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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 4. Primer extension using template T2. 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 and T1 (19mer and 43mer).
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Figure 4: Figure 4. Primer extension using template T2. 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 and T1 (19mer and 43mer).

Mentions: Next, consecutive incorporation of α-L-LNA-T nucleotides was investigated (Fig. 4). KOD, Phusion and HIV RT were unable to extend the primer beyond the first incorporation of α-L-LNA-T (lane 2). 9°Nm DNA polymerase was able to incorporate consecutive α-L-LNA-T nucleotides, but full-length extension product was not observed. It appears that 9°Nm did not move forward methodically. Rather, 9°Nm seemed to quickly incorporate several α-L-LNA-T nucleotides before stopping extension. On comparison with a known DNA marker (not shown), we could conclude that a major product of the extension was 31 nucleotides long corresponding to consecutive incorporation of five α-L-LNA-T nucleotides, though some of the shorter products were also present in trace amounts.


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 4. Primer extension using template T2. 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 and T1 (19mer and 43mer).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Figure 4. Primer extension using template T2. 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 and T1 (19mer and 43mer).
Mentions: Next, consecutive incorporation of α-L-LNA-T nucleotides was investigated (Fig. 4). KOD, Phusion and HIV RT were unable to extend the primer beyond the first incorporation of α-L-LNA-T (lane 2). 9°Nm DNA polymerase was able to incorporate consecutive α-L-LNA-T nucleotides, but full-length extension product was not observed. It appears that 9°Nm did not move forward methodically. Rather, 9°Nm seemed to quickly incorporate several α-L-LNA-T nucleotides before stopping extension. On comparison with a known DNA marker (not shown), we could conclude that a major product of the extension was 31 nucleotides long corresponding to consecutive incorporation of five α-L-LNA-T nucleotides, though some of the shorter products were also present in trace amounts.

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.

Show MeSH