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At least 19 recordsLinked to original sources
PubMed2000

Chromosomal localization of single copy genes SRY and SOX3 by primed in situ labeling (PRINS).

Primed in situ labeling (PRINS) is a sensitive and specific technique that can be used for the localization of single copy genes and DNA segments that are too small to be detected by conventional FISH. With PRINS, we physically localized the SRY gene to Yp11.31p11.32 and the SOX3 gene to Xq26q27. Locus-specific oligonucleotide primers were annealed in situ and extended on chromosome preparations fixed on microscope slides, in the presence of dATP, dCTP, dGTP, dTTP, biotin-16-dUTP, Tris-HCl, KCl, MgCl2, BSA, and Taq DNA polymerase. Fluorescent signals were detected in metaphase spreads and interphase nuclei. Our method may prove valuable for use with single copy genes in general.

Centromere↗
PubMed1997

Rapid identification of marker chromosomes using primed in situ labeling (PRINS).

Primed in situ labeling (PRINS) is a relatively new technology with wide-ranging applications in clinical cytogenetics. Using PRINS, we have identified the chromosomal origin of marker chromosomes in three patients. In the first patient with primary amenorrhea, we were able to confirm the marker chromosome as originating from an X. In the second (prenatal) case, PRINS allowed us to determine rapidly the origin of the marker as a Y chromosome. In the third patient with minor anomalies, the marker was identified as derived from a chromosome 18. In all three cases, application of PRINS permitted us to characterize the marker chromosomes within 1 hour after the slides were prepared. The methodology is simple, has added advantages over conventional fluorescence in situ hybridization (FISH), and can be used as a viable and effective alternative to FISH in clinical cytogenetic diagnosis.

Abnormalities, Multiple↗
PubMed1994

Fast, sensitive multicolor detection of nucleic acids in situ by PRimed IN Situ labeling (PRINS).

PRimed IN Situ labeling (PRINS) has become an alternative to traditional fluorescence in situ hybridization (FISH) methods for detection of nucleic acids in situ. PRINS is based on sequence-specific annealing in situ of an unlabeled DNA probe. The probe serves as a primer for chain elongation in situ, catalyzed by a suitable DNA polymerase that uses labeled nucleotides as substrate. The fact that the probe is unlabeled means that high probe concentrations can be utilized, making the hybridization very fast. We describe here a fast method for detection of three different target sequences visualized in different colors with PRINS. An advantage, relative to FISH, is that even probes with different melting temperatures can be detected in the same metaphase with optimal stringency for each probe.

DNA Probes↗
PubMed1999

Interphasic analysis of aneuploidy in cancer cell lines using primed in situ labeling.

The primed in situ (PRINS) labeling technique has been adapted to chromosomal screening of interphasic tumoral cells. A panel of ten chromosome-specific alpha-satellite DNA primers was used to evaluate numerical chromosome abnormalities in two colon cancer cell lines (Caco-2 and HT-29) and in three of their subpopulations (PF11, TC7, and HT29-MTX). In each cell line, the copy number distribution for different chromosomes showed different patterns. The observation of significant variations in the chromosome constitutions between subpopulations derived from the same original tumor suggests the common occurrence of chromosome copy number heterogeneity in tumoral cell lines. This study demonstrates that the PRINS procedure offers a simple and reliable method for in situ chromosomal screening, which could be efficiently used for karyotypic analysis of tumoral cells.

Aneuploidy↗
PubMed1991

An improved method for chromosome-specific labeling of alpha 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.

Autoradiography↗
PubMed1996

Primed in situ labeling (PRINS). A fast method for in situ labeling of nucleic acids.

PRimed IN Situ labeling (PRINS) is a fast and sensitive alternative to fluorescence in situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.

Chromosomes, Human↗
PubMed1999

Detection of human papilloma virus (HPV) genomes by the primed in situ (PRINS) labelling technique.

Primed in situ Labelling, a technique based on primer mediated DNA synthesis, has become a useful tool in cytogenetics, especially for chromosome mapping, banding and the investigation of sequence organization in fresh metaphase preparations. Its application in the routine surgical pathology laboratory has been hampered by the fact that the technique did not work on paraffin-embedded, formalin-fixed tissue. We investigated cervical biopsies (n = 20) with morphological signs of HPV infection and found that the PRINS method is at least as sensitive as a classical in situ hybridization assay for detecting HPV DNA in paraffin-embedded, formalin-fixed tissue. In all investigated cases (n = 20), HPV DNA was found by both methods. The PRINS method was able to demonstrate HPV DNA not only in superficial koilocytotic squamous cells but also in non-koilocytotic cells in the deeper spinous cell layers, and even in some basal cells. We describe an economical protocol using conventional consensus HPV oligonucleotide DNA primers. The described method is rapid (approximately 3 hours) and easy to perform for screening and subtyping HPV infection in the routine surgical pathology laboratory.

DNA Primers↗
PubMed1996

Preimplantation embryo chromosome analysis by primed in situ labeling method.

