Site-specific scission of lambda phage genomic DNA by Ce(IV)/EDTA-based artificial restriction DNA cutter

Chemistry Letters

Volumn 35, Issue 6, 2006, Pages 594-595

  Yamamoto, Yoji ^a   Miura, Kazuyuki ^a   Komiyama, Makoto ^a  

^a UNIVERSITY OF TOKYO   (Japan)

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EID: 33748125140     PISSN: 03667022     EISSN: None     Source Type: Journal    
DOI: 10.1246/cl.2006.594     Document Type: Article

References (19)

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13. Lambda DNA was incubated with PNA1 and PNA2 for 1 h at 50 C in pH 7 buffer in the presence of 10 mM NaCl. Then, H buffer and EcoRI (both from TAKARA) were added and incubated for 1 h at 37 C.
14. Similar assay was performed to investigate the invasion of PNA to plasmid DNA: K. I. Izvolsky, V. V. Demidov, P. E. Nielsen, M. D. Frank-Kamenetskii. Biochemistry 2000, 39, 10908.
15. Lambda DNA (from TAKARA) was purified by ethanol precipitation, and dissolved in 10 mM TRIS buffer (pH 8.0) before use.
16. According to the literature, invasion rate decreases with increasing the concentration of non-targeted DNA since PNA somewhat binds DNA nonspecific-ally by electrostatic interaction: A. Abibi, E. Protozanova, V. V. Demidov, M. D. Frank-Kamenelskii, Biophys. J. 2004, 86, 3070. However, 1 h incubation was sufficient for the invasion in our experiments.
17. M buffer (from TAKARA) was used for EcoRI digestion.
18. At pH 7.0 buffer (containing 10 mM NaCl). lambda DNA was incubated with 1:1 mixture of PNA1 and PNA2 (300 nM each) at 50 °C for 1 h. Then. NaCl was added to a final concentration of 100 mM. The DNA hydrolysis was started by adding aqueous solution of Ce(IV)/EDTA. The reaction was stopped by adding ethylenediaminetetramethylenephosphonic acid (aqueous solution adjusted to pH 7.0) to a final concentration of 1 mM. The mixture was further incubated for 1 h at 50 °C before agarose gel electrophoresis.
19. The other scission fragment (39.2 kbp) is too long and hardly separated from the substrate DNA (48.5 kbp).