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PubMed1997

Primed in situ (PRINS) labeling for rapid detection of numeric and structural chromosome anomalies.

Primed in situ (PRINS) labeling has been applied to replace the traditional fluorescence in situ hybridization (FISH) method for the detection of specific sequences in situ in several numerical and structural chromosome anomalies. PRINS is based on sequence-specific annealing in situ of an unlabeled DNA probe or oligonucleotide primer. The probe serves as a primer for chain elongation in situ, using the labeled nucleotides as substrate. An oligonucleotide, (CCCTAA) representing human telomeric sequences, was mixed with nucleotides, biotin-16-dUTP, and Taq DNA polymerase, and applied on metaphase slides with ring chromosomes 4, 13, 18, X and Y. Primers for alpha-satellite sequences specific for the centromeric regions of human chromosomes 13, 15, 18, X and Y were also used to characterize the nature and origin of unidentifiable supernumerary marker chromosomes. The specificity of PRINS in differentiating centromeric sequences of chromosome [3 from 21 which is not possible with FISH, was demonstrated. Absence of the telomeric sequences in all of the ring chromosomes was noted in normal and abnormal phenotypes. The results suggest a mechanism of ring formation, an end-to-end fusion after loss of the palindromic nucleotide sequences at the telomeres PRINS, a fast and sensitive method of detecting nucleic acid sequences in situ, may be a reliable technique for detecting chromosomal aneuploidies and some structural rearrangements.

Chromosome Aberrations↗
PubMed1999

Chromosomal localization of zebrafish AluI repeats by primed in situ (PRINS) labeling.

Two zebrafish AluI repeats were localized in metaphase chromosomes by means of the primed in situ (PRINS) labeling technique, using oligonucleotide primers based on published sequences. An AT-rich, tandemly repeated, long AluI restriction fragment (RFAL1) labeled the (peri)centromeric regions of all chromosomes. The GC-rich short fragment (RFAS) was found to be localized in the paracentromeric regions of 17 chromosome pairs, which were mostly subtelocentric. The RFAS labeling pattern generally fits the previously described chromomycin A3 (CMA3) staining pattern. The differential composition of heterochromatin in zebrafish chromosomes is discussed.

Alu Elements↗
PubMed1997

Primed in situ (PRINS) labelling with Alu and satellite primers for rapid characterization of human chromosomes in hybrid cell lines.

The primed in situ (PRINS) labelling method was developed as an alternative to classical cytogenetics and fluorescence in situ hybridization (FISH) for the characterization of interspecific somatic hybrids. Full karyotypes were performed by PRINS using Alu-specific primers to generate the painting of all human material associated with R-like banding. The representativity of individual human chromosomes was established using primers specific for discriminent alpha-satellite DNA sequences providing specific signals on the centromeres of the targeted chromosomes and corresponding spots in interphase nuclei. Using this methodology, a somatic hybrid clone was shown to be monochromosomal for the der(11) from a t(11;22) patient.

Animals↗
PubMed1995

Translocation (1;16) identified by chromosome painting, and PRimed IN Situ-labeling (PRINS). Report of two cases and review of the cytogenetic literature.

We used the molecular cytogenetic in situ techniques chromosome painting and PRimed IN Situ labeling (PRINS) to elaborate the cytogenetic observations in two cases of the rare aberration der(16)t(1;16), which occurs in a wide variation of hematologic and nonhematologic malignancies [1-3]. Review of the literature showed that, in contrast to the chromosome 1 breakpoint, the breakpoint on chromosome 16 is associated with diagnosis as well as patient age.

Adult↗
PubMed1997

Swine centromeric DNA repeats revealed by primed in situ (PRINS) labeling.

