Development and Adaptation of the Prins Technology

An Overview

Franck Pellestor

Summary

The advent of molecular genetic techniques has brought forth new procedures for in situ chromosomal analysis. One of these techniques is the primed in situ labeling (PRINS) procedure, which constitutes a fast and efficient alternative to conventional fluorescence in situ hybridization for nucleic acid detection. Based on the use of chromosome-specific primers, the PRINS method combines the high sensitivity of the PCR reaction with the cytological localization of DNA sequences. Since its introduction, the PRINS protocol has been optimized, and numerous applications have been developed. The technique has thus proved to be a useful tool for in situ chromosomal screening, and has become a simple and efficient complement to conventional and molecular cytogenetic methods.

Key Words: PRINS; primer; a-satellite sequences; chromosomal aneuploidy; unique sequence detection.

1. Introduction

The advent of molecular genetic techniques has led to the development of a new approach for cytogenetic screening in interphase nuclei, called interphasic cytogenetics. This strategy has become an important tool for chromosome research and cytogenetic diagnosis because it bypasses the culture phase required to generate metaphases in conventional cytogenetic procedures and to perform analysis on rarely dividing or nondividing cells that previously were not available to cytogenetic investigations. One of these techniques is the primed in situ labeling (PRINS) procedure, which constitutes an efficient alternative to conventional fluorescence in situ hybridization (FISH) technique for chromosomal screening. Both FISH and PRINS were developed around the same time (1,2). Because it is relatively easy to implement and perform and because a large variety of DNA probes and in situ hybridization kits are

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed.

Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

commercially availability, FISH has become the technique of choice for chromosomal investigations in both medical institutions and research centers. During the past decade, the parallel development of in situ detection systems and sophisticated microscopy has resulted in the introduction of multiple advanced FISH techniques, which have revolutionized the practice of human cytogenetic (3,4).

Compared with such a well-established technology and its innovative techniques, the PRINS procedure might appear of minor interest and of limited usefulness. However, the PRINS reaction presents several advantages, such as rapidity, high sensitivity, and high specificity, making the PRINS technique an attractive complement to FISH for in situ aneuploidy detection and study of subtle chromosomal rearrangements.

2. Principles and Methods of PRINS

The PRINS reaction combines the high sensitivity of the polymerase chain reaction (PCR) with the cytological localization of specific DNA sequences. The key to the PRINS technique is the use of unlabeled, short, and specific oligonucleotides. Oligonucleotides are annealed in situ to complementary DNA targets on denaturated chromosome spreads, nuclei, or tissue sections and then act as primers for chain elongation catalyzed by a Taq DNA polymerase in the presence of free nucleotides. The visualization of generated fragments results from the incorporation of one labeled nucleotide.

2.1. Definition of PRINS Primers

In their original report, Koch et al. (2) reported the in situ labeling of consensus centromeric repeated sequences. Subsequent studies identified oligonucleotides useful in identifying each type of repeat DNA sequences spreading over the human genome, that is, satellite, telomeric, or Alu DNA sequences (5). A great advantage of primers is their ability to differentiate between closely related sequences. This feature has been used for generating chromosome-specific primers from the a-satellite DNA motif (6,7). a-Satellite (or alphoid) DNA is a family of tandemly repeated sequences present at the centromere of all human chromosomes. These centromeric repeats are made up of a variable number of monomeres of 171 base pairs (bp) in length and are organized as a-satellite subfamilies. The DNA sequences of momomeres slightly deviate among subfamilies and individual chromosomes. The chromosome specificity of PRINS is based on the use of primers generated from these chromosome-specific a-satellite DNA sequences. The complementation process between the primer and its centromeric target will be so specific that a simple mismatch between the 3'-end of the primer and the genomic sequence will prevent initiation of the in situ elongation by the Taq DNA polymerase. Thus, it has been possible to define specific a-satellite primers for most of the human chromosomes, involving some chromosomes undistinguishable by conventional FISH with centromeric probes. This is the case with chromosomes 13 and 21, which share 99.7% homology in their a-satellite DNA sequences (8). To date, specific primers have been defined for 20 human chromosomes (see Chapter 6). Efficient primers have also been defined for in situ labeling of telomeres and studying variant telomeric repeats (9,10).

Using an automatic DNA synthesizer, both the preparation and the purification of oligonucleotides are now fast and rather inexpensive. The length of the PRINS primers ranges from 18 to 35 nucleotides. Compared with the size of DNA repetitive probes (250-600 bp), this small size greatly facilitates their in situ accessibility to their genomic target sequences, which is particularly significant in cells with highly condensed nuclei, such as spermatozoa. The use of oligonucleotide primers eliminates the need for probe preparation and can also circumvent the lack of resolution of DNA probes (approx 5-10 kb in interphase nuclei), because every known DNA sequence can be a potential primer source for PRINS reaction. As an alternative to oligonucleotides, cloned probes fragmented by restriction enzyme digestion also can been used as primers (11).

