Composition of the primer extension reactions. 2.00 ml 10X buffer 2 KOD (TOYOBO, supplied by Novagen, 71085-3). 1.50 ml Triphosphate mixture (2.50 mM each) 2.50 ml 10X KOD buffer 1 0.60 ml Primer:template complex (5.0:10.0 mM) 1.00 ml MgCl2 (25 mM) 0.60 ml Klenow fragment (1.0 U/ml) 1.00 ml MnCl2 (50 mM) 15.3 ml Twice distilled water 1.50 ml Triphosphate mixture (2.50 mM each) Dpo4 (TACS). 0.60 ml Primer:template complex (5.0:10.0 mM) 2.00 ml 10X Dpo4 buffer 15 0.60 ml KOD polymerase (2.50 U/ml) 2.00 ml MgCl2 (100 mM) 12.8 ml Twice distilled water 1.00 ml MnCl2 (50 mM) 9°Nm (NEB, supplied by Medinova, M0260S). 1.50 ml Triphosphate mixture (2.50 mM each) 2.00 ml 10X Thermopol buffer 0.60 ml Primer:template complex (5.0:10.0 mM) 1.50 ml Triphosphate mixture (2.50 mM each) 0.60 ml Dpo4 (0.40 mg/ml) 0.60 ml Primer:template complex (5.0:10.0 mM) 12.3 ml Twice distilled water 0.60 ml 9°Nm polymerase (2000 U/ml) HIV RT (Worthington biochemical company, LS05003). 15.3 ml Twice distilled water 4.00 ml 5X HIV RT buffer (250 mM TRIS-HCl, 300 mM Phusion (Finnzymes, F-530S). KCl, 12.5 mM MgCl2) 4.00 ml 5X Phusion HF buffer 1.50 ml Triphosphate mixture (2.50 mM each) 1.50 ml Triphosphate mixture (2.50 mM each) 0.60 ml Primer:template complex (5.0:10.0 mM) 0.60 ml Primer:template complex (5.0:10.0 mM) 0.60 ml HIV RT (2.7 U/ml) © 0.60 2012 ml Phusion (2000 U/ml) Landes 13.3 BDiscloisure ml Twice distilled oofPostential water cConfilicts eofIntnerest ce. 13.3 ml Twice distilled water Human polymerase β (Trevigen, 4020–500-EB). 2.00 ml 10X reaction buffer 8 No potential conflicts of interest were disclosed. 1.50 ml Triphosphate mixture (2.50 mM each) 0.60 ml Primer:template complex (5.0:10.0 mM) Acknowledgments 0.60 ml Human polymerase β (3.3 U/ml) The authors wish to thank Lykke Haastrup Hansen. This research Klenow Fragment (BDoehringoerMannnheim; NoEB, Bt7002Sd).i sDanisthNrationial bResearuchFoutndaetion. . 15.3 ml Twice distilled water was made possible by funding for the Nucleic Acid Center by The 20 Artificial DNA: PNA & XNA Volume 3 Issue 1 References 1. Singh SK, Nielsen P, Koshkin AA, Wengel J. LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem Commun (Camb) 1998; 4:455-6; http://dx.doi.org/10.1039/a708608c 2. Koshkin AA, Singh SK, Nielsen P, Rajwanshi VK, Kumar R, Meldgaard M, et al. LNA (locked nucleic acids): synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerization, and unprecedented nucleic acid recognition. Tetrahedron 1998; 54:3607-30; http://dx.doi.org/10.1016/S0040-4020(98)00094-5 3. Obika S, Nanbu D, Hari Y, Andoh J, Morio K, Doi T, et al. Stability and structural features of the duplexes containing nucleoside analogs with a fixed N-type conformation, 2'-O,4'-C-methyleneribonucleosides. Tetrahedron Lett 1998; 39:5401-4; http://dx.doi.org/ 10.1016/S0040-4039(98)01084-3 4. Rajwanshi VK, Håkansson AE, Dahl BM, Wengel J. LNA stereoisomers: xylo-LNA (β-D-xylo configured locked nucleic acid) and a-L-LNA (a-L-ribo configured locked nucleic acid). Chem Commun (Camb) 1999; 15:1395-6; http://dx.doi.org/10.1039/a903189h 5. Rajwanshi VK, Håkansson AE, Babu R, Wengel J. High-affinity nucleic acid recognition using ‘LNA’ (locked nucleic acid, β-D-ribo configured LNA), ‘xylo-LNA’ (β-D-xylo configured LNA) or ‘a-L-LNA’ (a-L-ribo configured LNA). Chem Commun (Camb) 