Non-isotopical labeling of murine heterochromatin in situ by hybridization with in vitro-synthesized biotinylated gamma (major) satellite DNA

Degenerate probe DNA, homologous to part of the 234-bp repeated mouse gamma (major) satellite DNA, was generated by primer-directed in vitro DNA amplification using the polymerase chain reaction with oligonucleotide primers that anneal in the most conserved parts of the repeat. Probe labeling with biotin was performed during DNA polymerization. In situ hybridization of probe DNA with metaphase chromosome preparations showed exclusive binding of probe molecules to the centromeric region of mouse chromosomes. We applied the probe DNA for labeling of mouse heterochromatin in metaphase chromosomes, as well as interphase cell nuclei, and compared results of probe visualization using avidin tagged with either fluorescein or alkaline phosphatase in combination with a chromogenic substrate.     

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.     

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.     

PRINS detection of 18S rDNA in pig, red fox and Chinese raccoon dog, and centromere DNA in horse

The fluorescence in situ hybridization (FISH) technique is widely used in animal cytogenetics. Contrary to FISH procedure, primed in situ DNA synthesis (PRINS) does not require the DNA probe preparation (design, synthesis, gel purification of PCR products and labeling). The PRINS method with primers used as 'DNA probes' is both PCR-sensitive and allows for chromosomal localization of DNA sequences. Here, we show the application of PRINS reaction with one unlabeled oligonucleotide pair to identify 18S rDNA loci in three different animal species: domestic pig (Sus scrofa), red fox (Vulpes vulpes) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides). We present the data of indirect labeling with the digoxigenin-PRINS using two different pairs of primers complementary to centromeric region of horse (Equus caballus) chromosomes. Our new PRINS application may be considered as a useful tool for chromosome investigation in the field of domestic and wild animal genetics and evolution.     

The labeling efficiency of human telomeres is increased by double-strand PRINS

Telomeres are composed of tandem repeated sequences, TTAGGG, that can be detected either by fluorescence in situ hybridization (FISH), more efficiently by using a peptide nucleic acid (PNA) probe, or by the primed in situ (PRINS) technique. However, the efficiency of human telomere labeling using PRINS is somewhat lower than the efficiency using PNA-FISH. To solve this problem, we developed a double-strand PRINS technique, which uses two primers, (TTAGGG)₇ and (CCCTAA)₇, to label both forward and reverse telomeric DNA strands. A total of 120 lymphocyte metaphases obtained from three normal adults were scored to evaluate the labeling efficiency based upon the telomere signal frequency present in chromatid ends and chromosome arms. As a comparison, 30 metaphases from the same three individuals were evaluated using PNA-FISH. The average labeling efficiency of PRINS was increased to a level very close to that obtained with PNA-FISH. Therefore, we demonstrated that the low labeling efficiency of human telomeres with regular PRINS was likely caused by uneven annealing of primers at the relatively short human telomere sequences, resulting in some telomere sites with very weak or absent labeling. We suggest that the present double-strand labeling protocol is critical to maximize the labeling efficiency of the human telomere sequence when using the PRINS technique.     

Primed in situ labeling for detecting single-copy genes

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.     

Primedin situ labeling (PRINS)

PRimedIn Situ labeling (PRINS) is a fast and sensitive alternative to fluorescencein 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.     

A combined PRINS-FISH technique for simultaneous localisation of DNA sequences on plant chromosomes

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.     

Single- and double-color oligonucleotide primed in situ labeling (PRINS): applications in pathology

 Molecular cytogenetics is mostly performed by fluorescence in situ hybridization using long DNA probes that are generated by vector cloning. Oligonucleotide primed in situ labeling (PRINS) is a recent method that has been established for the detection of the centromeric or telomeric region in metaphase chromosomes. In this overview, we demonstrate the possible applications of PRINS and provide elaborated protocols for its use in intact interphase cells of routine cytological preparations, e.g., cell smears, touch preparations, and cytospins of non-neoplastic and neoplastic tissues. Moreover, the various modifications of the PRINS method, such as multi-color PRINS for targetting different chromosomes within one cell or the enzymatic detection of the PRINS product instead of the more commonly used fluorochromes, are discussed. Accepted: 4 July 1997     

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. Received: 5 February 1998; in revised form: 8 May 1998 / Accepted: 11 May 1998     

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.     

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.     

In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations

 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     

PRINS as a method for rapid chromosomal labeling on human spermatozoa

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.     

Chromosomal localization of single copy genes SRY and SOX3 by primed in situ labeling (PRINS)

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.     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

Application of the primer in situ DNA synthesis (PRINS) technique to titer recombinant virus and evaluation of the efficiency of viral transduction

Titration is an important and critical step in dosing recombinant virus for gene therapy. We present a relatively fast, convenient, and sensitive method that allows for precise quantification of recombinant retrovirus. The method is based on PCR amplification of a foreign gene by the PRINS (primer in situ DNA synthesis) technique. The PRINS technique is based on the sequence-specific annealing of unlabeled oligonucleotide DNA in situ. This oligonucleotide operates as a primer for in situ chain elongation catalyzed by the Taq I polymerase. Using digoxygenin-labeled nucleotides as a substrate for chain elongation, the neo-synthetic DNA is labeled by an FITC-conjugated anti-digoxygenin antibody. To avoid the possibility of false positives, we amplified the puromycin-resistance gene, which is associated with the transgene in the same viral vector and is not normally present in mammalian cells. The retroviral titer was evaluated by counting fluorescein isothiocyanate-positive cells after PRINS labeling, while knowing the number of plated cells that were transduced with different amounts of viral supernatant. A comparable viral concentration of 1 x 10(7) infectious units/mL was found among the retroviruses.     

Primed in situ labeling: sensitivity and specificity for detection of alpha-satellite DNA in the centromere regions of chromosomes 13 and 21

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.     

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.     

Digoxigenin-labeled probes can detect single-copy genes in human metaphase chromosomes

A technique of in situ hybridization on metaphases of chromosomes by a digoxigenin-labeled probe is described. This technique was able to detect single DNA sequences of 2 and 7 kilobases. The results obtained were compared with those of a biotin streptavidin alkaline phosphatase-based detection system. The digoxigenin method was at least as efficient and sensitive as the biotin-streptavidin method.