Methods

The following protocol largely corresponds to the recently published procedure for the genotyping of individual mitochondrial genomes in situ (2) and was derived in collaboration with Mats Nilsson and Ulf Landegren at Uppsala University. It should be pointed out that it is an experimental protocol, and some considerations should be made before applying it:

• The detection efficiency for a given target is, for now, 1-10%. We are working to increase it, but for the time being, there is this limiting factor to consider.

• The protocol is for detection on standard chromosome slides. Preliminary results suggest that enhanced reactions can be obtained in some sort of well format. This

Fig. 2. Detection of a gene in the long arm of human chromosome 6. The efficiency of the reaction is not 100%, so only one chromosome 6 presents with a signal. This signal consists of a well-defined spot on one chromatid and a long thread of DNA looping out from the other chromatid. This long thread of DNA was the product of a reaction where the rolling-circle PRINS proceeded for an extended period of time, and the rolling circle at some point wandered out of the chromatin. However, even in this extreme case, it is clear at which locus the signal originated. (Please see color insert following p. 48.)

would be in agreement with findings in other in situ reactions that consume large amounts of reagents locally, such as the cycling PRINS (9).

• All possible steps have been included. We have not been able to document an effect of the RNase treatment, the pepsin may not be relevant for chromosome spreads (Fig. 2), preformed circles do not require ligation in situ, and at least some of the dehydrations may be replaced by equilibration in the subsequent reaction buffer. Furthermore, detection of endogenous 3'-ends does not require a restriction digest. Thus, although the full procedure can be completed within a single working day, it may still be possible to cut it down for individual applications.

Decide how large a region of the slide is to be used and choose a cover slip and a volume of reaction mixture that fits the area. In general use, 1 pL of mixture for each millimeter in cover slip length. To cover a standard slide completely, prepare 60 pL of each mixture and cover with a 24 x 60-mm cover slip. To cover a smaller area, prepare less mixture (and use a smaller cover slip). Rinse the slide in a fairly neutral, low-salt buffer without excessive buffering capacity (e.g., PBS or SSC).

Put slide into preheated pepsin solution and incubate 1 min at 37°C. The duration of this step is critical. Try to avoid over- or underdigestion of samples. Rinse the slide in the same buffer used in step 2. Dehydrate the specimen by immersing the slide successively in an ethanol series (3 min each in 70%, 90%, and 99% ethanol). Drain off the ethanol and air-dry the slide. The reaction can be paused after this and all subsequent dehydrations.

Incubate the slide with the RNase A mixture under the cover slip for 30 min at

37°C in a humidified incubator.

Rinse, dehydrate, and air-dry the slide as in step 4.

Digest the target DNA on the slide and under cover slip with a suitable restriction enzyme so that the hybridization target will be in the vicinity of a free 3'-end by incubating with a restriction enzyme mixture under cover slip for 30 min at 37°C (or other temperature suitable for the particular enzyme). Rinse, dehydrate, and air-dry the slide as in step 4.

Remove the target complementary DNA strand by incubating with ^-exonuclease mixture under cover slip for 30 min at 37°C. This step provides an enzymatic "denaturation" of the genomic DNA, where one strand of the DNA is removed to leave the other as a single-stranded DNA, ready for hybridization of the probe. Standard denaturation procedures have also been useed (1) but, for the time being, the enzymatic denaturation is our favorite. Rinse, dehydrate, and air-dry the slide as in step 4.

Hybridize the padlock probe(s) at a concentration of 10 to 200 nm (e.g., 100 nM) in hybridization mixture under cover slip for 30 min at 37°C. Rinse away excess probe by incubating 5 min each in two changes of wash buffer at 37°C, then dehydrate and air-dry the slide as described in step 4. Incubate the slide with DNA ligase mixture under cover slip for 30 min at 37°C. Remove padlock probes not turned into closed circles by washing 15 min each in two changes of padlock removal solution at 42°C. Rinse, dehydrate, and air-dry slide as in step 4.

Incubate the slide with the rolling-circle PRINS reaction mixture under cover slip for 30 min at 37°C.

Rinse, dehydrate, and air-dry the slide as in step 4.

Incubate with identifier oligonucleotides at a concentration of 10 to 250 nm in hybridization mixture for 30 min at 37°C. The identifier oligonucleotides are used for the identification of the rolling-circle product from a given padlock probe (which may be applied as a padlock probe within a mixture of padlock probes that are all hybridized and rolled in parallel; see Notes 2 and 3).

18. Rinse away excess probe by incubating in two changes of wash buffer for 5 min each at 37°C.

19. Counterstain and mount slides. For blue counterstaining of DNA, mount slide in 20 pL antifade mounting medium containing 0.4 pM of DAPI. For red counter-staining of DNA, Mount slide in 20 pL of antifade mounting medium containing 0.5 pg/mL propidium iodide.

Examine slides by fluorescence microscopy using standard excitation and emission filters.

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