Quantitative PRINS

With the PRINS design, high concentrations of small probes that penetrate the specimen well can be used, making targets effectively saturated and the reaction quantitative. This is particularly true for the dideoxy-PRINS, which is used for measuring the size of telomeric repeat domains at individual chromosome ends, as well as for sizing of other simple repeat domains that vary in size, such as trinucleotide repeats.

Evaluation of quantitative PRINS reactions can be done in either of two ways. One way is by counting signals and calculating the fraction of potential targets actually stained (the author typically counts 100 potential targets): The higher the staining frequency, the larger the target (8). With dideoxy-PRINS, this often becomes impractical because the staining efficiency in many cases is close to 100% (e.g., 99.8% for telomeric repeats in normal blood lymphocytes). In such cases, it is preferable to measure the light intensities of the PRINS signals instead and average those over 10 metaphases. I use the pre-existing image analysis program QUIPS from VYSIS. This program was designed for CGH and compares the intensity of a test sample to the intensity of a reference sample. For PRINS analysis, the signals are designated as "tester" and the DAPI coun-terstain as "reference," and the result is thus a signal-to-counterstain ratio that is directly proportional to the amount of target sequence (1,2). The adaptation to the program has the derived advantage that some of the factors causing artificial variation in signal intensities (e.g., uneven illumination) have a similar influence on the counterstain, leaving the ratio less affected than the absolute values (see Note 6).

4. Notes

1. The PRINS technique relies strongly on the intact nature of the target sequences— nicked or broken DNA is a poor template for chain elongation in situ—and freshly prepared slides are the preferred starting point for an optimal reaction. If good slides cannot be prepared, accessibility can be increased by treatment with a pro-teinase (DNase-free), vulnerable chromatin can be stabilized by fixation with paraformaldehyde, and damaged DNA can be repaired with a DNA ligase (10). The chromatin in "aged" slides may be so hardened that it requires long incubations to denature it by heat, for example, 10 min for a slide aged 1 mo at room temperature. An occasional problem in PRINS is "self-labeling" of certain regions of satellite DNA. In human chromosomes, satellite III on chromosome 9 and, more rarely, satellite II on chromosomes 1 and 16 may self-label. This self-labeling is somewhat dependent on the primer but may occur with any primer. Some primers always induce the self-labeling, but with most primers it is only seen in low quality chromosome spreads.

2. Glycerol prevents evaporation and enhances the reaction in concentrations of 5 to 20%. If the nucleotides, and possibly the buffer, are stored in 50% glycerol, that concentration may be reached without the addition of extra glycerol (the oligonucleotide probes also may be stored in 50% glycerol, in which case all reagents in the reaction mixture (except for the water) can be pipetted directly from -20°C).

3. Reaction mixture without polymerase may be stored at -20°C for months.

4. When in use, it is preferable to store the blocking solution at 4°C and not use it for more than 1 wk (the milk turns sour with time, which may also happen to the milk powder if it sucks water from the air).

5. Probes for simple repeats anneal rapidly, more complex probes more slowly, and chain elongation is virtually instantaneous; therefore, the annealing time determines the incubation time.

6. In collaboration with Peter Lansdorp, DAKO has released an analysis program that is tailor-made for telomere signal quantification. This program operates with absolute intensity values.

References

1. Krejci, K. and Koch, J. (1998) Improved detection and comparative sizing of human chromosomal telomeres in situ. Chromosoma 107, 198-203.

2. Serakinci, N. and Koch, J. (1999) Detection and sizing of human telomeric repeat DNA in situ. Nat. Biotech. 17, 200-201.

3. Krejci, K. and Koch, J. (1999) An in situ study of variant telomeric repeats. Genomics 58, 202-206.

4. Serakinci, N. Krejci, K., and Koch, J. (1999) Telomeric repeat organization — a comparative in situ study between man and rodent. Cytogenet. Cell Genet. 86, 204-211.

5. Koch, J., K0lvraa, S., Gregersen, N., and Bolund, L. (1989) Oligonucleotide-prim-ing methods for the chromosome-specific labeling of alpha satellite DNA in situ. Chromosoma 98, 259-265.

6. Kadandale, J. S., Tunca, Y., and Tharapel, A. T. (2000) Chromosomal localization of single copy genes SRY and SOX3 by primed in situ labeling (PRINS). Microb Comp Genomics 5, 71-74.

7. Kadandale, J. S., Wachtel, S. S., Tunca, Y., Wilroy R. S. Jr., Martens, P. R., and Tharapel, A. T. (2000) Localization of SRY by primed in situ labeling in XX and XY sex reversal. Am. J. Med. Genet. 95, 71-74.

8. Therkelsen, A. J., Nielsen, A., Koch, J., Hindkjaer, J., and K0lvraa, S. (1995) Staining of Human telomeres with primed in situ labeling (PRINS). Cytogenet. Cell Genet. 68, 115-118.

9. Koch, J., Hindkjœr, J., K0lvraa, S., and Bolund, L. (1995) Construction of a panel of chromosome specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ. Cytogenet. Cell Genet. 71, 142-147.

10. Koch, J., Hindkjœr, J., Mogensen, J., K0lvraa, S., and Bolund, L. (1991) An improved method for chromosome-specific labelling of alpha satellite DNA in situ using denatured double stranded DNA probes as primers in a PRimed IN Situ labelling (PRINS) procedure. Genet. Anal. 8, 171-178.

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