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Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides.

Højland T, Veedu RN, Vester B, Wengel J - Artif DNA PNA XNA (2012 Jan-Mar)

Bottom Line: It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT.Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides.In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

ABSTRACT
We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

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Figure 5. Primer extension using template T3. Lane 1: positive control (dATP, dGTP, dCTP and TTP); lane 2: incorporation of α-L-LNA-T nucleotides (dATP, dGTP, dCTP and α-L-LNA TTP); lane 3: negative control (dATP, dGTP and dCTP); lane 4: P1 (19mer).
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Figure 5: Figure 5. Primer extension using template T3. Lane 1: positive control (dATP, dGTP, dCTP and TTP); lane 2: incorporation of α-L-LNA-T nucleotides (dATP, dGTP, dCTP and α-L-LNA TTP); lane 3: negative control (dATP, dGTP and dCTP); lane 4: P1 (19mer).

Mentions: We also investigated whether the polymerases needed a running start in order to incorporate α-L-LNA-T nucleotides. We designed template T3 to direct the extension of the primer with α-L-LNA TTP as the first triphosphate to be used as substrate. The results in Figure 5 show that KOD, 9°Nm and HIV RT were able to extend the primer to full length. In fact, KOD was so efficient with template T3 that misincorporation bands are seen in the positive and negative control reactions (Fig. 5, lanes 1 and 3). Phusion DNA polymerase proceeded with difficulty in extending the primer to afford only trace amounts of full-length product.


Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides.

Højland T, Veedu RN, Vester B, Wengel J - Artif DNA PNA XNA (2012 Jan-Mar)

Figure 5. Primer extension using template T3. Lane 1: positive control (dATP, dGTP, dCTP and TTP); lane 2: incorporation of α-L-LNA-T nucleotides (dATP, dGTP, dCTP and α-L-LNA TTP); lane 3: negative control (dATP, dGTP and dCTP); lane 4: P1 (19mer).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368812&req=5

Figure 5: Figure 5. Primer extension using template T3. Lane 1: positive control (dATP, dGTP, dCTP and TTP); lane 2: incorporation of α-L-LNA-T nucleotides (dATP, dGTP, dCTP and α-L-LNA TTP); lane 3: negative control (dATP, dGTP and dCTP); lane 4: P1 (19mer).
Mentions: We also investigated whether the polymerases needed a running start in order to incorporate α-L-LNA-T nucleotides. We designed template T3 to direct the extension of the primer with α-L-LNA TTP as the first triphosphate to be used as substrate. The results in Figure 5 show that KOD, 9°Nm and HIV RT were able to extend the primer to full length. In fact, KOD was so efficient with template T3 that misincorporation bands are seen in the positive and negative control reactions (Fig. 5, lanes 1 and 3). Phusion DNA polymerase proceeded with difficulty in extending the primer to afford only trace amounts of full-length product.

Bottom Line: It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT.Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides.In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

ABSTRACT
We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

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