Simplified Probe Prep - aration Facilitates S 1 Nuclease Analysis

S1 nuclease analysis is used to (i) locate the 5′ and 3′ ends of an mRNA transcript on DNA, (ii) map the location of intron-exon junctions in primary mRNA transcripts, (iii) determine the direction of transcription and (iv) quantitate the amount of a specific mRNA extracted from cells. The original procedure described by Berk and Sharp (1) employed labeled, doublestranded (ds) DNA as probes for hybridization to mRNA. Problems associated with probe renaturation required that a hybridization temperature be established that would suppress the formation of DNA:DNA hybrids and favor the formation of RNA:DNA hybrids. To circumvent this problem, singlestranded (ss) probes complementary to DNA fragments cloned into bacteriophage M13mp vectors have been used (2). Alternatively, probes have been prepared by asymmetric PCR from ds linear templates (3). The ss probes are then purified from the DNA templates, generally by electroelution from agarose or polyacrylamide gels. Purification of ss probes by adsorption onto glass beads has also been described (5). These purification steps are time-consuming and result in substantial losses of the probe. The protocol for the probe preparation described here results in greatly improved yields and simplifies S1 nuclease analysis. S1 nuclease analysis of the 5′-untranslated region for the leukocyte integrin gene, CD11c, has been previously described (4) and is used as an example in our modified protocol. The region spanning -89 to +88 (with respect to the transcription initiation site, +1) was first amplified from a CD11c genomic clone by the polymerase chain reaction (PCR). The ds PCR product was then purified from a 4% NuSieve (FMC BioProducts, Rockland, ME, USA) low melting point agarose minigel (LMP minigel) by adsorption onto USBioclean glass beads according to manufacturer’s instructions (United States Biochemical, Cleveland, OH, USA) and used as the template for the probe preparation. In a separate reaction, 20 pmol of one of the primers used to prepare the ds template were end-labeled in a 10-μL reaction mixture with [γ32P]ATP (6000 Ci/mmol; Du Pont NEN, Boston, MA, USA) to a specific activity of 2–4 × 108 dpm/μg. Approximately 50–100 ng of ds template and 20 pmol of labeled primer (no need to remove unincorporated counts) were then subjected to 20–25 cycles of asymmetric PCR in a standard 100-μL reaction volume. The labeled ss PCR product (along with the ds template) was concentrated by ethanol precipitation and then electrophoresed in one lane of a 4% LMP minigel containing ethidium bromide. The ss DNA, which runs at approximately one-half the molecular weight of the ds template, was visualized on a UV transilluminator. Alternatively, ss DNA can be localized following autoradiography of the gel. A thin segment of agarose containing the ss DNA was excised from the gel and trimmed of excess agarose with a razor blade. The agarose block containing the ss DNA was transferred to a centrifuge tube and melted at 55°C for 1 min. An equal volume (generally 30–40 μL) of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was added to the melted agarose, and 1–2 μL of the melted agarose was counted in a scintillation counter. The yield of ss product is dependent on the efficiency of asymmetric PCR, but generally 5–10 × 105 dpm of ss product are easily obtained. For S1 nuclease analysis, 25 μg of total RNA from phorbol 12-myristate 13-acetate (PMA)-stimulated HL60 cells (promyeloblastic leukemic cell) were ethanol-precipitated and resuspended in 20 μL 80% deionized formamide (molecular biology grade; Fisher Biotech, Fair Lawn, NJ, USA), 0.4 M NaCl, 1 mM EDTA, 50 mM piperazine-N,N′,-bis(2-ethane-sulfonic acid) (PIPES), pH 6.4. Approximately 2 × 104 dpm of ss probe DNA, still in molten agarose, were added to the resuspended RNA and hybridized at 50°C overnight. As much as 3 μL of molten agarose can be added without affecting the hybridization efficiency. After hybridization, 150 μL of 0.5 M NaCl, 0.1 M sodium-acetate (pH 4.5), 9 mM ZnSO4, 5 μL of 10 mg/mL ss