1999; 20:2073-4; http://dx.doi.org/10.1039/a906713b 6. Rajwanshi VK, Håkansson AE, Sørensen MD, Pitsch S, Singh SK, Kumar R, et al. The eight stereoisomers of LNA (locked nucleic acid): a remarkable family of strong RNA binding molecules. Angew Chem Int Ed Engl 2000; 39:1656-9; PMID:10820467; http://dx.doi.org/10.1002/(SICI)1521-3773(20000502) 39:9,1656::AID-ANIE1656.3.0.CO;2-Q 7. Håkansson AE, Koshkin AA, Sørensen MD, Wengel J. Convenient syntheses of 7-hydroxy-1-(hydroxymethyl)- 3-(thymin-1-yl)-2,5-dioxabicyclo. J Org Chem 2000; 65:5161-6; PMID:10993341; http://dx.doi.org/10. 1021/jo000232g 8. Håkansson AE, Wengel J. The adenine derivative of a-L-LNA (a-L-ribo configured locked nucleic acid): synthesis and high-affinity hybridization towards DNA, RNA, LNA and a-L-LNA complementary sequences. Bioorg Med Chem Lett 2001; 11:935-8; PMID:11294395; http://dx.doi.org/10.1016/S0960- 894X(01)00110-X 9. Sørensen MD, Kvaernø L, Bryld T, Håkansson AE, Verbeure B, Gaubert G, et al. a-L-ribo-configured locked nucleic acid (a-L-LNA): synthesis and pro- perties. J Am Chem Soc 2002; 124:2164-76; PMID: 11878970; http://dx.doi.org/10.1021/ja0168763 10. Gaubert G, Wengel J. Synthesis of a base-protected a-L-LNA guanine nucleoside. Nucleosides Nucleotides Nucleic Acids 2003; 22:1155-7; PMID:14565368; http://dx.doi.org/10.1081/NCN-120022824 11. Petersen M, Wengel J. LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol 2003; 21:74-81; PMID:12573856; http://dx.doi.org/10. 1016/S0167-7799(02)00038-0 12. Vester B, Wengel J. LNA (locked nucleic acid): high- affinity targeting of complementary RNA and DNA. Biochemistry 2004; 43:13233-41; PMID:15491130; http://dx.doi.org/10.1021/bi0485732 13. Jepsen JS, Sørensen MD, Wengel J. Locked nucleic acid: a potent nucleic acid analog in therapeutics and biotechnology. Oligonucleotides 2004; 14:130- 46; PMID:15294076; http://dx.doi.org/10.1089/ 1545457041526317 14. Kaur H, Babu BR, Maiti S. Perspectives on chemistry and therapeutic applications of Locked Nucleic Acid (LNA). Chem Rev 2007; 107:4672-97; PMID: 17944519; http://dx.doi.org/10.1021/cr050266u 15. Veedu RN, Vester B, Wengel J. In vitro incorporation of LNA nucleotides. Nucleosides Nucleotides Nucleic Acids 2007; 26:1207-10; PMID:18058567; http://dx. doi.org/10.1080/15257770701527844 16. Veedu RN, Vester B, Wengel J. Enzymatic incorpora- tion of LNA nucleotides into DNA strands. Chembiochem 2007; 8:490-2; PMID:17315250; http://dx.doi.org/10.1002/cbic.200600501 17. Veedu RN, Vester B, Wengel J. Polymerase chain reaction and transcription using locked nucleic acid nucleotide triphosphates. J Am Chem Soc 2008; 130: 8124-5; PMID:18533656; http://dx.doi.org/10.1021/ ja801389n 18. Veedu RN, Vester B, Wengel J. Efficient enzymatic synthesis of LNA-modified DNA duplexes using KOD DNA polymerase. Org Biomol Chem 2009; 7:1404-9; PMID:19300826; http://dx.doi.org/10.1039/b819946a 19. Kuwahara M, Obika S, Nagashima J-I, Ohta Y, Suto Y, Ozaki H, et al. Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleo- sides. Nucleic Acids Res 2008; 36:4257-65; PMID: 18583360; http://dx.doi.org/10.1093/nar/gkn404 20. Veedu RN, Wengel J. Locked nucleic acid nucleoside triphosphates and polymerases: on the way towards evolution of LNA aptamers. Mol Biosyst 2009; 5:787-92; PMID:19603111; http://dx.doi.org/10.1039/b905513b