OBJECTIVE: To present the use of primed in situ labeling method in preimplantation diagnosis. DESIGN: Double- and triple-primed in situ labeling were performed on 10 morphologically abnormal preimplantation embryos, using combinations of specific primers for chromosomes 9, 13, 16, 18, 21, X, and Y. SETTING: Embryos were obtained from patients at the Montpellier University Hospital. PATIENT(S): Seven women undergoing IVF at the Montpellier University Hospital. INTERVENTION(S): Isolated interphase nuclei from poor quality preimplantation embryos were prepared for primed in situ labeling technique. MAIN OUTCOME MEASURE(S): Numerical abnormalities assessed by primed in situ labeling analysis. RESULT(S): Using directly fluorescent-labeled nucleotides, the labeling reaction for three chromosomes did not exceed 2.30 hours. Only three analyzed embryos appeared to be chromosomally normal. Mosaicism, aneupoidy, and haploidy were observed in the seven other embryos. CONCLUSION(S): The primed in situ labeling method offers a simple and reliable screening tool for gender determination and aneuploidy detection. The use of this technique may contribute to significantly improve the procedure of preimplantation diagnosis.

Aneuploidy↗
PubMed1998

[Px gene of rice-field eels primed in situ labeling].

Peroxidase (Px) gene of rice-field eels (Monopterus albus) was successfully localized to the segments of two bivalents, 8q15-q26 and 11q32-q37 with digoxigenin labeled primed in situ labeling (PRINS) technique, which is utilized for localization of single copy gene of fish for the first time. Our results demonstrated the possibility of mapping genes in fish by the DNA probes of other species and PRINS technique.

Animals↗
PubMed2001

[Primed in situ labelling of human metaphase chromosomes].

Primed in situ labelling (PRINS) contributes to the elucidation of the organization of eukaryotic chromosomes and provides an alternative way to localize gene on chromosomes. Here we present the results of random primer and SOX degenerate primer PRINS on human metaphase chromosomes. The particular band pattern of dark and light was observed by using random primer PRINS. SOX degenerate primer PRINS revealed more SOX gene loci and proved further that SOX genes were not clustered on human chromosomes.

Chromosome Mapping↗
PubMed1997

Rapid characterization of human chromosomes in hybrid cell lines by primed in situ (PRINS) labeling.

The primed in situ (PRINS) labeling technique allows a rapid and specific labeling of human chromosomes in situ. This method is based on annealing of specific oligonucleotide primers and subsequent primer extension by a Taq DNA polymerase. We have developed a PRINS protocole for the cytogenetic analysis of somatic hybrid cell lines. Painting of human chromosomes is performed using Alu specific primers. Individual human chromosomes are identified using chromosome-specific alpha-satellite primers. The method was successfully tested to 3 different human-hamster hybrid cell lines. This approach provides an interesting alternative to classical cytogenetic and in situ hybridization techniques for the characterization of the human content of hybrid cell lines.

Animals↗
PubMed2002

Prader Willi/Angelman and DiGeorge/velocardiofacial syndrome deletions: diagnosis by primed in situ labeling (PRINS).

A recently developed methodology-primed in situ labeling (PRINS)-can be used in place of fluorescence in situ hybridization (FISH) to diagnose microdeletions. To demonstrate the efficiency, sensitivity, and specificity of PRINS in the diagnosis of microdeletions, we studied groups of patients with Prader Willi/Angelman (PWS/AS) syndrome and DiGeorge/velocardiofacial syndrome (DGS/VCFS). Results obtained by PRINS were then confirmed with the results obtained with FISH. Oligonucleotide primers specific for SNRPN and GABRB3 were used for PWS/AS syndromes. For DGS/VCFS, the primers used were DGCR2/TUPLE1 loci. Labeling patterns obtained by PRINS and FISH were analyzed and scored under a fluorescence microscope. Five normal subjects served as controls and were used for standardization of the PRINS protocol. In all, 20 study patients were involved: 10 PWS/AS and 10 DGS/VCFS. Five of the 10 patients referred with the clinical diagnosis of PWS/AS showed absence of labeling for SNRPN and GABRB3 on one chromosome 15, confirming deletion of the two loci. Similarly, 6 of the 10 patients referred for DGS/VCFS showed deletion for the DGCR2/TUPLE1 loci on one chromosome 22. The remaining patients and controls had normal patterns for all the loci as indicated by FISH and PRINS. Concordant FISH and PRINS results were obtained in all patients and controls studied.

Angelman Syndrome↗
PubMed2000

Localization of SRY by primed in situ labeling in XX and XY sex reversal.

