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PubMed2005

Simultaneous identification of chromosomes 18, X and Y in uncultured amniocytes by using multi-primed in situ labelling technique.

The aim of this study is to validate the multi-PRINS (primed in situ labelling) technique for simultaneous detection of chromosomes 18, X and Y in uncultured amniocytes for prenatal diagnosis of aneuploidy. The sites of the newly synthesized DNA sequences were showed as fluorescent signals by using immunochemistry. A multi-PRINS technique was specifically performed for simultaneous detection in the same cells of three chromosome targets, e.g. chromosomes 18, X and Y. Fluorescent signals corresponding to chromosomes 18, X and Y were showed as yellow, red and green colour spots, respectively. A multi-FISH technique using chromosome 18, X and Y probes was performed for comparison. Sixty cases were analysed using both multi-PRINS and multi-FISH. Fifty to two hundred nuclei were scored for each case for each technique. In all cases, there was no significant difference in the detection of chromosomes 18, X and Y regarding the sensitivity, the specificity and the efficiency; multi-PRINS and multi-FISH yield a similar distribution of the number of spots per nucleus. Both techniques were able to identify aneuploid cases without any ambiguity. Both multi-PRINS and multi-FISH can accurately and reliably detect aneuploidies involving chromosomes 18, X and Y in uncultured amniocytes. Finally, multi-PRINS represents a faster and more cost-effective alternative to FISH for prenatal testing of aneuploidy in uncultured amniocytes.

Amniotic Fluid↗
PubMed1998

CpG islands detected by self-primed in situ labeling (SPRINS).

We have studied the distribution and methylation of CpG islands on human chromosomes, using the novel technique of self-primed in situ labeling (SPRINS). The SPRINS technique is a hybrid of the two techniques primed in situ labeling (PRINS) and nick translation in situ. SPRINS detects chromosomal DNA breaks, as in nick translation in situ, and not annealed primers, as is the case in PRINS. We analyzed in situ-generated DNA breaks induced by the restriction enzymes HpaII and MspI. These restriction enzymes enable the detection of chromosomal CpG islands. Both HpaII- and MspI-SPRINS produce a banding pattern resembling R-banding, indicating a higher level of CpG islands in R-positive bands than in R-negative bands. Our SPRINS banding observations also indicate differences in sequence copy number in the satellites of homologous acrocentric chromosomes. Furthermore, a comparison of homologous HpaII-SPRINS-banded X chromosomes of females from lymphocyte cultures grown without methotrexate or bromodeoxyuridine revealed methylation difference between them. The same comparison of homologous X chromosomes from the cell line GM01202D, which has four X chromosomes, one active and three inactive, revealed the active X chromosome to be hypermethylated.

Cells, Cultured↗
PubMed1995

Use of the primed in situ labelling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y.

The primed in situ labelling (PRINS) technique is an alternative to in situ hybridization for chromosomal screening. We have developed a semi-automatic PRINS protocol, using a programmable thermocycler. The method has been successfully tested with specific primers for chromosomes, 13, 16, 18, 21, X and Y. Specific chromosome detection has been obtained on both metaphases and interphase nuclei. This suggests that PRINS may be a reliable technique for detecting aneuploidies and some chromosomal aberrations.

Base Sequence↗
PubMed1993

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↗
PubMed2004

[Rapid detection of 18-trisomy syndrome using primed in situ labeling technique].

OBJECTIVE: To develop a rapid method for the detection of chromosomes and try to verify the feasibility of using modified primed in situ labeling (PRINS) technique for rapid detection of the interphase nuclei of uncultured amniocytes. METHODS: Chromosome 18 was detected and analyzed by the modified PRINS in 262 amniotic fluid samples. RESULTS: The specific chromosomes were obtained on both metaphase and interphase nuclei. In more than 95% of the samples, PRINS reactions with primer 18cen were successfully induced. Two samples were properly identified and correctly scored as trisomic 18. CONCLUSION: The results suggest that PRINS technique is simple, rapid and cost-effective. It is sensitive, specific, and thus can enhance the accuracy of standard cytogenetic analysis.

Amniocentesis↗
PubMed1996

Primed in Situ Labeling as a Fast and Sensitive Method for the Detection of Specific DNA Sequences in Chromosomes and Nuclei

Primed in situ labeling (PRINS) has become a routine technique for the microscopical staining of specific DNA sequences in cells and nuclei. For this purpose, the technique has the general advantage of being fast, simple, and sensitive. Furthermore, the reaction characteristics of the technique enable it to discriminate efficiently among closely related sequences-sometimes even when these differ by only one base. This high selectivity is obtained partly because the technique works optimally with oligonucleotide probes, which select more efficiently among closely related sequences than cloned probes do. This selectivity is further enhanced because no labeling occurs subsequent to probe binding unless the hybridized probe can function as primer for DNA synthesis, a process that is efficient only when the probe (primer) matches the target perfectly. Should this in itself provide insufficient selectivity, the involved chain elongation of the probe can be used in various ways to increase the selectivity of the staining further. These features of the technique have made it attractive for the detection of repeated DNA sequences, which now can be detected with a one-step procedure of a few minutes' duration, and make it a good candidate for the future detection of single-base variations in single-copy sequences in situ.

