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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 7. Primer extension using templates T4-T7. Lanes 1, 3, 5 and 7: positive controls (dATP, dGTP, dCTP and TTP); lanes 2, 4, 6 and 8: negative controls [dGTP, dCTP and dTTP (for incorporation across α-L-LNA-T); or dATP, dCTP and dTTP (for incorporation across α-L-LNA-5-methyl-C)]; lanes 1 and 2: template T4; lanes 3 and 4: template T5; lanes 5 and 6: template T6; lanes 7 and 8: template T7; lane 9: P1 and T1 (19mer and 43mer).
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Figure 7: Figure 7. Primer extension using templates T4-T7. Lanes 1, 3, 5 and 7: positive controls (dATP, dGTP, dCTP and TTP); lanes 2, 4, 6 and 8: negative controls [dGTP, dCTP and dTTP (for incorporation across α-L-LNA-T); or dATP, dCTP and dTTP (for incorporation across α-L-LNA-5-methyl-C)]; lanes 1 and 2: template T4; lanes 3 and 4: template T5; lanes 5 and 6: template T6; lanes 7 and 8: template T7; lane 9: P1 and T1 (19mer and 43mer).

Mentions: KOD, 9°Nm, Phusion and HIV RT which performed well at α-L-LNA-T incorporations were investigated for their ability to use templates containing α-L-LNA nucleotides. The results of primer extension experiments using templates T4-T7 are shown in Figure 7. All four polymerases demonstrated difficulties in extending the primer using templates T4-T7. However, template T6 which contained a single α-L-LNA-5-methyl-C nucleotide afforded the full-length extension product by all four polymerases (Fig. 7, lane 5) with KOD as the more efficient.


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 7. Primer extension using templates T4-T7. Lanes 1, 3, 5 and 7: positive controls (dATP, dGTP, dCTP and TTP); lanes 2, 4, 6 and 8: negative controls [dGTP, dCTP and dTTP (for incorporation across α-L-LNA-T); or dATP, dCTP and dTTP (for incorporation across α-L-LNA-5-methyl-C)]; lanes 1 and 2: template T4; lanes 3 and 4: template T5; lanes 5 and 6: template T6; lanes 7 and 8: template T7; lane 9: P1 and T1 (19mer and 43mer).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3368812&req=5

Figure 7: Figure 7. Primer extension using templates T4-T7. Lanes 1, 3, 5 and 7: positive controls (dATP, dGTP, dCTP and TTP); lanes 2, 4, 6 and 8: negative controls [dGTP, dCTP and dTTP (for incorporation across α-L-LNA-T); or dATP, dCTP and dTTP (for incorporation across α-L-LNA-5-methyl-C)]; lanes 1 and 2: template T4; lanes 3 and 4: template T5; lanes 5 and 6: template T6; lanes 7 and 8: template T7; lane 9: P1 and T1 (19mer and 43mer).
Mentions: KOD, 9°Nm, Phusion and HIV RT which performed well at α-L-LNA-T incorporations were investigated for their ability to use templates containing α-L-LNA nucleotides. The results of primer extension experiments using templates T4-T7 are shown in Figure 7. All four polymerases demonstrated difficulties in extending the primer using templates T4-T7. However, template T6 which contained a single α-L-LNA-5-methyl-C nucleotide afforded the full-length extension product by all four polymerases (Fig. 7, lane 5) with KOD as the more efficient.

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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