Primed in situ labeling (PRINS) can be used to localize DNA segments too small to be detected by fluorescence in situ hybridization. By PRINS we identified the SRY gene in two XX males, a woman with XY gonadal dysgenesis, and an azoospermic male with Xp-Yp interchange. Because PRINS has been used generally in the study of repetitive sequences, we modified the technique for study of the single copy 2. 1-kb SRY sequence. SRY signals were identified at band Yp11.31p11.32 in normal XY males and in the woman with XY gonadal dysgenesis. SRY signals were identified on Xp22 in one XX male but not in the other. They were identified in the corresponding region (Xp22) of the der(X) in the azoospermic male with Xp-Yp interchange. SRY signals were not observed in normal XX females. Presence of SRY in DNA samples from the various subjects was confirmed by polymerase chain reaction. We conclude that PRINS is ideal for rapid localization of single copy genes and small DNA segments in general.

Adult↗
PubMed1998

Primed in situ labeling for rapid prenatal diagnosis.

OBJECTIVE: Our purpose was to assess the feasibility of primed in situ labeling for analysis of prenatal diagnostic specimens. STUDY DESIGN: Prenatal diagnostic specimens were chosen at random for analysis without knowledge of clinical indication. Primed in situ labeling with primers for chromosomes 18, 21, X, and Y was performed separate from conventional cytogenetic analyses. All clinical management considerations were based solely on conventional cytogenetic analyses. RESULTS: Forty-one samples were analyzed by primed in situ labeling: 35 direct preparations of chorionic villi and 6 uncultured amniotic fluid samples. In all cases analysis confirmed the particular chromosome number determined by conventional cytogenetic analysis. CONCLUSIONS: Although conventional metaphase studies remain the standard for prenatal cytogenetic analyses, the preliminary feasibility study finds primed in situ labeling to be a rapid and reliable adjunctive diagnostic technique applicable for prenatal diagnosis in certain clinical situations. Further study is needed to assess the efficacy of primed in situ labeling in comparison to fluorescent in situ hybridization and conventional cytogenetic analyses for prenatal diagnoses.

Amniotic Fluid↗
PubMed1996

[Detection of chromosome aberrations in brain gliomas by primed in situ labelling].

To investigate the possibility of applying the primed in situ labelling (PRINS) method to detect chromosome aberrations in brain gliomas and eventually for pathological diagnosis, PRINS with chromosome 10 and 12 centromere specific primers as well as chromosome 1 telomere specific primer was carried out on tissues from 5 brain glioma cases, of which 4 cases were found to have monosomy 10 or loss of one copy of chromosome 10. The remaining case was found to have trisomy 12 and multi-copies of chromosome 1. It is suggested that PRINS is a new molecular cytological bio-technique which is faster, simpler and more efficient than conventional fluorescence in situ hybridization, and which may be clinically applied to detect chromosome aberrations in brain gliomas in the future.

Brain Neoplasms↗
PubMed1997

Incidence of chromosome 1 disomy in human sperm estimated by the primed in situ (PRINS) labeling technique.

The primed in situ (PRINS) labeling technique was used to determine the rate of disomy of chromosomes 1 and 16 in sperm of two normal subjects. Two different but specific primers (alpha-satellite and satellite II) for chromosome 1 were used in parallel experiments to test the efficiency of PRINS labeling in sperm nuclei. A minimum of 10,000 sperm nuclei per chromosome primer was analyzed, leading to a total number of 41,651 scored spermatozoa. Similar rates of chromosome 1 disomy (mean values, 0.18% and 0.20%) were found in both donors when the alpha-satellite and satellite II primers were used, demonstrating the reliability of PRINS labeling on sperm nuclei. For chromosome 16, the disomy rate among the two donors ranged from 0.20% to 0.24%. This study confirms that PRINS provides a rapid and efficient method for in situ chromosomal screening of sperm nuclei.

Adult↗
PubMedDate not supplied

Validation of primed in situ labeling for interphase analysis of chromosomes 18, X, and Y in uncultured amniocytes.

OBJECTIVE: Primed in situ labeling (PRINS) is an interesting alternative to the traditional fluorescence in situ hybridization (FISH) for the in situ detection of specific sequences in chromosome anomalies. It combines the sensitivity and specificity of the polymerase chain reaction with the specific in situ signal detection capability of FISH. METHODS: We performed PRINS on uncultured amniocytes and compared the results with standard cytogenetic analysis. In a prospective study, a total of 262 independent samples were analyzed for numerical aberrations of chromosome 18. RESULTS: In more than 95% of the cases PRINS reactions were successfully achieved. Neither false-positive nor false-negative results were obtained. 62 of the 262 cases were in parallel examined for chromosome 18 aneuploidies by FISH. Although there were significant differences in signal distribution, these did not lead to a different overall classification of the respective cases, i.e., the end results of disomic and trisomic cases for chromosome 18 were identical between FISH and PRINS. In 205 of 262 cases PRINS was performed for chromosomes X and Y. 97.6% of these samples were properly sex differentiated. CONCLUSIONS: The PRINS assay is a simple and cheap alternative to detect numerical aberrations of chromosome 18. However, the rate of false-positive results for chromosome X was calculated as 1% and the rate of false-negative ones for chromosome Y as 2%. Further investigations are required to transform PRINS into an alternative to conventional methods for routine rapid prenatal diagnosis of gonosomes.

Amnion↗