Separation of DNA Strands Facilitates Detection of Point Mutations by PCR-SSCP

Single-strand conformation polymorphism (SSCP) is a powerful technique used to identify mutations in short pieces of DNA (1). In this technique, the DNA of interest is often amplified by the polymerase chain reaction (PCR) and then denatured by heat or alkali treatment before electrophoresis on a non-denaturing polyacrylamide gel. Differences in mobility of either of the single strands, compared to the control DNA, indicate mutations that have affected the secondary structure and altered the mobility of the DNA. We applied PCR-SSCP analysis for the detection of mutations in the rpoB gene of Mycobacterium tuberculosis. These mutations are associated with resistance to the drug rifampicin (3). In our experience using denaturation by alkali or heating, the denatured PCR product most often re-annealed to form a large proportion of double-stranded DNA during the electrophoresis. After visualization by staining with ethidium bromide (EtdBr) or silver, most of the DNA was in the double-stranded form, with very little or no single-stranded DNA. The single strands that could be observed often ran very close together, making analysis of any differences in mobility difficult. We therefore investigated the feasibility of using a biotinylated PCR primer to generate a PCR product that was biotinylated in one strand. This enabled the two strands of DNA to be separated before electrophoresis using streptavidin paramagnetic beads, eliminating the problem of strand-annealing during SSCP and emphasizing any differences in mobility of the two strands. A nested PCR was used to amplify the region of the ropB gene in which mutations have been shown to determine the rifampicin resistance (4). A 293-bp region of the rpoB region of M. tuberculosis was amplified by the first PCR. The PCR mixture (20 μL) contained 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 5% dimethyl sulfoxide (DMSO), 200 μM nucleotides, 10 pM each of a forward primer (rpoBFO; 5′ CGT TGA TCA ACA TCC GGC CGG TGG 3′) and a reverse primer, (rpoBRO; 5′ TTT CGA TGA ACC CGA ACG GGT TGA C 3′) and 1 U of Taq DNA polymerase. The reaction mixture was denatured at 93°C for 2 min, followed by 35 cycles each of denaturation at 93°C for 1 min, and annealing and extension at 72°C for 2 min. The reaction was terminated after a final extension at 72°C for 10 min. Of the first PCR product, 103 bp were amplified in a second 20-cycle nested PCR containing biotinylated forward primer (rpoB FIBio; 5′ GT TCT TCG GCA CCA GCC AG 3′) and nonbiotinylated reverse primer (rpoB SRI; 5′ CAG ACC GCC GGG CCC 3′). In this PCR, the annealing temperature was 52°C for 30 s. To separate the DNA strands, the biotinylated PCR product was captured using streptavidin paramagnetic beads following the instructions given by the manufacturer. Briefly, 40 μL of the beads (Promega, Southampton, England, UK), suspended in 2× Buffer A (1× Buffer A is 10 mM Tris-HCl, pH 7.5, 1 mM EDTA and 2 M NaCl), were mixed with 20 μL of PCR product and 20 μL of H2O. The mixture was incubated at room temperature for 30 min. After washing the beads twice with 1 mL of Buffer A, the captured DNA was denatured by adding 8 μL of 0.1 M NaOH and incubating for 10 min at room temperature. The alkali containing the denatured nonbiotinylated DNA strand was aspirated, made up to a volume of 50 μL in sterile distilled water and then precipitated by a standard ethanol-sodium acetate method. The precipitate was resuspended in 5 μL of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The beads with the captured biotinylated strand were washed with 50 μL of 0.1 M NaOH and washed an additional three times with Buffer A and finally suspended in 5 μL of TE buffer. The separated strands were heated at 95°C for 5 min with an equal volume of 0.05% bromophenol blue, 2 mM EDTA, 95% (vol/vol) formamide and then cooled on ice and analyzed immediately by non-denaturing polyacryl-