Primed in situ labeling (PRINS) and fluorescence in situ hybridization (FISH).
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Base Sequence↗
Biomedical subjects
J Hindkjaer
Publications and source records attributed to J Hindkjaer.
Pseudodicentric chromosome 18 diagnosed by chromosome painting and primed in situ labelling (PRINS).
We report on a newborn white male infant with marked dysmorphic features and various congenital malformations. The initial clinical evaluation showed Crouzon-like features as well as some features of trisomy 18 syndrome and trisomy 13 syndrome. The results from conventional cytogenetic analysis showed a structurally abnormal chromosome replacing one normal chromosome 18, but only by applying molecular cytogenetic methods could the architecture of this abnormal chromosome be characterised clearly. The primed in situ labelling (PRINS) technique, using a newly synthesised alpha 18 oligonucleotide, showed the dicentric pattern and direct chromosome painting established the origin to be from chromosome 18. The combination of conventional cytogenetics and molecular cytogenetics showed the karyotype in the proband to be 45,XY,-14,-18,-21,+t(14;21),+psu dic(18) (qter-->cen-->p11.3: :p11.3-->psu cen-->qter). This was supported by molecular analysis using chromosome 18 specific DNA markers, which showed the paternal origin of the abnormal chromosome.
Abnormalities, Multiple↗
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↗
Reverse chromosome painting.
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Base Sequence↗
Primed in situ (PRINS) labeling of RNA.
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Animals↗
Primed in situ (PRINS) labeling of DNA.
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Biotin↗
Aneuploid malignant T cells from a patient with Sézary syndrome can be visualized by in situ hybridization.
BACKGROUND AND DESIGN: Enumeration of malignant cells in Sézary syndrome often relies on the identification of the Sézary cell nucleus. This morphologic method is, however, nonspecific and unreliable in the enumeration of the proportion of malignant lymphocytes in peripheral blood of patients with Sézary syndrome. Malignant lymphocytes of patients with mycosis fungoides and Sézary syndrome are often characterized by multiple chromosome aberrations. Herein, we demonstrate that fluorescent in situ hybridization can visualize and accurately enumerate malignant aneuploid mononuclear cells in a patient with Sézary syndrome. RESULTS: Fluorescent in situ hybridization demonstrated that 90% of the mononuclear cells in the patient with Sézary syndrome showed numerical aberrations for both chromosome 7 and X, a figure confirmed by flow cytometry. CONCLUSION: Fluorescent in situ hybridization may be a valuable tool to visualize and enumerate aneuploid tumor cells in patients with cutaneous T-cell lymphoma.
CD8 Antigens↗
Molecular cytogenetics: applications in clinical genetics.
To illustrate the advantage of fluorescence in situ hybridization (FISH) in clinical genetics, we have chosen six clinical cases from our routine laboratory where we have used molecular cytogenic techniques to optimise the cytogenetic analysis. Using centromere-specific DNA probes and whole chromosome DNA libraries either obtained from somatic cell hybrids or generated from flow-sorted chromosomes, we have been able to identify small marker chromosomes, chromosomal duplications and inversions, to determine the ploidy in interphase nuclei, and to characterize subtle chromosomal translocations. We conclude that molecular cytogenetics is a valuable technique that should be used as a supplement to conventional cytogenetics to reduce the number of unresolved karyotypes in clinical genetics.
Adolescent↗
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↗
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↗
A chromosomal basis for the differential organization of a porcine centromere-specific repeat.
Digestion of porcine DNA with the restriction enzymes SnoI and BamHI and subsequent gel electrophoresis revealed the presence of a repeat fragment of approximately 340 bp in length. When this fragment was cloned and used to probe a Southern blot containing pig DNA that had been digested with various enzymes, a ladder pattern of bands based on a unit of 340 bp was observed. Three individual BamHI repeat units were sequenced; each monomer unit was approximately 90% identical in sequence to the other two monomers. Double digestion did not alter the ladder pattern, indicating a differential distribution of restriction enzyme sites within the repeat family. We demonstrate by Southern blotting of DNAs from a panel of pig x rodent hybrid cell lines that the differential restriction site distribution has a chromosomal basis. We show that this repeat family hybridizes to the centromeres of all pig chromosomes other than the Y chromosome. However, when stringency was raised, hybridization persisted only to certain metacentric chromosomes.
