Introduction

In the last 20 yr, the positional cloning approach has allowed the identification of the majority of the genes involved in the most important human genetic diseases. Studies conducted on large samples of affected patients have disclosed specific mutations associated to the diseases, allowing us to identify specific genotype-phenotype correlations. Consequently, screening of mutations of these genes is now conducted to confirm clinical diagnosis or to identify healthy carriers of genetic diseases.

So far, several techniques have been developed that are capable of performing large-scale screening of gene point mutations; however, the search for gene deletions still poses a challenge in diagnostic laboratories. In fact, although deletions of a few base pairs are easily detectable by polymerase chain reaction (PCR) amplification and DNA sequencing, and very large deletions are evidenced by cytogenetic investigations, great difficulties arise in detecting deletions of a few kilobases, which can be missed by both techniques (1). Because

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

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

several single-gene disorders are caused mainly by deletions or duplications of part or all the gene (e.g., Duchenne muscular dystrophy [DMD], spinal muscular atrophy, Charcot-Marie-Tooth disease, Fanconi anemia, and others), diagnostic analysis of these diseases can be time-consuming and often inconclusive. Among these, DMD is of particular relevance because of the frequency and severity of the disease. DMD (MIM #310200) and Becker muscular dystrophy (MIM #300376) are the most common form of dystrophinopathy, with a reported incidence of 1:3500 and 1:18000 per birth males, respectively. These diseases are due to mutations of the DMD gene (Xp21.2 [2]). DMD, which produces the protein dystrophin, is the largest human gene known so far, spanning 2.4 mb on the human genome, containing 79 exons, and encoding a 14-kb messenger RNA (3). Both diseases show progressive symmetrical muscular weakness, with DMD characterized by an earlier onset and a more severe progression. Approximately 98% of DMD deletions are easily detectable in affected men and boys using a multiplex PCR approach able to simultaneously amplify exons mapped within two hot-spot regions (exons 2-20 and 44-53 [4-6]). Because no effective treatment is so far available for these diseases, the identification of carrier females is critical to prevent the birth of affected males. However, the multiplex PCR approach is not able to detect heterozygous female carriers because, in these cases, the normal X chromosome masks the presence of deletions. Different strategies must thus be used for the identification of female carriers.

In the last two decades, fluorescence in situ hybridization (FISH) analysis has represented a reliable and powerful tool in physical gene mapping and in the detection of gene rearrangements such as deletions, translocations, and duplications. The increased availability of specific probes for the most relevant human genes and the use of fluorescence instead of isotopic elements for the detection of the in situ hybridization have made this technique as a tool of choice in the molecular diagnosis of several genetic diseases. However, the typical FISH approach still has some limits. In fact, small gene rearrangements, such as deletions involving one or few exons, cannot be evidenced by this technique. Moreover, FISH analysis remains a low throughput analysis as compared with molecular genetics techniques.

Consequently, great efforts have been made to develop new strategies able to combine the benefits of cytogenetic analysis with those provided by the use of molecular techniques. The primed in situ labeling (PRINS) technique is based on the specific hybridization of a short unlabeled olinucleotide (approx 20-30 pb) with a denaturated template and synthesis of a single strand in situ, catalysed by a Taq polymerase. During primer elongation, labeled nucleotides are incorporated into the newly synthesized DNA, allowing their detection by

Fig. 1. (A) Example of DMD gene and X centromer labeling in chromosome and nucleus using PRINS and FISH protocol. Two oligonucleoties for exon 1 dystrophin gene: MD15 5'-TCTGGGAGG CAATTACCT TC-3' and MD16 5'-ACAGTCCTCT ACTTCCC-3', have been used. The image was obtained by using confocal laser microscopy. (B) DMD gene: position of exons and primers respect to X centromer (Cen [7,8]). (Please see color insert following p. 48.)

Fig. 1. (A) Example of DMD gene and X centromer labeling in chromosome and nucleus using PRINS and FISH protocol. Two oligonucleoties for exon 1 dystrophin gene: MD15 5'-TCTGGGAGG CAATTACCT TC-3' and MD16 5'-ACAGTCCTCT ACTTCCC-3', have been used. The image was obtained by using confocal laser microscopy. (B) DMD gene: position of exons and primers respect to X centromer (Cen [7,8]). (Please see color insert following p. 48.)

means of specific fluorescent antibodies. This method is able to produce multiple independent signals within the same gene segment, thus allowing the detection of partial deletions within a single gene.

