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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Prins and C-PRINS: Promising tools for the physical mapping of the lupin genome

Two molecular cytogenetics methods, PRINS (primed in situ DNA labeling) and C-PRINS (cycling PRINS), were optimized for the physical mapping of several types of DNA sequences on the mitotic chromosomes of the narrow-leafed lupin (Lupinus angustifolius L.). The fragment of the FokI element from Vicia faba was localised by indirect PRINS reaction. Two other sequences, fragments of the coding sequences of L. luteus and of L. angustifolius, were localised by indirect C-PRINS. These techniques are faster and more sensitive than FISH, and they allowed the mapping of short DNA fragments. The data obtained shows that both types of PRINS are valuable tools for chromosome identification in lupin.     

应用引物原位标记技术检测21号染色体

应用原位引物标记技术（PRINS）检测了21号染色体着丝粒,在外周血和绒毛细胞的标记效率分别为91％和93％,实验过程可以在2h之内完成,证明这一检测方法是一种快速、灵敏、特异性良好的染色体数目检测方法,有可能用于21号染色体数目异常的快速诊断。     

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。     

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     

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.     

From the conception of the PRINS to its coronation

As a non-isotopic molecular cytogenetic technique, the primed in situ (PRINS) labelling reaction represents a major technological progress achieved in the past decade. It has become a routine technique for the microscopic visualization of specific DNA sequences in cells and nuclei and constitutes a good alternative to the fluorescence in situ hybridization (FISH) procedure. Among the multiple advantages that characterize the PRINS technique, specificity, rapidity, reliability, reproducibility, and cost-effectiveness can be mentioned. PRINS can be in addition associated with other techniques like FISH, indirect immunofluorescence, and nick translation. The most recent developments show the great potential of this technique. Now PRINS can be used to study single-copy genes and, consequently, can be routinely used to investigate deletions associated with microdeletion syndromes. Therefore, the PRINS technique has the potential to become a widely used molecular cytogenetic tool in clinics and research. This short review presents how the PRINS technique contributed to further the understanding of biological phenomena and describes the different possibilities and applications of the PRINS method in several biological and clinical fields (pre-implantation testing, prenatal, constitutional and oncologic genetic diagnosis).     

A comparison of sequence resolution on plant chromosomes: PRINS versus FISH

 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     

Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification

Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 °C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus.     

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.     

Creating a new color by omission of 3′ end blocking step for simultaneous detection of different chromosomes in multi-PRINS technique

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.     

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.