John M. S. Bartlett.pdf - Bio-Nica.info

Cycling Primed In Situ Amplification 427
most severe when the cytoplasm has been stripped away, as in typical methanol/acetic
acid fixed metaphase spread preparations used in cytogenetic analysis (6). The naked
nucleus provides no barrier to diffusion, and although PRINS is very efficient, cycling
PRINS confers no advantage as a steady state of signal is reached after the first cycle.
Many elaborate schemes can be imagined to overcome this, involving crosslinking
agents, looped or circular structures, and photoactivatable anchors. The method
described here simply uses ethanol fixation of whole cells. When ethanol is used,
signal retention in cycling PRINS is markedly improved (7). Background labeling
of the nuclear DNA is increased with this fixative, but this is easily counteracted by
microwave boiling of the slides before the PRINS reaction is set up. Using an extended
initial denaturation step also helps reduce background.
The method described here is applicable to cell preparations, such as blood smears,
cytospin, or gravity-settled cell preparations. Once dry, the slides can be fixed and stored
for several months before cycling PRINS analysis. Although it is presently limited
to whole cell preparations and seems incompatible with the analysis of metaphase
spreads, it is simple, robust, and effective.
2. Materials
2.1. Slide Preparation
1. Glass slides and coverslips: high-grade, dust-free slides are a sound investment and
require no further preparation (e.g., Menzel Glaser Super Premium, Fisher Scientific,
Loughborough, UK). Coated slides can be useful if there are doubts around loss of valuable
cells (see Note 1). Coverslips can be 22 × 22 mm or bigger.
2. Cell preparation (see Subheading 3.2.).
3. Ethanol (100%).
4. Slide staining jar, for example, a Coplin jar.
5. 10 mM Tris-HCl, 5 mM EDTA, pH 7.0.
6. Anti-bumping granules (Merck).
7. 800-W Microwave oven (e.g., Matsui M162TC).
8. Graded ethanol solutions: 100%, 90%, and 70% v/v with water.
2.2. Cycling PRINS
1. AmpliTaq Gold DNA polymerase (PE Biosystems).
2. 10× PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 200 mM MgCl2 (see Note 2).
3. dNTPs (Amersham Pharmacia Biotech Inc., Uppsala, Sweden). Dilute to make a 50× stock
solution of 10 mM each dATP, dCTP, dGTP; and 1.8 mM dTTP.
4. Digoxygenin-11-dUTP (DIG-dUTP; 10 nmol/µL, Roche Molecular Biochemicals, Lewes,
UK) (see Note 3).
5. Oligonucleotide primer (50 µM, see Subheading 3.1.).
6. Double distilled water.
7. High-grade clean coverslips and rubber solution (see Note 4) or Amplicovers and
Ampliclips (PE Biosystems).
8. Flat-bed or slide-adapted thermal cycling (“PCR”) machine (e.g., Hybaid, Ashford, UK
or PE Biosytems).
9. Stop buffer: 500mM NaCl, 50 mM EDTA, pH 7.0.
10. Water bath at 65°C.