In swine, distinct centromeric satellite DNA families have been described that correspond to either all the metacentric chromosomes except the Y (Mc1) or all the acrocentric chromosomes (Ac2). Using primed in situ (PRINS) labeling, we show here that primers derived from various sequences specifically label the centromeres of different subgroups of chromosomes. Among five primers derived from centromeric sequences of acrocentric chromosomes reported to be very homogeneous, four recognize all the acrocentric chromosomes, whereas one labels prominently chromosome 17. For the metacentric chromosomes, six primers have been derived from several divergent sequences. Among these primers, two recognize all the metacentric chromosomes except 5, 10, and 12. Three other primers label small subsets of metacentric chromosomes, including the X and one or two additional chromosomes. The last primer is specific to chromosome 1. These preliminary results suggest that it should be possible to define specific primers for almost every swine chromosome. Already, some of the primers reported here permit a distinction between swine chromosomes difficult to differentiate without banding, such as the X chromosome and chromosome 9. Therefore, the PRINS technique using centromeric motifs constitutes an additional tool for cytogenetic studies in swine.

Animals↗
PubMed2006

PRINS combined with peptide nucleic acid labeling.

Both primed in situ labeling (PRINS) and peptide nucleic acid (PNA) technologies have emerged as research techniques, but they have quickly evolved to applications in biological diagnosis assays. The two procedures present several features (specificity, discriminating ability, rapidity) that make them very attractive for cytogenetic purposes. The combined use of PRINS and PNA for in situ chromosomal detection on a same cell preparation is described in this chapter.

Chromosomes, Human↗
PubMed2003

Mouse telomere analysis using an optimized primed in situ (PRINS) labeling technique.

Telomeres are chromosomal elements composed of variable numbers of a TTAGGG repeated DNA sequence required for genomic stability. Telomeric length is correlated with the number of copies of this repeated DNA sequence and is an important property relevant to telomeric function. Recently, it has been demonstrated that the length of the shortest telomere, not average telomeric length, is important for cell viability and chromosomal stability. Consequently, assays permitting assessment of telomeric length are important for the analysis of genomic instability disorders. The length of individual telomeres can be analyzed using the primed in situ (PRINS) labeling reaction, which produces a labeled copy of the telomeric DNA repeats in situ. In this study, we tested different variables to optimize the PRINS reaction to enable it to be applied to the detection of mouse telomeric DNA and the study of telomeric length. The specificity, efficiency and uniformity of staining were evaluated using digital fluorescence microscopy. Labeling efficiency is dependent upon the conditions used to denature the telomeric DNA and reaction duration. Staining uniformity is increased at higher annealing and elongation temperatures as well as when a fluorescently labeled nucleotide is incorporated during the elongation step. Our results also indicate that chromosomal background staining is observed when a fluorochrome-labeled nucleotide is used as opposed to a hapten-labeled nucleotide. From this study, we conclude that an optimized PRINS technique can be reliably employed to analyze mouse telomeres and, compared with the FISH (fluorescence in situ hybridization) technique, presents advantages including greater cost efficiency and reduced processing time. These advantages may encourage wider use of the PRINS technique for quantitative evaluation of the length of individual telomeres in situ.

Animals↗
PubMed1996

Rapid bright-field detection of oligonucleotide primed in situ (PRINS)-labeled DNA in chromosome preparations and frozen tissue sections.

We describe a new application of a bright-field microscopic procedure for rapid enzyme cytochemical detection of repeated DNA sequences in metaphase preparations and frozen tissue sections. Various chromosome-specific oligonucleotide primers were used in up to three sequential primed in situ (PRINS) labeling reactions together with Taq DNA polymerase and biotin, digoxigenin and/or fluorescein isothiocyanate (FITC)-modified nucleotides. DNA target sequences were localized simultaneously by the precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) and horseradish peroxidase-teramethylbenzidine (PO-TMB, green color) reaction in hematoxylin counterstained metaphases and interphase nuclei using a standard bright-field microscope. In addition, a protocol is reported for the application of PRINS to frozen tissue sections from normal colon and bladder epithelium. Methanol/acetic acid fixation in combination with a pepsin digestion before performing the PRINS reaction proved to be critical steps in the total procedure that permits access of the PRINS reactants, while preserving the morphology of the nuclei in the tissue. Quantification of PRINS signals showed the majority of epithelial cells with the expected two chromosome copies. The described procedures can be considered valuable tools for application in molecular cytogenetics, cell biology and pathology.

Biotin↗
PubMed1998

Characterization of reciprocal translocations in pigs using dual-colour chromosome painting and primed in situ DNA labelling.