2.2. Coming of Age and Advanced PRINS Procedures

Initially, PRINS reactions were performed either on a hotplate or a water bath. The required changes of temperature were achieved by moving the sides as often as needed. The weakness of these methods was in the lack of stringency of primer annealing and high background. These procedures did not allow precise and durable temperature control. Indeed, although the range of Taq DNA polymerase activity is large enough, the optimization of the annealing and its stringency to increase specificity need accurate temperature control. The protocol has been considerably improved and simplified by the introduction of programmable temperature cyclers equipped with a flat plate block. With this equipment, the precision of temperature control may reach 0.2°C and the required temperature changes are both easy to program and rapidly conducted. The use of automatic thermocyclers allows an optimization of both annealing and extension conditions. Thus, semiautomatic PRINS protocols were developed offering a high reproductibility in labeling reaction. An additional improvement was the direct use of various fluorochromes and the introduction of the multiple-color PRINS reaction (7,12), allowing the sequential labeling of several chromosomal targets on a same cell preparation. By incorporating directly fluorochrome-labeled nucleotides into newly synthetized DNA fragments, the turnaround time of the PRINS reaction was reduced to no more than a few minutes, leading to the development of ultrafast PRINS protocols (13). Attempts to increase sensitivity also involved the use of serial cycles of PRINS reactions for accumulating labeled copies of the target sequence at the site of synthesis (14,15). However, this process, called cycling PRINS, also could alter the specificity of the signal and quality of the chromosome preparation (16).

In practice, a PRINS mix is prepared in a final volume of 50 |L containing the oligonucleotide (50-250 pM); the nucleotide mixture, including a labeled dUTP (biotine, digoxigenine, fluoresceine, coumarine, rhodamine, cyanine, etc.), the Taq polymerase buffer; and 1 to 2 U of the enzyme. Because they are unlabeled, high amounts of primers can be used in PRINS reaction without inducing background signals. Both chemical and thermal denaturations of the preparation slide can be used. Thus, a classical PRINS procedure consists of two programmed steps (annealing at the specific annealing temperature of the used primer, and nucleotide chain elongation at 72°C) in case of preliminary chemical denaturation (in 70% formamide, 2X standard saline citrate [SSC] at 72°C), whereas the thermic denaturation of the preparation is directly involved in the PRINS reaction, then consisting of three programmed steps (denaturation at 94°C, annealing, and elongation). Usually, slides and cover slips are not sealed. Both the volume of the mix and the short incubation time prevent the slide from drying during the reaction. In conventional multitarget PRINS reaction, a blocking step is used between each chromosome-specific PRINS reaction to prevent the mixing of labeling and to ensure a good recognition of each targeted site. The slide is thus treated at 37°C with a dideoxynucleotides mix and Klenow enzyme, which block the free 3'-ends of the elongation fragments generated by the previous PRINS reaction. Detection of the labeling sites is performed by fluorescence microscopy. Initial PRINS procedures used primers labeled with biotine or digoxigenine, and the detection of the labeling sites required immunocytochemistry procedure for detecting reporter molecules. Now, most of the PRINS reactions are performed using directly fluorochrome-labeled nucleotides, which allows the direct visualization of targeted sequences on fluorescence microscope.

Recently, a new multicolor PRINS protocol has been reported, allowing the performance of ultra-rapid detection of several chromosomes only by mixing the different fluorochromes during the chain elongation reaction (17). In this sequential procedure, each PRINS reaction consists of a unique short step (4-6 min) for annealing and elongation of each chromosome-specific primer. This ingenious strategy, which eliminates the intermediate blocking reaction, greatly simplifies the PRINS protocol and facilitates its adaptation to various cytogenetic applications (see Chapter 1 and 6).

Compared with the conventional FISH technique, the PRINS method has proven to be equally accurate for detecting chromosome, quicker, and less expensive. Werner et al. (18) estimated that the PRINS reaction costs approximately one-tenth as much as FISH.

3. Applications of PRINS

The PRINS procedure combines several features that make it very attractive for a number of cytogenetic purposes. Although PRINS initially was limited to the detection of repeat sequences, numerous applications of PRINS technology have been developed in mammals, fish, insects, and plants, demonstrating that PRINS could be easily adapted to various types of cells.

In Drosophilia, PRINS was used for gene mapping on polytene chromosomes (19), and in fish, the localization of Alu repeat and nuclear organizer regions was performed by PRINS (20,21). In plants, PRINS was used for labeling telomeres (22) and gene clusters (23). The technique also was adapted to suspensions of bean chromosomes to facilitate the flow sorting and karyotyping of similar-sized chromosomes (24). In mammalian cells, a large variety of PRINS applications have been described, and the technique has been integrated into various protocols of research. For instance, Russo et al. (25) used PRINS for studying mechanisms of aneuploidy in mouse splenocytes. Because of its high sensitivity, the PRINS technique was also chosen for telomeric studies in various species (26,27).