In this chapter, we describe the use of the combined PRINS and FISH methods for the in situ detection of single DMD exon in fixed metaphase chromosomes and interphase nuclei (Fig. 1A). This approach could allow the identification of female carries of small intragenic deletions of the DMD gene.

2. Materials

2.1. Preparation of Metaphase Spreads

2.1.1. Preparation From Whole Peripheral Blood Cells

1. Histopaque-1077 Lymphocyte separation medium (Sigma-Aldrich, St. Louis, MO).

2. Peripheral blood karyotyping medium (Gibco/BRL, Bethesda, MD; see Note 1).

3. Phosphate-buffered saline (PBS): Prepare 10X stock with 1.37 MNaCl, 27 mM KCl, 100 mMNa2HPO4, and 18 mMKH2PO4 (adjust to pH 7.4 with HCl if necessary). Prepare working solution by dilution of one part with nine parts distilled water and filtration with a 0.2-pMfilter.

2.1.2. Preparation From Cultured Cells

1. Appropriate culture medium, supplemented with 10% fetal bovine serum (Gibco/ BRL) containing 100 U/mL penicillin and 100 pg/mL streptomycin (Gibco/BRL).

2. Solution of trypsin (0.25%) and EDTA (1 mM; Gibco/BRL).

3. 1X Sterile PBS.

2.2. Preparation of Metaphase Chromosomes

1. Colcemid® Solution, liquid (10 pg/mL), in 1X PBS (Gibco/BRL; see Notes 2 and 3).

2. Hypotonic solution: 0.075 Mpotassium chloride (see Note 2).

3. Fixative solution: mix precooled (-20°C) absolute methanol (Sigma-Aldrich) and glacial acetic acid (Sigma-Aldrich) in a 3:1 ratio (v/v).

2.3. PRINS Reaction

1. Formamide solution: 70% deionized formamide, 10% 20X standard saline citrate (SSC), 10% 0,1 M Sorensen buffer, pH 7.4, 10% distilled water (see Note 4).

2. 20X SSC solution: 0.3 Msodium citrate trihydrate, 3 MNaCl, adjust to pH 7.0 with citric acid. 2X SSC solution is prepared by dilution of one part of 20X SSC with nine parts distilled water.

3. 0.1 MSorensen buffer: 12.0 mMNaH2PO4, 69.0 mMNa2HPO4, adjust to pH 7.4 with HCl.

4. PRINS-modified mixture: 1X PCR buffer with 1.5 mM MgCl2 (QIAGEN, Valencia, CA), 0.5 U/pL Taq DNA polymerase (QIAGEN); DIG-dNTP labeling mix (40 pM each of 2'-deoxyadenosine 5'-triphosphate, 2'-deoxycytosine 5'-triph-osphate, 2'-deoxyguanosine 5'-triphosphate and 14 pM DIG-2'-deoxyuridine 5'-triphosphate, 26 pM 2'-deoxythymidine 5'-triphosphate in 5% glycerol), 4 pM oligonucleotide primers, that is, for exon 1 dystrophin gene (MD15 5'-TCTGGGAGG CAATTACCT TC-3' and MD16 5'-ACAGTCCTCTAC TTCCC-3'; Fig. 1B ¡7,81), 2 ng/mL human a-satellite chromosome X-specific DNA probe, digoxigenin labeled (Q-Biogene, Irvine, CA; see Notes 5 and 6).

5. Stop solution: 50 mM NaCl, 50 mM EDTA, pH 8.0 (see Note 7).

6. Washing buffer: 0.05% Triton X-100 in 4X SSC (see Note 8).

7. Blocking solution: 0.5% bovine serum albumin (Sigma-Aldrich) in 4X SSC, pH 7.0.

8. Antidigoxigenin antibody: monoclonal antidigoxigenin-FITC (Roche Molecular Biochemicals, Mannheim, Germany) dilute 1:100 in 4X SSC, 0.5% bovine serum albumin, pH 7.0 (see Note 9).

9. Propidium iodide solution: 1 |g/mL propidium iodide (PI; Sigma-Aldrich) in distilled water.

10. Antifading reagent-DABCO, 2% (w/v) in 50% glycerol/lX PBS. 3. Methods

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