely being unusually rich in coding sequences. The low microsatellite density in the genome in general and on the microchromosomes in particular imposes an obstacle for the development of marker-rich genetic maps of chicken and other birds, and for the localization of quantitative trait genes.

Animals↗

PubMed1993

Ring chromosome 20 with loss of telomeric sequences detected by multicolour PRINS.

A ring chromosome 20 in a male infant with epileptic seizures, mental and somatic growth retardation, and behavioural disturbances is described. Conventional cytogenetics revealed the karyotype to be 46,XY,r(20)(pter-->qter) and no signs of mosaicism were found. Fluorescence in situ hybridisation using the clone p20Z1 identified the ring to be derived from chromosome 20. By counting 111 metaphases, only 7% were found to be missing the ring. The absence of telomeric sequences in the ring chromosome was demonstrated by multicolour PRINS: a three-step PRimed IN Situ labelling technique, using unlabelled primers. A terminal deletion of both arms thus seems to be the cause of the ring formation in the proband. Bivariate flow-analysis of chromosomes verified a deletion of the ring chromosome. The clinical and cytogenetic findings are compared with previous cases. A specific ring 20 syndrome seems justified.

Abnormalities, Multiple↗

PubMed1993

Identification of a supernumerary der(18) chromosome by a rational strategy for the cytogenetic typing of small marker chromosomes with chromosome-specific DNA probes.

A case of a supernumerary der(18) marker chromosome is presented. The chromosomal origin of the marker chromosome was not evident by traditional chromosome analysis, but was determined by PRimed IN Situ labelling (PRINS) with chromosome specific centromere probes as primers for chain elongation in situ. For this purpose a strategy was developed which, in a few simple reactions, makes it possible unequivocally to determine the origin of any small marker chromosome. The approach does not require any hints about the origin of the chromosome prior to the analysis, since the chromosomal origin of the marker is established through PRINS reactions with pooled and single chromosome-specific centromere probes. Identification, mosaic screening and structural analysis require a total of 8-9 such reactions and may, due to the extreme speed of the PRINS reaction, be obtained within a single working day.

Chromosome Aberrations↗

PubMed2006

Quantification of all fetal nucleated cells in maternal blood in different cases of aneuploidies.

We quantified all fetal nucleated cells (FNCs) per unit volume of maternal blood in different aneuploid pregnancies using molecular cytogenetic techniques. Seven cases of male trisomy 18, two triploidies (69,XXX), two 47,XXX, one 47,XXY, one 47,XYY, one male trisomy 13, and one case of 47,XY,r(22),+r(22) were analyzed. Whole blood samples were obtained from 15 women between 17 and 29 gestational weeks and harvested without using fetal cell enrichment procedures. Fluorescence in situ hybridization and primed in situ labeling were performed to identify the FNCs. All slides were manually scanned to quantify those cells. We have identified 4-20 FNCs/ml of maternal blood in the cases of trisomy 18; 10 and 25 FNCs/ml in the two cases of triploidy; 16 and 14 FNCs/ml, respectively, in the two X trisomies; 19 FNCs/ml in the 47,XXY; 26 FNCs/ml in the 47,XYY; nine FNCs/ml in the trisomy 13; and 10 FNCs/ml in the case of r(22). To detect all FNCs in all aneuploid pregnancies, we have used a very simple method that minimizes the manipulation steps to avoid losing fetal cells. The number of FNCs identified in aneuploid pregnancies was 2-5 times higher than in normal pregnancies. This higher number of FNCs will favor the design of a non-invasive pre-natal test.

Adult↗

PubMed2001

Centromeric tandem repeat from the chaffinch genome: isolation and molecular characterization.