In humans, PRINS method has been successfully adapted not only in the assessment of aneuploidy in lymphocytes, amniocytes, and preimplantation embryos (28-30) but also in the analysis of structural aberrations, such as translocations, marker chromosomes, and ring chromosomes (31). More recently, the PRINS protocol has been adapted to detect fetal cells among separated maternal nucleated cells from peripheral venous blood of pregnant women, demonstrating the efficiency of the technique for noninvasive prenatal chromosome analysis (32,33). As the PRINS reaction with Alu primers gives high quality R-like banding on human chromosomes, the procedure has been adapted for the cytogenetic screening of somatic hybrid cell lines (34) and identification of euchromatin in aberrant short arms of acrocentric chromosomes and small ring chromosomes (35). Further applications have been found in tumoral cytogenetics. To date, preliminary studies involve the detection of host cells after sex-mismatched bone marrow transplantation in patients grafted for leukemia and the cytogenetic screening of tumoral cell lines (36). The PRINS technique also can be performed in both frozen and paraffin-embedded tissue sections or to localize RNA within cells, which generates new possibilities for cytogeneticists and pathologists (37). All these applications point out the potential efficiency of PRINS method for diagnostic use.

Taking advantage of the high sensitivity and specificity of PRINS reaction, some laboratories have successfully adapted the technique to the in situ detection of RNA (38) and the quantitation of RNA species in cell suspensions by flow cytometry (39). In the same way, Andersen et al. (40) use a variation of PRINS (the self-PRINS) to study the methylation status of CpG islands in human chromosomes. By using variant telomeric primers, Krejci and Koch (41,42) clearly demonstrated the superiority of PRINS for measuring telomeric length and identifying polymorphic telomeric repeats. Studies have also shown that PRINS and FISH could be used in concert for simultaneous detection of chromosome targets (43,44).

One of the most remarkable and efficient applications of the PRINS technique has been conducted on human spermatozoa. The adaptation of PRINS technology to male gametes constituted an interesting challenge because of the particularities of sperm nucleus in term of genomic compaction and accessibility of DNA sequences. The PRINS methodology has been combined with NaOH treatments, which allows the simultaneous decondensation and denaturation of sperm nuclei. In PRINS reaction, the decondensation of the sperm head is a less limiting factor than in FISH because of the small size of the oligonucleotide primers. This facilitates their penetration into sperm nuclei and their access to the genomic target sequences, resulting in a more efficient and rapid labeling of sperm nuclei. Using standart PRINS technique, diploidy and disomy frequencies have been estimated for most of the human chromosomes in sperm samples from several men (45), and studies of meiotic segregation pattern were performed in sperm samples from reciprocal translocation carriers (46). Recently, the new multicolor PRINS protocol has been adapted successfully on human spermatozoa (47), as well as on human oocytes and blastomeres (48).

The adaptation of PRINS technique to the in situ detection of unique sequences has constituted an important challenge because this procedure could allow to rapidly map genes on chromosomes by simply using synthetic oligo-nucleotides derived from sequenced DNA. The advantage of the PRINS approach is that it does not rely on the possession of a cloned probe for the target. As long as the sequence of the gene is known, oligonucleotides can be synthesized for use as primers. Several teams have worked on this new application of PRINS. Preliminary results have been obtained on porcine chromosomes where unique sequences as short as 100 to 300 bp were localized by using pairs of specific primers (49). More recently, the procedure has been optimized to detect single copy loci on human chromosomes. Thus, Kadandale et al. (50) first localized the SRY gene in situ, and several further reports showed that PRINS could be used for gene mapping (16,51) but also for the diagnosis of microdeletion syndromes and cancer abnormalities (52,53), opening news and promising perspectives for PRINS in the field of physical mapping.

4. Conclusion

PRINS has emerged as a research technique, and since its introduction, the technique has undergone many stages of development to improve the sensitivity, the efficiency, and the versability of the process. The PRINS technique has moved from research benches to diagnostic laboratories, and numerous studies have demonstrated that PRINS is as reliable and more efficient than FISH for detecting chromosomes in metaphase and interphase nuclei. With the development of rapid and simplified protocols producing reliable and reproducible results, PRINS has become a powerful tool for cytogenetic investigations and diagnosis. During the last few years, PRINS has progressed from the labeling of repeat sequences to the use for the detection of unique sequences. New innovative variations to PRINS, such as padlock probes or rolling circle PRINS (see Chapter 5), have been introduced (54,55), which leads to think that the PRINS technology could provide an efficient complement to FISH and PCR in various diagnostic and research situations.

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