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1.
M. Menke J. Fuchs I. Schubert 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(8):1314-1320
The resolution of the chromosomal positions of six high- and one low-copy sequences by oligonucleotide-primed in situ (PRINS)
labelling was compared with corresponding data obtained after fluorescent in situ hybridization (FISH) on field-bean and barley
chromosomes. While PRINS proved to be suitable for the rapid detection of high-copy tandem repeats at the same loci as those
revealed by FISH, no clear PRINS signal was obtained for the low-copy family of vicilin genes at their locus on field-bean
chromosome II. This indicates that localization of short target sequences by primer extension via Taq polymerase in situ does not yet provide a resolution equal, or superior, to FISH on plant chromosomes. Therefore, the use
of a cocktail of chromosome-specific single-copy sequences as primers for PRINS is no alternative for the not as yet feasible
chromosome painting in plants.
Received: 21 April 1998 / Accepted: 12 May 1998 相似文献
2.
Use of the primed in situ labelling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y 总被引:6,自引:0,他引:6
Franck Pellestor Anne Girardet Geneviève Lefort Brigitte Andréo Jean Paul Charlieu 《Human genetics》1995,95(1):12-17
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. 相似文献
3.
Assessment of aneuploidy for chromosomes 8, 9, 13, 16, and 21 in human sperm by using primed in situ labeling technique. 总被引:4,自引:0,他引:4
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F. Pellestor A. Girardet L. Coignet B. Andro J. P. Charlieu 《American journal of human genetics》1996,58(4):797-802
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. 相似文献
4.
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.
Received: 5 February 1998; in revised form: 8 May 1998 / Accepted: 11 May 1998 相似文献
5.
A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS) 总被引:8,自引:0,他引:8
Franck Pellestor Anne Girardet Brigitte Andréo Jean-Paul Charlieu 《Human genetics》1994,94(4):346-348
The centromeric alpha satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence and cannot be easily distinguished by mean of probes for Southern blot or in situ hybridisation. We have used the oligonucleotide-primed in situ (PRINS) labelling technique with primers defined from the alpha satellite sequence of chromosome 13. One primer was found to label specifically the centromeric region of chromosomes 13 and allowed the detection of a polymorphism between two chromosome 13 homologues in one individual. 相似文献
6.
7.
J Koch J Hindkjaer J Mogensen S K?lvraa L Bolund 《Genetic analysis, techniques and applications》1991,8(6):171-178
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. 相似文献
8.
Primed in situ labeling: sensitivity and specificity for detection of alpha-satellite DNA in the centromere regions of chromosomes 13 and 21 总被引:4,自引:0,他引:4
The centromeric alpha-satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence. So far it has proven difficult to discriminate between sequence variations in the chromosome 13 and 21 alpha-satellite regions using in situ techniques. To analyze whether the method of modified single-color and double-color PRINS could be used to detect single nucleotide polymorphisms within this region, we used previously published primers D13Z and D21Z that differ in the terminal 3'-nucleotide and an additionally constructed primer "D13/21-test" lacking the final nucleotide at the 3' end. The results show that a one-base pair mismatch at the 3' end is sufficient to be detected by PRINS. Surprisingly, only about 35% of our samples exhibited the expected combination of two chromosomes 13 specifically labeled with only primer D13Z and two chromosomes 21 specifically labeled with only primer D21Z. The rest of the samples showed a polymorphic distribution of the target sequence for the primers, therefore these primers are not suited for routine detection of chromosomes 13 and 21 during interphase. Our data indicate that an interchromosomal exchange of alpha-satellite DNA takes place between chromosomes 13 and 21, possibly due to a concerted evolution process. 相似文献
9.
M. Kubaláková J. Macas J. Dolez˘el 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,94(6-7):758-763
The primed in situ DNA labelling (PRINS) procedure was optimised for the rapid physical mapping of several types of repetitive
DNA sequences on the mitotic chromosomes of Vicia faba, Pisum sativum and Secale cereale. A localization of the highly repeated FokI sequence on V. faba chromosomes was achieved after a 7-min total reaction time. In addition, we report a procedure for direct cycling-PRINS (C-PRINS),
a variation of PRINS which involves a sequence of thermal cycles analogous to the polymerase chain reaction. Compared to PRINS,
C-PRINS was more sensitive. Further work is needed to improve the sensitivity of the reaction to allow for the reliable detection
of low-copy DNA sequences.
Received: 17 September 1996 / Accepted: 18 October 1996 相似文献
10.
Franck Pellestor Anne Girardet Genevive Lefort Brigitte Andro Jean Paul Charlieu 《Molecular reproduction and development》1995,40(3):333-337
Direct in situ labeling of human spermatozoa was performed using the PRINS method. This technique is based on annealing of specific oligonucleotide primers, and subsequent primer extension by a Taq DNA polymerase. The reaction was carried out on a programmable temperature cycler, and labeling was obtained in a 1-hr reaction. The method was successfully tested with specific primers for chromosomes 13, 16, and 21. This suggests that PRINS may be a fast and reliable technique for detecting aneuploidies. © 1995 Wiley-Liss Inc. 相似文献
11.
