e100 Nucleic Acids Research, 2007, Vol. 35, No. 15 Their sequences are: 50-GCT-30, 50-GCTCGTA-30, 50-GC TCGTAGAGTGGTC-30, 50-CGGTATCTTGGCTATG ACGGC-30, 50-GCTCGTAGAGCGGTCATAGTCAA GATACCG-30 and 50-ATGTGAATCAGTATGGTTAC TATCTGCTGAAGGAAAT-30, respectively. They were puriﬁed after synthesis by high-performance liquid chromatography (HPLC) and their concentrations were determined from their absorbance at 260 nm. The 50-phosphorylated or 50-hydroxylated single-stranded oli- godeoxynucleotides, 50-ATGTGACTCAGTATGGG-30, were also synthesized and puriﬁed by HPLC. Then, they were annealed to their complementary 50- phosphorylated or 50-hydroxylated single-stranded oligodeoxynucleotides (50-CCCATACTGAGTCACAT-30) to obtain double- stranded oligodeoxynucleotides in TE buﬀer. Annealing was performed in a thermal cycler according to the following temperature proﬁle: 5 min at 958C, followed by a slow decrease from 958C to 378C over 60 min and from 37 to 48C over 30 min. Oligonucleotide which contains the locked nucleic acid (LNA) at 30-terminal end was also synthesized. Nuclease reaction The C-terminal domain of ttMutS2 (CTD) was over- expressed and puriﬁed as described previously (17). The substrate oligonucleoitdes (25 mM) were incubated with 0, 50, 100 or 200 nM CTD in a buﬀer containing 50 mM Tris–HCl (pH 7.5), 100 mM KCl and 5 mM MgCl2 at 378C for 16 h. The total volume of reaction mixture was 30 ml. Reactions were quenched by addition of an equal volume of phenol–chloroform solution and the mixtures were then centrifuged at 15 000 r.p.m for 10 min. Super- natants were loaded onto 5 ml of SuperQ resin equilibrated with water in a 0.6 ml Eppendorf tube. After washing with 30 ml of water three times, DNAs were eluted with 30 ml of 0.75 M ammonium acetate (pH 7). The elutants were dried in a centrifugal evaporator. ESI–FT ICR MS analysis The elutants were resuspended to a concentration of 1 mM in 50% methanol containing 25 mM imidazole and 25 mM piperidine. The 10 ml aliquots were loaded into quartz nanospray emitters. All measurements were performed on an Apex IV Fourier transform mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with a 9.4-T shielded superconductive magnet. The oligonucleotide solutions were infused into an external Apollo electro- spray ion source at a ﬂow rate of 13 ml/min with the assistance of N2 nebulizing gas. The oﬀ-axis sprayer was grounded, and the inlet capillary was set to 1.7 kV for generation of oligonucleotide anions. N2 drying gas was applied to assist desolvation of ESI droplets. Ions were accumulated in the hexapole for 0.2 s. All data were acquired in negative ion mode and processed using XMASS 6.0.1 (Bruker Daltonics). Electrophoretical analysis Single-stranded DNAs were radiolabeled at the 50-end with [g-32P]ATP using polynucleotide kinase before PAGE 2 OF 6 annealing. The 50-labeled duplexes (10 nM) were incu- bated with CTD or king of DNA (KOD) polymerase (TOYOBO, Osaka, Japan) in a 50 mM Tris–HCl (pH 7.5) containing 100 mM KCl, 5 mM MgCl2 and 25 mM non- labeled substrate dsDNA for 16 h at 378C. The enzyme concentrations are indicated in the legends to ﬁgures. Reactions were stopped with the addition of equal volume of phenol–chloroform solution and the solutions were centrifuged at 15 000 r.p.m. for 10 min. The supernatants were mixed with the sample buﬀer (5 mM EDTA, 80% deionized formamide, 10 mM NaOH, 0.1% bromophenol blue and 0.1% xylene cyanol) and heat-treated at 958C for 5 min. They were loaded onto 11% polyacrylamide sequencing gels (8 M urea and 1 TBE buﬀer, 89 mM Trisborate and 2 mM EDTA) and electrophoresed with 1 TBE buﬀer. The gels were dried and placed in contact with an imaging plate. The bands were visualized and analyzed using a BAS2500 image analyzer (Fuji Film, Tokyo, Japan). The substrate 32P-labeled DNA was base-speciﬁcally modiﬁed and digested according to the Maxam–Gilbert method (21). In order to determine the length of the product DNA, these fragments were mixed with the sample buﬀer and electrophoresed along with the products on DNA sequencing gels. RESULTS AND DISCUSSION First, we examined the validity of our method for the puriﬁcation of DNA samples. Contamination of non- volatile salts such as sodium or potassium, which would otherwise prevent ionization of the biomolecules and cause the formation of series of metal adduct ions (22), must be avoided. The single-stranded DNAs (ssDNAs) (3–30-mer) were prepared and analyzed as described in MATERIALS AND METHODS section. Raw mass spectra consisted of a series of peaks, corresponding to multiply charged ions of intact ssDNA having a speciﬁc number of protons removed from the phosphodiester groups (Figure 1A). As shown in inset, few metal adduct ion species were observed, showing the eﬃcacy of the puriﬁcation method. When the same sample was puriﬁed by ethanol precipitation, even the charged ions of ssDNA were hardly detected (data not shown) probably because non-volatile salts were poorly removed. In Figure 1B, signals representing the same DNA were deconvoluted to yield the molecular mass of the corresponding DNA. Comparison of the measured molecular mass against theoretical molecular mass enabled the length and sequence of the DNA species to be identiﬁed. A deviation between the measured and theoretical molecular mass of within 2 p.p.m. was deemed acceptable. Our results show that the DNA desalting method can be applied to the analysis of extremely short oligonucleotides, at least to a 3-mer. The 50-phosphorylated 17-bp dsDNA incubated with CTD were analyzed as described above. We chose the oligonucleotide sequence shown in MATERIALS AND METHODS section as a substrate since it has left–right asymmetric nucleotide content. Symmetric distribution of