Introduction

Primed in situ labeling (PRINS) is a novel method that can be used as a specific and rapid alternative to fluorescence in situ hybridization (FISH) in the detection of subtle chromosome rearrangements (1,2). According to the PRINS method, laboratory-synthesized oligonucleotide probes ("PRINS primers") are used instead of cloned DNA probes for the in situ localization of individual genes or unique sequences. The primers are annealed in situ to complementary target sequences on slide-fixed chromosomes and extended in the presence of labeled nucleotides and Taq DNA polymerase (3). Chromo-

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

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

somal DNA acts as template for the extension, and the labeled nucleotides serve as substrate for the DNA polymerase. The labeled extended sequences are visualized by fluorescence microscopy.

In its early days, PRINS was used to detect repetitive sequences such as a-satellite, Alu, and telomeric repeats (1,2,4). However the method now is used to identify single copy sequences—the X-linked factor IXgene (5), for example, and, more recently, the SRY sex-determining gene (see ref. 6 and Chapters 4 and 5). This shift represents a significant biological milestone because any gene with a published sequence is now amenable to study by PRINS.

After modification of the original method, our group used PRINS with the TSA™ Biotin System to evaluate RBM and DAZ gene deletions in azoospermia (7), and SNRPNand GABRB3 deletions in the Prader Willi and Angelman syndromes (8). Deletion in these cases was indicated by absence of signal in experimental samples as compared with its presence in normal control subjects. Here, we describe the modified PRINS method in the study of RB1 and p53 gene deletions in cultured bone marrow cells from patients with hematopoietic cancers (9).

2. Materials

2.1. Bone Marrow Culture

1. RPMI-1640 culture medium (RPMI-1640; Gibco Invitrogen, Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; Invitrogen-Life Technologies, Carlsbad, CA). Store at 4°C.

2. Gentamycin sulfate (Irvine Scientific, Santa Ana, CA) as a supplement to RPMI-1640 medium. Store at 4°C and add to medium at 10 mg/mL.

3. Sodium heparin (Elkins-Sinn, Cherry Hill, NJ) as a supplement to RPMI-1640. Store at 4°C and added to cultures at 1000U/mL.

4. L-Glutamine (Gibco Invitrogen, Bethesda, MD) as a supplement to RPMI-1640. Store at -20°C and add to medium at room temperature (4 mL per 500 mL of medium).

5. KCl-Na citrate hypotonic solution (Fisher Scientific, Pittsburgh, PA): add 0.560 g of KCl to 100 mL of distilled or deionized water to make a 0.075 M solution. Add 0.8 g of Na citrate to 100 mL of distilled or deionized water to make a 0.8% Na citrate solution. Mix the two solutions just before use: 2:1, KCl:Na citrate. Store at room temperature and prepare a fresh mixture every 7 d.

2.2. Cell Harvest and Preparation of Slides

1. Colcemid (Gibco Invitrogen). Purchase in lyophilized form and store at 4°C. Use at room temperature after reconstitution in deionized or distilled water. Discard after 1 mo of use.

2. Glass slides and covers (Fisher Scientific).

3. Fixative solution: 3:1 methanol:glacial acetic acid (Fisher Scientific).

2.3. Primed In Situ Hybridization

1. Programmable thermal cycler equipped with a flat plate for holding slides (MISHA, Shandon Lipshaw, Pittsburgh PA; see Note 1).

2. Dimethyl sulfoxide (DMSO; Sigma Genosys, St. Louis, MO) molecular biology or high-performance liquid chromatography grade.

3. Ethanol (Fischer Scientific).

4. Oligonucleotide primers (Research Genetics, Huntsville AL; see Note 2). The following probes for the RB1 and p53 genes were HPLC purified, and stored at -20°C:

RB1: 5'-TGGTAGGCTTGAGTTTGAAGA-3' 5'-CAGCTATTTGGGAAGCTGAG-3' 5'-TCCAGCCTGGGCAACACAG-3' 5'-AACTTGACCAAGGTCCCTAG-3' 5'-ACACAGCGGAATGTGTTTATG-3' p53: 5'-TGCTTTCCACGACGGTGA-3' 5'-AACTGGCCAAGACCTGCCC-3' 5'-AGCTCCTCTCCCCAGCCA-3' 5'-ACTGTGAGGGATGTTTGGGA-3'

5. dATP, dCTP, dGTP, dTTP: from TSA™ kit; tyramide signal amplification, for chromogenic and fluorescence in situ hybridization and immunohistochemistry (NEN Life Science Products, Boston MA).

6. Biotin-16-2'-deoxyuridine 5'-triphosphate (dUTP; Roche Molecular Systems, Alameda, CA; formerly Boehringer-Mannheim).

8. Magnesium chloride (MgCl2) (Fischer Scientific).

9. Potassium chloride (KCl; Fisher Scientific).

10. Bovine serum albumin (Invitrogen Life Technologies, Carlsbad, CA).

11. Taq DNA polymerase (Amplitaq, Perkin-Elmer, Foster City, CA) with TaqStart antibody (Clontech Laboraories, Palo Alto, CA).

12. 20X SSC: 3 MNaCl, 0.3 MNa citrate, pH 7.0 (Invitrogen Life Technologies).

13. Formamide-SSC (Invitrogen Life Technologies).

14. TSA Biotin System. The kit should be kept at 4°C until used, although the blocking reagent may be kept at room temperature. With proper storage, the kit is useful for 6 mo, after which the contents may become unstable.

a. Biotinyl tyramide stock solution. Reconstitute biotinyl tyramide (amplification reagent) by addition of 0.3 to 1.2 mL of DMSO (the amount of DMSO will depend on the particular NEN kit used: 0.3 mL for 50 to 150 slides; 1.2 mL for 200-600 slides). Since DMSO freezes at 4°C, it is necessary to thaw the stock solution after removal from refrigerator.

b. Prepare TNT washing buffer: 0.1 MTris-HCl, pH 7.5, 0.15 MNaCl, 0.05% Tween-20. The manufacturer advises that PBS may be used as an alternative buffer, and that 0.3% Triton X-100 may be used instead of 0.05% Tween-20.

c. TNB blocking buffer: 0.1 MTris-HCl, pH 7.5, 0.15 MNaCl, 0.5% blocking reagent (in kit). According to the manufacturer's protocol, the blocking reagent should be added slowly to buffer in small amounts while stirring. The mixture should be heated to 60°C with continuous stirring (see Note 3). The blocking reagent should be dissolved completely (this may take several hours). After preparation, store at -20°C.

15. Tween-20 (Sigma Genosys).

16. Triton X-100 (Sigma Genosys).

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