Journal Article↗
PubMed1999

Validation of primed in situ labeling (PRINS) for interphase analysis: comparative studies with conventional fluorescence in situ hybridization and chromosome analyses.

Primed in situ labeling (PRINS) is a rapidly developing new technology with wide ranging clinical applications. To assess the sensitivity, specificity, and accuracy of PRINS, we carried out a retrospective study on cultured bone marrow cells to detect aneuploidy for chromosomes 7, 8, and 12. The results were then compared to the results of previous fluorescence in situ hybridization (FISH) and chromosome analyses (CA). In patients who showed aneuploidy with CA, both FISH and PRINS confirmed the aneuploidy in interphase cells. FISH and PRINS also showed excellent correlation with conventional cytogenetic analysis for the detection of mosaic aneuploidies. However, both FISH and PRINS showed significantly higher sensitivity in the detection of abnormal clones compared to CA. In 9 of the 17 cases, there were no significant differences in the detection rates between the two methods. Based on our studies, we conclude that PRINS is as sensitive as FISH in most cases for aneuploidy detection; and that PRINS, like FISH, is more sensitive than conventional CA for aneuploidy detection.

Aneuploidy↗
PubMed1996

Primed IN situ labelling (PRINS) as a rational procedure for identification of marker chromosomes using a panel of primers differentially tagging the human chromosomes.

PRimed IN Situ labelling (PRINS) is a highly specific and sensitive technique for detecting DNA sequences on human chromosomes in situ. PRINS is currently being introduced for research and routine analysis in clinical and cancer genetics. In this paper, we report a rational PRINS procedure for the rapid identification of marker chromosomes. Using this method it is possible to test a sample from a patient with up to eight different primers simultaneously on one slide. We have synthesized oligonucleotide primers that can differentially tag the human chromosomes, and with the protocol presented in this report we are able to identify the chromosomal origin of a marker chromosome within 2 hours.

Amniocentesis↗
PubMed1997

Localisation of the classical DNA satellites on human chromosomes as determined by primed in situ labelling (PRINS).

The chromosomal localisation and relative amounts in humans of the classical DNA satellites I, II and III have been determined by using the primed in situ labelling reaction with a variety of oligonucleotide primers. The centromeres of seven of the human chromosomes, viz. nos 6, 8, 11, 12, 18, 19 and X, are not identifiably marked by any of the primers. A possible phylogenetic explanation of this is suggested and the possible relationship of the classical satellites to the function of the centromere is discussed.

Chromosome Mapping↗
PubMed1998

[Use of the primed in situ labeling technique for a rapid detection of chromosomes X, 18].

OBJECTIVE: To develop a rapid method for detection of chromosomes. METHODS: Chromosomes X, 18 were detected by the primed in situ labeling(PRINS) in eight samples of female peripheral blood cultures. RESULTS: Specific chromosomes were obtained on both metaphase and interphase nuclei. The efficiency of labeling was 84%-92%(mean=89%) in chromosome 18, and 73%-87%(mean=84%) in chromosome X. The pretreatment of slides by proteinase K markedly raised the efficiency of labeling and increased the intensity of signals. PRINS reaction could be performed automatically with a programmable thermocycler in less than one hour. CONCLUSION: The results suggest that PRINS is a fast and specific method for identifying chromosomes. It may be a reliable technique for detecting aneuploid in prenatal diagnosis.

Chromosomes, Human, Pair 18↗
PubMed1993

MULTIPRINS: a method for multicolour primed in situ labelling.

With this fast and simple MULTIPRINS protocol we show simultaneous mapping of multiple repetitive sequences by sequential cycles of primed in situ (PRINS) labelling with various primers and reporter molecules. Differentially labelled DNA sequences synthesized in situ were visualized by directly incorporated rhodamine-4-dUTP, by fluorescein isothiocyanate and Cy5 using digital imaging microscopy. This further development of the PRINS method enhances its potential as an alternative to traditional in situ hybridization.

Base Sequence↗
PubMed2005

Localization of repetitive DNA sequences on in vitro Xenopus laevis chromosomes by primed in situ labeling (PRINS).