Pellestor F Paulasova P Andréo B Lefort G Hamamah S 《Cytogenetic and genome research》2006,114(3-4):263-269
Both PRimed IN Situ (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 now constitute efficient alternatives to the conventional fluorescence in situ hybridization (FISH) procedure for in situ chromosome identification and aneuploidy detection. They present several advantages (specificity, speed, discriminating ability) that make them very attractive for a number of cytogenetic purposes. Multicolor PRINS and PNA protocols have been described for the specific identification of human chromosomes. Various applications have already been developed in human genetics and new adaptations are ongoing. 相似文献
12.
In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations 总被引:2,自引:0,他引:2
Ethanol fixation combined with microwave pretreatment allows rapid and simple detection of signals produced by cycling primed
in situ (PRINS) amplification, which uses a single primer, and in situ polymerase chain reaction (ISPCR) in intact cells.
After thermal cycling, signals remain as discrete subnuclear spots in the region of amplification and are clearly distinguishable
from non-specific background labelling. These methods are applicable to routine blood smears, even after Giemsa staining or
immunocytochemistry, and cellular morphology is retained. Chromosome enumeration by cycling PRINS is demonstrated using primers
for repeat DNA sequences, whilst single copy sequence detection is demonstrated using bcl-2, CFTR and chromosome 21 specific
primer pairs in ISPCR. We show that ethanol fixation supports efficient extension of cycling PRINS products to approximately
550 bp using up to 70 rounds of thermal cycling.
Accepted: 15 February 1999 相似文献
13.
Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ 总被引:21,自引:0,他引:21
Jørn E. Koch Steen Kølvraa Kirsten B. Petersen Niels Gregersen Lars Bolund 《Chromosoma》1989,98(4):259-265
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. 相似文献
14.
Creating a new color by omission of 3′ end blocking step for simultaneous detection of different chromosomes in multi-PRINS technique 总被引:5,自引:0,他引:5
In the multiple color primed in situ labeling (multi-PRINS) technique, the blocking step using ddNTPs, incorporated by a DNA polymerase, is an important procedure that blocks the free 3' end generated in the previous PRINS reaction, thus avoiding the next PRINS reaction using it as a primer to perform spurious elongation at non-desired sites. However, we found that omission of the blocking step never affected the correct identification of two chromosomes because the signals from the second PRINS reaction site always showed the pure original color. Nevertheless, taking advantage of the color mixing, we successfully used a multi-PRINS technique to create a third color using the two most common forms of labeled dUTP (biotin- and digoxigenin-labeled dUTP) and two fluorochromes (fluorescein and rhodamine) in order simultaneously to detect three chromosomes in the same cell. By arranging the labeling either in bio-dig-bio or in dig-bio-dig order in the sequential PRINS reaction, then detecting with a mixture of avidin-fluorescein/anti-dig-rhodamine or a mixture of anti-dig-fluorescein/avidin-rhodamine, the signals at the centromeres of three different chromosomes displayed perfect yellow, red and green colors, respectively. The entire procedure could be completed in less than 90 min because the blocking step was omitted. We showed that this is a practical and efficient way to carry out multiPRINS so that even more than three chromosome targets could be detected in the same cell. 相似文献
15.
Chromosomal localization of single copy genes SRY and SOX3 by primed in situ labeling (PRINS) 总被引:1,自引:0,他引:1
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. 相似文献
16.
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. 相似文献
17.
Use of primed in situ labeling (PRINS) for the detection of telomeric deletions associated with mental retardation 总被引:5,自引:0,他引:5
Bonifacio S Centrone C Da Prato L Scordo MR Estienne M Torricelli F 《Cytogenetics and cell genetics》2001,93(1-2):16-18
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. 相似文献
18.
In order to analyze male sterility caused by deletion of SRY and DAZ, we examined the accuracy and cost-effectiveness of a modified primed in situ labeling (PRINS) technique for detection of single-copy genes. Peripheral blood samples were collected from 50 healthy men; medium-term cultured lymphocytes from these samples were suspended in fixative solution and then spread on clean slides. We used four primers homologous to unique regions of the SRY and DAZ regions of the human Y-chromosome and incorporated reagents to increase polymerase specificity and to enhance the hybridization signal. PRINS of SRY and DAZ gave bands at Yp11.3 and Yq11.2, respectively, in all 50 metaphase spreads. The PRINS SRY signals were as distinct as those obtained using traditional fluorescence in situ hybridization (FISH). This new method is ideal for rapid localization of single-copy genes or small DNA segments, making PRINS a cost-effective alternative to FISH. Further enhancement of PRINS to increase its speed of implementation may lead to its wide use in the field of medical genetics. 相似文献
19.
A novel approach for simultaneous localization of two DNA sequences on plant chromosomes is described. The approach is based
on a combined use of primed in situ DNA labelling (PRINS) with fluorescent in situ hybridization (FISH). Traditionally, this
has been done using FISH with two probes labelled by two different marker molecules. Compared to this method, the combined
PRINS-FISH procedure is faster. Furthermore, because one of the DNA sequences is localized by PRINS with specific primers,
only one labelled probe is needed.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
20.
Sex genotypes in mature androgenetic and control rainbow trout Oncorhynchus mykiss were determined using primed in situ labelling (PRINS) detection of 5S rDNA sequences on the X chromosomes. Three sex genotypes, corresponding to phenotypic sex, were revealed: female XX, male XY and supermale YY. 相似文献