Fig. 3.

Characterization of reaction products by MALDI-TOF mass spectrometry. a Mass spectrum of 100-mer PCR product. The peak around m/z of 64,000 represents singly charged molecular ion (M⁺), and the peak around 32,000 represents a doubly charged molecular ion (M²⁺ ). The broad peaks may have resulted from Na ion adducts and/or wide isotope distributions. b Mass spectrum of annealed adapter. The peak at m/z around 13,600 shows M⁺ of the adapter and M²⁺ appears at around 6,800. M⁴⁺ is also detected around 3,400. Other peaks (m/z around 10,000 and in between 4,000 and 6,000) are non-specifically bound products during annealing process. c–e Mass spectra of ligated product without gel selection. Strong signal of unreacted adapters was present (c), and M⁺ around m/z 90,000 showed the dominant presence of 150-mer-ligated product (e). M²⁺ of 150-mer product appeared around m/z 45,000, and only M²⁺ of 125-mer one-end-ligated product was observed around m/z 38,000 (d). No significant peak around 64,000 or 32,000 was detected, confirming that the initial 100-mer PCR product was mostly converted to 125-mer- or 150-mer-ligated products