Xenopus laevis is an important reference model organism used in many vertebrate studies. Gene mapping in X. laevis, in comparison to other reference organisms, is in its early stages. Few studies have been conducted to localize DNA sequences on X. laevis chromosomes. Primed in situ labeling (PRINS) is a recently developed innovative tool that has been used to locate specific DNA sequences in various organisms. PRINS has been reported to have increased sensitivity compared to other in situ hybridization techniques. In the present study, PRINS was first used to label the location of telomeres at the ends of in vitro X. laevis chromosomes. The terminal location was as expected from in vivo reports, however, the overall amount seemed to decrease in the in vitro chromosomes. Once the PRINS technique was optimized, this technique was used to determine the chromosomal location of the satellite 1 repetitive sequence, which is an important sequence in X. laevis development. The sequence was observed on the interstitial regions of the majority of the chromosomes similar to the in vivo locations reported. In contrast to the telomeric sequence, the amount of sequence appeared to increase in the satellite 1 sequence. PRINS was found to be useful in the localization of repetitive DNA sequences in the X. laevis genome.

Animals↗
PubMed1996

Assessment of aneuploidy for chromosomes 8, 9, 13, 16, and 21 in human sperm by using primed in situ labeling technique.

The incidence of aneuploidy was estimated for chromosomes 8, 9, 13, 16, and 21 in mature human spermatozoa by primed in situ (PRINS) labeling technique. This method allows us to perform a chromosome-specific detection by in situ annealing of a centromeric specific primer. A dual color PRINS protocol was adapted to human sperm. The decondensation and the denaturation of sperm nuclei were simultaneously performed by 3-M NaOH treatment. Double labeling of spermatozoa was obtained in <2 h. A total of 96,292 sperm nuclei were analyzed by two independent observers. The estimates of disomy were 0.31% for chromosome 8, 0.28% for chromosome 9, 0.28% for chromosome 13, 0.26% for chromosome 16, and 0.32% for chromosome 21. These homogeneous findings suggest an equal distribution of aneuploidies among autosomal chromosomes in males.

Adult↗
PubMed2004

[Detection of chromosomal aberrations in nasopharyngeal carcinoma by rapid primed in situ labeling].

BACKGROUND & OBJECTIVE: The chromosomal aberration is common in nasopharyngeal carcinoma (NPC). The currently-used methods for detecting chromosomal abnormalities are complicated and of limited clinical value. The purpose of the present study was to explore the feasibility of detecting chromosomal abnormalities in NPC by rapid primed in situ labeling (RPRINS). METHODS: Using RPRINS technique with specific oligonucleotide primers of chromosome 3 and 7, the abnormalities of chromosome 3 and 7 in the frozen section tissues of 15 cases of NPC and 5 cases of normal nasopharyngeal mucosa (NNM) were detected. Loss of chromosome was defined when the percentage of cells with labeling signals </=1 was >/=65%, and the increase in chromosomal copy number was defined when the percentage of cells with labeling signals >/=3 was >/=6.5%. RESULTS: In 15 cases of NPC tissues, the chromosome 3 had a labeling rate of 88.6% and increasing copy numbers in ten cases (66.7%), and the chromosome 7 had a labeling rate of 87.4% and loss of chromosome in five cases (33.3%). Four cases coexisted with increasing chromosome 3 copy numbers and loss of chromosome 7. In contrast, the labeling rates of chromosome 3 and 7 in NNM were 92.0% and 91.8%, respectively, and the percentage of diploid cells were 43.2% and 43.6%, respectively, with absence of triploid. There was a significant difference in the percentage of diploid cells between NPC and NNM (P< 0.05). CONCLUSION: The technique of rapid PRINS could be used to detect chromosomes in frozen section tissues, and the chromosomal abnormalities would be helpful in diagnosis of NPC.

Chromosome Aberrations↗
PubMed1998

Six-year-old archival chromosome preparations are still good biological reagents for repeated primed in situ labelling (rPRINS).

Six-year-old chromosome preparations of Eulemur coronatus were used for in situ localization of highly repetitive DNA sequences. The application of the repeated primed in situ labelling (rPRINS) technique allowed the detection of positive signals whereas conventional fluorescence in situ hybridization gave negative results on the same archival preparations. This paper reveals the possibility of rescuing archival chromosome preparations for both clinical and comparative cytogenetics. Moreover, the chromosomal localization of the ECO-SAT 503 bp highly repetitive DNA sequences were found to localize in the pericentromeric region of every chromosome, with the exception of chromosome 9.

Animals↗
PubMed2001

Use of primed in situ labeling (PRINS) for the detection of telomeric deletions associated with mental retardation.

Rearrangements involving the telomeric regions of human chromosomes are often associated with mental retardation. These rearrangements, however, are difficult to detect using conventional cytogenetic techniques. We propose the use of primed in situ (PRINS) labeling as an alternative to fluorescence in situ hybridization because it is very fast, reproducible, and simple to perform. Sixty-five children with unexplained mental retardation were studied using PRINS technology; two of them were shown to have a telomeric deletion.

Base Sequence↗