We report the use of dual-colour chromosome painting to determine the exact nature of certain chromosome rearrangements observed in the pig (Sus scrofa domestica). The chromosomal abnormalities were detected by GTG- and RBG-banding techniques. The initially proposed interpretations were: (1) rcp(6;13)(p1.5;q4.1); (2) rcp(11;16)(p1.4;q1.4); (3) rcp(6;16)(p1.1;q1.1); (4) rcp(13;17)(q4.1;q1.1); (5) rcp(6;14)(q2.7;q2.1); (6) rcp(3;5)(p1.3;q2.3); (7) rcp(2; 14)(q1.3;q2.7); (8) rcp(15;17)(q1.3;q2.1). Hybridizations were carried out with biotin- and digoxigenin-labelled probes obtained by priming authorizing random mismatches polymerase chain reaction (PARM-PCR) amplification of porcine flow-sorted chromosomes. In some cases, i.e. (1), (4), (5), (6), (7) and (8), the fluorescence in situ hybridization (FISH) results allowed confirmation of the interpretations proposed with classical cytogenetic methods. Chromosome painting proved the reciprocity of the translocation in cases (1), (6) and (8), whereas modifications of the formula were proposed for case (2). Primed in situ DNA labelling (PRINS) experiments have also been carried out in case (3) using a primer specific for the centromeres of acrocentric chromosomes (first experiment) or a primer specific for the centromeres of a subset of meta- and submetacentric chromosomes including chromosome 6 (second experiment). It allowed us to demonstrate that the breakpoints occurred in the centromeric region of chromosome 16 and in the p. arm of chromosome 6, just above the centromere.

Animals↗
PubMed1998

Fetal cells in maternal blood: the use of primed in situ (PRINS) labelling technique for fetal cell detection and sex assessment.

Prenatal diagnosis is presently performed following invasive procedures with variable risks of fetal loss; non-invasive procedures using fetal cells in maternal blood would be welcome for the early detection of fetal sex or aneuploidy. We describe a simple and rapid protocol to detect fetal cells and thus to assess fetal sex. In a first step, nucleated blood cells were separated into mononuclear and polynuclear cells using a double density gradient centrifugation. In a second step primed in situ (PRINS) labelling technique was performed to label Y-chromosomes. 15 samples were studied and correct gender assignment was made in 13/15. The number of labelled nuclei was higher in polynuclear cell phases than in mononuclear cell phases. Moreover, the polylobular aspect of labelled nuclei from polynuclear cell phases strongly suggested that they could belong to fetal polynuclear cells. The PRINS technique combines some advantages of FISH, such as visual assessment of in situ chromosome labelling and the powerful specificity and sensitivity of PCR. In association with a simple enrichment procedure it constitutes a rapid protocol for fetal cell detection, non-invasive early prenatal sex assessment, and could further be applied to detect the main viable aneuploidies.

Adult↗
PubMed2001

Study of the occurrence of interchromosomal effect in spermatozoa of chromosomal rearrangement carriers by fluorescence in-situ hybridization and primed in-situ labelling techniques.

The possibility that a chromosomal rearrangement might disturb the meiotic behaviour of chromosomes not involved in the rearrangement and favour non-disjunction is a controversial issue in human cytogenetics. Using two-colour fluorescence in-situ hybridization and primed in-situ labelling techniques, we have investigated the segregation pattern of 10 chromosomes (chromosomes 1, 4, 9, 13, 15, 16, 20, 21, X and Y) in spermatozoa from nine carriers of balanced structural rearrangements and three normal men. The patients were divided into two groups according to their semen parameters. In rearrangement carriers and normal subjects, sex chromosomes and chromosome 21 displayed a higher rate of disomy than the other chromosomes. No evidence for the occurrence of interchromosomal effect was found in the spermatozoa of fertile rearrangement carriers, but significant variations were observed for all chromosomes tested in the group of infertile translocation carriers, suggesting a direct correlation between poor quality spermatozoa and increased aneuploidy rate in this group. In fertile carriers of chromosomal rearrangements, the occurrence of non-disjunction of chromosomes not involved in the rearrangement might therefore be considered as fortuitous, whereas in infertile carriers, the risk for interchromosomal effect appears to be real and should be taken into consideration in the genetic counselling of infertile couples with a male partner carrying a chromosomal rearrangement.