A new family of avian centromeric satellites is described. The highly repeated sequence, designated FCP (Fringilla coelebs PstI element), was cloned from the 500-bp PstI digest fraction of the chaffinch (Fringilla coelebs L.) genomic DNA, sequenced, and characterized. The FCP repeat was found to have 505-506 bp length of monomer, 57% content of GC, to compose about 0.9% of the chaffinch genome, and to be highly methylated. Results of Southern-blot hybridization of cloned FCP element onto genomic DNA digested with different restriction enzymes, and sequencing directly from total genomic DNA using FCP-specific primers and ThermoFidelase enzyme (Fidelity Systems Inc.) were in agreement with a tandem arrangement of this repeat in the chaffinch genome. Five positions of single-nucleotide polymorphism (SNP) were found in the FCP monomers using direct genomic sequencing. Fluorescence in situ hybridization (FISH) with FCP probe and primed in situ labelling (PRINS) with FCP specific primers showed that the FCP elements occupy pericentric regions of all chaffinch chromosomes. On chromosome spreads, the fluorescent signals were also observed in the intercentromeric connectives between nonhomologous chromosomes. The results suggest that the centromeric FCP repeat is responsible for chromosome ordering during mitosis in chaffinch.

Animals↗

PubMed2000

15(S)-HETE modulates LTB(4) production and neutrophil chemotaxis in chronic bronchitis.

We evaluated the levels of 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] and the expression of 15-lipoxygenase (15-LO) mRNA in induced sputum obtained from 10 control and 15 chronic bronchitis subjects. 15(S)-HETE was evaluated by reverse phase high-performance liquid chromatography separation followed by specific RIA. 15-LO mRNA expression was determined by primed in situ labeling. The levels of both soluble and cell-associated 15(S)-HETE resulted significantly higher in chronic bronchitis than in control subjects. The percentage of cells expressing 15-LO mRNA was significantly higher in chronic bronchitis than in control subjects (P < 0.01). Double staining for specific cell type markers and 15-LO mRNA showed macrophages and neutrophils positive for 15-LO, whereas similar staining of peripheral blood neutrophils did not show evidence for 15-LO expression, suggesting that expression of 15-LO in neutrophils takes place on migration into the airways. Because 15(S)-HETE inversely correlated with the percentage of neutrophils in sputum of chronic bronchitis subjects, we studied the effect of 15(S)-HETE on leukotriene B(4) (LTB(4)) production in vitro and evaluated the concentration of LTB(4) in induced sputum and the contribution of LTB(4) to the chemotactic activity of induced sputum samples ex vivo. The results obtained indicate that macrophages and neutrophils present within the airways of chronic bronchitis subjects express 15-LO mRNA; increased basal levels of 15(S)-HETE may contribute to modulate, through the inhibition of 5-lipoxygenase metabolites production, neutrophil infiltration and airway inflammation associated with chronic bronchitis.

Adult↗

PubMed1992

Assignment of a porcine male-specific DNA repeat to Y-chromosomal heterochromatin.

Primers were designed to amplify by PCR a 509-bp genomic fragment from male pig DNA, using the porcine male-specific repeat sequence described by McGraw et al. (1988). This PCR product showed male-specific hybridization in Southern blots. Nonradioactive in situ hybridization localized it to the entire length of the heterochromatic portion of Yq. The assignment was confirmed using the PCR primer pDYZ1-S for primed in situ labeling.

Animals↗

PubMed1995

Selection of chromosome-specific primers and their use in simple and double PRINS techniques for rapid in situ identification of human chromosomes.

The PRimed IN Situ labeling (PRINS) technique is an alternative to in situ hybridization for rapid chromosome screening. We have defined and tested new specific oligonucleotide primers for alpha-satellite DNA of several chromosomes. When using a semiautomatic PRINS protocol, specific labeling was obtained in both metaphase cells and interphase nuclei in a 1-h reaction. PRINS may be a simple and reliable technique for rapidly detecting aneuploidies.

Base Sequence↗

PubMed1996

Spontaneous and aphidicolin-sensitive fragile site 3cen co-localizes with the (TTAGGG)n telomeric sequence in Chinese hamster cells.

Aphidicolin-sensitive fragile sites were analyzed in immortalized Chinese hamster embryonal fibroblast cells (CHEF18) at three different passages along their spontaneous progression toward tumorigenicity. Five fragile sites (viz., 12q22, 3cen, 3p21, 3q31, and Xq21) were detected. Three of these sites carry spontaneous aberrations and are thus regions of chromosomal instability; however, they were not involved in the formation of the clonal rearrangements that are characteristic of CHEF 18 cells. The presence of the (TTAGGG)n telomeric sequence in chromosome bands associated with fragile sites was investigated using fluorescence in situ hybridization and primed in situ labeling. A common location of fragile sites and telomeric sequence was found at the centromere of chromosome 3.

Animals↗