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.     

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.     

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.     

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     

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.     

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.     

Rapid detection of yeast rRNA genes with primed in situ (PRINS) labeling

In yeast, rRNA genes can be detected with the FISH technique using rRNA gene probes. This technique yields reliable, reproducible and precise results, but is time-consuming. Here, the primed in situ DNA synthesis (PRINS) procedure has been optimized for rapid detection of yeast rRNA genes. PRINS, which is as sensitive as PCR and allows cytological localization of analyzed sequences, can be adapted for various screening tests requiring fast labeling of rRNA genes.     

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.     

引物原位标记技术快速检测人类染色体非整倍性

染色体数目异常是人类染色体疾病的重要类型, 经常导致胚胎丢失、胎儿流产、婴儿死亡、先天畸形和神经发育异常等出生缺陷。文章应用引物原位标记(Primed in situ labeling, PRINS)技术快速检测人类染色体非整倍性, 率先采用更新的非ddNTP阻断的多色PRINS技术, 对人类外周血淋巴细胞和精子等多种样本进行标记; 然后对不同靶标序列的标记效率及不同荧光色素的发光特点通过实验进行评估, 获得关于PRINS技术的多项反应原理参数, 并筛选标记顺序以获得均一稳定的标记效果, 最后进行临床FISH探针与PRINS的标记比较实验。通过实验比较PRINS技术与传统FISH技术之间的标记特点与差别, 评估PRINS的实际应用效果。在2.5 h内标记了同一精子核内的多条染色体, 单色以上标记达到99％。同时在人类外周血淋巴细胞中也得到较好的标记效果。与FISH技术相比, PRINS的这些优点使得它成为诊断染色体非整倍性变异的首选技术。     

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.     

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. Received: 5 November 1996 / Accepted: 25 March 1997     

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.     

A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS)

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.     

Nonradioactive labeling and high-sensitive detection of PCR products

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactivc labeled nucleotides during PCR byTaq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin-(DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin-labeled amplification products as hybridization probes, sensilivities in the 0.1-pg range are obtained in Southern blot procedures.     

Rapid detection of chromosome X,Y, 13, 18, and 21 aneuploidies by primed in situ labeling/synthesis technique

AIMS AND OBJECTIVE:

Primed in situ labeling/synthesis (PRINS) technique is an alternative to fluorescent in situ hybridization for chromosome analysis. This study was designed to evaluate the application of PRINS for rapid diagnosis of common chromosomal aneuploidy.

MATERIALS AND METHODS:

We have carried out PRINS using centromere specific oligonucleotide primers for chromosome X, Y, 13, 18 and 21 on lymphocyte metaphase and interphase cells spread. Specific primer was annealed in situ, followed by elongation of primer by Taq DNA polymerase in presence of labeled nucleotides. Finally, reaction was stopped and visualized directly under fluorescent microscope.

RESULTS:

Discrete centromere specific signals were observed with each primer.

CONCLUSION:

PRINS seems to be a rapid and reliable method to detect common chromosome aneuploidy in peripheral blood lymphocyte metaphase and interphase cells.     

Detection of Klinefelter syndrome by G-banding analysis combined with primed in situ labeling technique

Primed in situ labeling (PRINS) technique is an alternative to in situ hybridization for rapid chromosome screening. We employed triple-color PRINS technique to detect chromosomal abnormalities in Klinefelter syndrome patients diagnosed by G-banding karyotype analysis. Among 1034 infertile male patients, 134 were found to be cytogenetically abnormal, including 70 with chromosomal number abnormalities and 64 with chromosomal structure abnormalities. Among these cytogenetically abnormal patients, 56 were diagnosed as having Klinefelter syndrome. PRINS technique was used on cultured lymphocyte metaphase cells of the Klinefelter syndrome patients; the same result was obtained with G-banding karyotype analysis. PRINS proved to be a rapid and reliable method to detect numerical chromosome abnormalities in peripheral blood lymphocytes in metaphase.     

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.     

Application of primed in situ DNA synthesis (PRINS) with telomere human commercial kit in molecular cytogenetics of Equus caballus and Sus scrofa scrofa

Recently, molecular techniques have become an indispensable tools for cytogenetic research. Especially, development of in situ techniques made possible detection at the chromosomal level, genes as well as repetitive sequences like telomeres or the DNA component of telomeres. One of these methods is primed in situ DNA synthesis (PRINS) using an oligonucleotide primer complementary to the specific DNA sequence. In this report we described application of PRINS technique with telomere human commercial kit to telomere sequences identification. This commercial kit may be use to visualization of interstitial telomeric signal in pig genome. PRINS is attractive complement to FISH for detection of DNA repetitive sequences and displays lower level of non-specific hybridization than conventional FISH.     

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