Adult↗
PubMed2001

Localisation of DNA sequences on plant chromosomes using PRINS and C-PRINS.

Localisation of DNA sequences to plant chromosomes in situ has traditionally been accomplished using fluorescence in situ hybridisation (FISH). Although the method is suitable for most applications it is time-consuming and requires labelled probes. Recently, primed in situ labelling (PRINS) has been developed as an alternative to FISH. PRINS is based on annealing of unlabelled oligonucleotide primer(s) to chromosome DNA and its elongation by DNA polymerase in the presence of labelled nucleotide(s). The method was found useful to detect high-copy tandem repeats on plant chromosomes. Low copy repeats were detected after a more sensitive variant of PRINS called cycling PRINS (C-PRINS), which involves a sequence of thermal cycles analogous to polymerase chain reaction. This paper describes protocols of PRINS and C-PRINS, which have been optimised for chromosome spreads and for chromosomes purified using gradient centrifugation and/or flow sorting. The methods result in clear signals with negligible non-specific labelling. Further work is needed to improve the sensitivity to allow for reliable detection of single- copy DNA sequences.

DNA, Plant↗
PubMed1997

Rapid detection of karyotype changes in interphase bone marrow cells by oligonucleotide primed in situ hybridization (PRINS).

Fluorescence in situ hybridization (FISH) using DNA probes of several hundred or thousand base pairs in length enables the visualization of chromosomal aberrations in interphase nuclei. A new method for in situ labelling of chromosomes is the oligonucleotide primed in situ labelling (PRINS) technique. So far, this has mainly been used to demonstrate subtle changes in metaphase spreads. The aim of the present study was to investigate the suitability of PRINS for detecting chromosome gains or losses in interphase nuclei. This technique was compared with FISH analysis by examining the bone marrow cells of ten patients in whom the karyotypes were known from conventional chromosome banding. Corresponding results by both PRINS and FISH were obtained for chromosomes 1, 3, 7, 8, and Y in five patients with normal chromosome patterns, as well as in five patients with clonal karyotype changes, e.g., monosomy 7, trisomy 8, or loss of the Y chromosome. Being faster and approximately ten times less expensive, PRINS can replace FISH for detecting numerical karyotype changes.

Adult↗
PubMed1989

Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ.

It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.

Base Sequence↗
PubMed1993

Flow cytometric quantitation of sequence-specific mRNA in hemopoietic cell suspensions by primer-induced in situ (PRINS) fluorescent nucleotide labeling.

We modified a technique of RNA detection based upon primed in situ labeling of RNA (PRINS) to quantitate poly(A) and histone mRNA in HL60 cell suspensions by flow cytometry. The direct PRINS technique was used to label poly(A) mRNA, based upon the incorporation of fluoresceinated nucleotides into cDNA chains generated by reverse transcription from the site of oligo(dT) primer-specific annealing on the poly(A) template. By this method, poly(A) fluorescent signals of up to 20-fold above background were obtained when primer concentrations were saturating. Nonspecific binding of the fluorochrome-conjugated nucleotide proved to be the greatest source of background fluorescence in cell suspensions. This was considerably reduced by preincubation of the cells in lysine and an excess of cold nucleotides prior to the PRINS reaction. The application of indirectly fluoresceinated antibodies to the FITC moiety of the conjugated nucleotide (indirect PRINS) enhanced detection of a fluorescent signal generated from cells by the direct PRINS technique after stringent histone priming. Cell cycle analysis, based upon DNA histograms obtained after PRINS labeling, showed the histone fluorescence to be primarily in S-phase cells of an exponentially growing population. Confocal laser scanning microscopy revealed good retention of cellular architecture, with cytoplasmic localization of poly(A) and histone mRNA fluorescence and heterogeneous mRNA expression among cells. Our modified PRINS labeling of RNA represents a useful advance in the precise quantitation of RNA species in cell suspension by flow cytometry.

Flow Cytometry↗