Heteroduplex Cleavage Analysis Using S1 Nuclease

A variety of methods have been developed to screen for genetic mutations. Most rely on the comparative analysis of polymerase chain reaction (PCR) products. Single-strand conformational polymorphism (4), the most widely used technique, relies on sequence-dependent conformational electrophoretic mobility differences between wild-type (WT) and mutant strands; whereas, heteroduplex analysis (5,8), the second most popular technique, is based on differential mobility between homoduplexes and heteroduplexes in gel electrophoresis. Both techniques require exacting electrophoretic conditions. Another approach to mutation screening relies on cleavage of unpaired nucleotides in self-folded DNA or heteroduplexes between WT and mutant forms as in RNase A cleavage of, for example, RNA/DNA heteroduplexes (1,3,9). None of the techniques is fully applicable to all situations. Each method has its own advantages and disadvantages; however, all require multiple steps subsequent to PCR and/or specialized electrophoretic conditions, size restrictions or do not provide information on the relative location of the mutation. Here, we describe a version of heteroduplex cleavage analysis of PCR products based on the ability of S1 nuclease to cleave base pair mismatches in DNA/DNA heteroduplexes (6,7). The procedure requires minimal sample, no purification steps, a short 30min incubation and can be visualized on simple agarose minigels in <20 min. As used in our laboratory, small aliquots of PCR product spanning the suspected site of mutation and reference (WT) PCR products are mixed, heated and cooled to effect annealing. An equal volume of S1 nuclease in digestion buffer is added to each tube. Following incubation, the samples are electrophoresed in agarose minigels to visualize digestion products. PCR amplification products were produced by incubating DNA template using standard PCR conditions and reagents [5 U Taq DNA Polymerase (Life Technologies, Gaithersburg, MD, USA) in the manufacturer’s PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl) with 1.5 mM MgCl2, 0.2 mM each dNTP mixture and 0.5 mM of each primer]. In the basic S1 nuclease digestion protocol, samples to be digested consisting either of WT (control reference) or mutant PCR amplification products, or a 1:1 mixture of reference and mutant products were incubated under mineral oil at 95°C for 5 min (denaturation) followed by a gradual cooling to 65°C (re-annealing stage) over 5 min. Four-microliter samples were deoiled by pipetting onto Parafilm, dragging the drop over the film with a clean pipet tip and placing the droplet into 0.5-mL microcentrifuge tubes placed on ice and containing 2.7 μL H2O, 0.8 μL 10× S1 buffer (80 mM NaCl, 0.5 M Na acetate, pH 4.5, 10 mM ZnSO4, 5% glycerol) and 0.5 μL of a 1:20 dilution (25 U) of S1 nuclease (Life Technologies) in the dilution buffer supplied by the manufacturer. Reactions were incubated 30 min at 37°C and immediately placed on ice. Samples were mixed with running buffer and electrophoresed in 1.5% agarose and stained with ethidium bromide. Gels were viewed by UV transillumination and photographed. Of the five reference/mutant amplification product combinations tested (Table 1), all showed a single band following amplification and after melting and re-annealing and subsequent incubation in the digestion buffer in the absence of the enzyme (data not shown). In the presence of S1 nuclease, the heteroduplex combination with a contiguous three-base mismatch (77/81) showed both the homodimer band (higher molecular weight [mol wt] and two lower mol wt bands) corresponding to the cleavage products of the heterodimer (Figure 1, lane C). Of the two heteroduplex combinations in which there was only a single base mismatch, one (KG2/KG4) digested using the standard protocol (Figure 1, lane B), and the other (77/93) did not. The pair differing by a single deletion (2131/213-2) also did not digest. In an attempt to increase the sensitivity of the reaction to single-base mismatches, we varied the digestion conditions and digestion buffer and tested these conditions on the various duplex combinations. The following observations were made: (i) Digestibility was not affected by additional glycerol or the addition of betaine or dimethyl sulfoxide (DMSO). (ii) Sensitivity was increased by lowering the NaCl concentration of the S1 buffer. (iii) Maximal digestion was achieved when the NaCl concentration in the reaction mixture was in the 8.0 to 0.0 mM range (data not shown). (iv) The addition of dioxBenchmarks