frog) 205 Mixophyes fasciolatus, M. schevilli (Australian ground frog) 239 mouse 7, 51, 62, 95, 100,

picta (painted turtle) 139 Drosophila melanogaster

Trisomy 10 as the only chromosome aberration is a rare phenomenon in malignant and premalignant hemopoietic disorders. We describe 7 new cases and have found another 12 in the literature. It appears that, whereas adult patients have myeloid disorders (acute myeloid leukemia, myeloproliferative, or myelodysplastic syndromes), in children the diagnosis is lymphocytic leukemia or lymphoma. The median survival was 122 months in the total material. Age above 60 years proved to be a significant adverse factor (median survival only 5 months; p = 0.003). None of the other clinical, cytogenetic, or hematological variables were of demonstrable prognostic importance. In contrast with the larger trisomy 10 clones, those of limited size were associated with nonleukemic diagnoses, normal or slightly elevated leukocyte counts, and few or no circulating blasts. This may suggest that expansion of the trisomy 10 clone is associated with clinical and hematological progression.

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.

Chapter 3 Detection of Nucleic Acids (DNA and RNA) In Situ by Single and Cyclic Primed In Situ Labelling (PRINS): Two Alternatives to Traditional In Situ Hybridization Methods J. Hindkjær, J. HindkjærSearch for more papers by this authorC. Terkelsen, C. TerkelsenSearch for more papers by this authorS. Kølvraa, S. KølvraaSearch for more papers by this authorJ. Koch, J. KochSearch for more papers by this authorL Bolund, L BolundSearch for more papers by this author J. Hindkjær, J. HindkjærSearch for more papers by this authorC. Terkelsen, C. TerkelsenSearch for more papers by this authorS. Kølvraa, S. KølvraaSearch for more papers by this authorJ. Koch, J. KochSearch for more papers by this authorL Bolund, L BolundSearch for more papers by this author Book Editor(s):Melody Clark, Melody ClarkSearch for more papers by this author First published: 13 January 1996 https://doi.org/10.1002/9783527615070.ch3Citations: 2 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Summary The chapter contains sections titled: Introduction Materials and Chemicals DNA-PRINS with Oligonucleotide Probes PRINS with Ddel Digested Cloned Probes Multicolour-PRI NS PRINS-painting PRINS-PCR and Repeated-PRINS of DNA PRINS-PCR of mRNA Visualization of Hapten-Labelled Nucleotides Troubleshooting Citing Literature In Situ Hybridization: Laboratory Companion RelatedInformation

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.

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.

In this report we describe a partial triplication (7q) and other structural aberrations found in a child with acute lymphoblastic leukemia (ALL), and we demonstrate the importance of PRimed IN Situ labeling (PRINS) and chromosome painting as a support to banding analysis for the clarification of complex structural chromosome rearrangements. Initially, the der(7) was interpreted as der(7)t(1;7;7). However, PRINS and chromosomes painting showed that der(7) consisted entirely of chromosome 7 material. Further, a derivative chromosome interpreted by banding analysis as a der(17)t(?1;17) was shown to be der(17)t(13;17) by the newly developed PRINS painting technique.

We present the sequences of a set of oligonucleotides that, when used as primers for PRimed IN Situ (PRINS) labeling, are diagnostic for repetitive sequences in specific human chromosomes. Combined, they enable identification of all human chromosomes except 6, 19, and 20. However, as is also the case with cloned centromeric hybridization probes, chromosomes 14 and 22 are stained together. Along with the sequences of these oligonucleotides we offer a simple, universal procedures for their use. We also present an oligonucleotide that binds to both strands of alpha-satellite DNA, making it possible to specifically amplify alpha-satellite DNA by PCR with this one oligonucleotide alone as primer. The origin of the alpha-satellite DNA in the starting material (whether somatic cell hybrids, flow-sorted chromosomes, or microdissected material) can then be determined on test metaphase spreads by in situ hybridization or PRINS with the PCR product.

As described, the PRINS method is a very rapid and reliable way of staining human telomeres. To obtain the maximum frequency of stained telomeres, the primer (CCCTAA)7 should be used, although the average frequency never quite reaches 100 %. The frequency is strongly dependent on the age of the individual, being significantly higher in children and newborns than in adults. A difference between the (CCCTAA)7 primer and the complementary primer is demonstrated and a possible explanation is proposed, namely, that gaps in the C-rich strand cause chain elongation termination after the addition of only one dTTP molecule.

Primed in situ labeling (PRINS) and fluorescence in situ hybridization (FISH) are methods for visualizing specific DNA sequences directly on chromosome spreads, thereby demonstrating the presence of a specific sequence in a certain cell and at the same time localizing the sequence at a specific site on the chromosome. This chapter describes protocols for the fluorescence labeling of specific sequences in chromosomes fixed to a microscope slide using either PRINS or FISH. For the labeling it uses either the biotin-avidin or the digoxigenin-antidigoxigenin system, conjugated with various fluorochromes. PRINS and in situ hybridization are both methods originally developed for microscopy of standard metaphase chromosome spreads. The two techniques have been used in many situations, in gene mapping, in the deciphering of complex chromosome aberrations, and in the simple determination of aneuploidy. For a number of applications PRINS and traditional in situ hybridization are equally effective. This applies to situations where cloned probes are used for detection of highly repeated sequences.

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.

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.