Animals↗
Repeated primed in situ labeling: formation and labeling of specific DNA sequences in chromosomes and nuclei.
A new version of our previously published PRINS (PRimed IN Situ labeling) method is presented. It represents a significant improvement in the detection of specific DNA sequences in situ. The idea is to perform the reaction repeatedly, rather than just once. This change of strategy results in a localized accumulation of sequence-specific labeled DNA, resulting in up to a 15-fold amplification of the signal as compared to the standard PRINS method. Interestingly, the retention of the labeled DNA is so good that it stays within the chromosomal band where it is synthesized, provided that the reaction is not performed an excessive number of times. The key trick is the performance of the procedure on small glass slides in PCR tubes, thereby avoiding the use of cover slips. In addition, use of the small glass slides seems to give less variance when the signals are quantified in a fluorescence microscope.
Base Sequence↗
[Identification of Y chromosome material in an XX male by means of fluorescent in situ hybridization].
This article describes a case of 46,XX male, the most frequent form of sex reversal syndromes in humans. A method of identifying Y chromosome material in these and other patients with structural chromosomal abnormalities involving chromosome Y is given. Chromosomes from a phenotypically normal male child without any congenital malformation, where prenatal diagnosis revealed the female karyotype 46,XX, were analysed using fluorescence in situ hybridisation (FISH). The analysis revealed an X chromosome, containing Y chromosome sequences on the tip of the short arm. The sequences are not normally visible in conventional cytogenetic analyses of XX males. The breakpoint on Y was determined to be in the region of Yp11.2, which is proximal for the putative sex determining gene on Y. The results are consistent with theories of abnormal crossing-over during the paternal meiotic cell division where meiotic recombination can give rise to structural abnormalities, which can then cause sex reversal syndromes. Prenatal diagnosis of structural sex chromosome abnormalities has become available using the FISH method.
Adult↗
Fast one-step procedure for the detection of nucleic acids in situ by primer-induced sequence-specific labeling with fluorescein-12-dUTP.
We provide fast, simple, one-step procedures for sequence-specific detection of nucleic acids in situ. Tandem repeat sequences in DNA are stained within 30 min, and mRNA is stained within 2 h. The procedures are based on the incorporation of the newly available fluorescein-labeled dUTP into DNA synthesized in situ by primed in situ labeling, with denatured fragments of cloned DNA or oligonucleotides as primers. The extreme speed and simplicity of the reaction make it attractive for automatization in routine laboratory procedures and opens up new diagnostic possibilities.
DNA↗
A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica).
Chromosomes were prepared from lymphocytes of a male domestic pig and flow-sorted on a dual-laser FACS. Twenty spots were observed, corresponding to the known pig karyotype of 18 pairs of autosomes plus the X and Y. DNA was isolated from 10,000 copies of the presumed chromosome 1 spot, restricted with Sau3A, ligated into the vector pGEM4z, and PCR amplified using universal primers; the products were then re-ligated into pUC18. After transformation into Escherichia coli, 210,000 independent colonies were obtained, 5% of which contained only vector DNA. The average insert size of the library was 405 bp. Southern blotting revealed that 36% of the clones contained single-copy DNA and that the remainder contained moderately or highly repetitive DNA. Screening with a (CA)n probe revealed that roughly 1% of the clones contained microsatellite sequences. A bulk insert of the library was biotinylated by PCR and used as a probe for chromosomal in situ suppression hybridization to pig chromosomes, which confirmed that the library is specific for chromosome 1. However, sequences from the centromeric and telomeric regions seem to be underrepresented in the library.
Animals↗
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↗
Nonradioactive, sequence-specific detection of RNA in situ by primed in situ labeling (PRINS).
A new method for the detection of RNA in situ is presented. It is based on sequence-dependent annealing of unlabeled specific oligonucleotide primers to intracellular RNA and subsequent chain elongation catalyzed by reverse transcriptase. Under the conditions described, biotin-labeled nucleotides can be incorporated and the cDNA synthesized in situ can thus be detected using fluorescein-conjugated avidin. Compared to traditional in situ hybridization the use of short oligonucleotide primers has the potential advantage of being better to discriminate between closely related RNA transcripts. Compared to in situ transcription with radioactive precursors we find it more attractive to use fluorescein-conjugated avidin as detection system because it allows a more detailed study of cell and signal simultaneously.
Animals↗
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↗