©FdiGgTuPr,ed3C.2TPPrimanedr 0eax-tLe-LnNsiAo1nTTuPsi)n;gla2nteem3p: lnateegTaL1ti.vLeacnoean1t:roplo(sdnitAivTeP,codnGdtTrPola(dnAdeTdPC,TdPG)T;sPla,ndeCT4P:PaB1ndanTdTPT)1;ila(1n9eom2e:irnacnosdrp4o3ramticeorn).of ia-L-eLNA-T nnucleotidces(dAeTP, . We also investigated whether the polymerases needed a running extending the primer to afford only trace amounts of full-length start in order to incorporate a-L-LNA-T nucleotides. We product. a-L-LNA TTP as the D first troiphosphate not to be used as substrate.inucleotides. d stri Next, bwe investigated ute. whether the four polymerases designed template T3 to direct the extension of the primer with Primer extension using templates containing a-L-LNA The results in Figure 5 show that KOD, 9°Nm and HIV RT are able to use a-L-LNA TTP as substrate that can tolerate a-L- were able to extend the primer to full length. In fact, KOD was LNA nucleotides in the template. The commercially available T so efficient with template T3 that misincorporation bands are and 5-methyl-C a-L-LNA phosphoramidites were used to pro- seen in the positive and negative control reactions (Fig. 5, lanes 1 duce templates T4-T7 (Fig. 6). In templates T5 and T7, a-L- and 3). Phusion DNA polymerase proceeded with difficulty in LNA nucleotides are placed one after another to produce a four Figure 4. Primer extension using template T2. Lane 1: positive control (dATP, dGTP, dCTP and TTP); lane 2: incorporation of a-L-LNA-T nucleotides (dATP, dGTP, dCTP and a-L-LNA TTP); lane 3: negative control (dATP, dGTP and dCTP); lane 4: P1 and T1 (19mer and 43mer). 16 Artificial DNA: PNA & XNA Volume 3 Issue 1