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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 3. Primer extension using template T1. 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 3: Figure 3. Primer extension using template T1. 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: The following seven polymerases were initially explored for their ability to incorporate α-L-LNA nucleotides: the Klenow fragment of E. coli DNA polymerase I (A-family polymerase); KOD, 9°Nm and Phusion DNA polymerases (B-family polymerase); human polymerase β (X-family polymerase); S. solfataricus DNA polymerase IV (Dpo4, Y-family polymerase); and HIV RT (reverse transcriptase family polymerase). It was found that the four most efficient polymerases for α-L-LNA nucleotide incorporation were KOD, 9°Nm, Phusion and HIV RT. Figure 3 shows the results of primer extension experiments on the T1 template for these four polymerases. The experiment demonstrated that KOD, 9°Nm and Phusion DNA polymerases can efficiently accept α-L-LNA TTP as a substrate and afford the full-length extension products (lane 2). In particular, KOD is very quick at extending the primer to full length, however accompanied by some product degradation. Although HIV RT could also produce the fully extended product in low yield, the reaction did not progress to completion in the time the other three polymerases required.


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 3. Primer extension using template T1. 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 3: Figure 3. Primer extension using template T1. 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: The following seven polymerases were initially explored for their ability to incorporate α-L-LNA nucleotides: the Klenow fragment of E. coli DNA polymerase I (A-family polymerase); KOD, 9°Nm and Phusion DNA polymerases (B-family polymerase); human polymerase β (X-family polymerase); S. solfataricus DNA polymerase IV (Dpo4, Y-family polymerase); and HIV RT (reverse transcriptase family polymerase). It was found that the four most efficient polymerases for α-L-LNA nucleotide incorporation were KOD, 9°Nm, Phusion and HIV RT. Figure 3 shows the results of primer extension experiments on the T1 template for these four polymerases. The experiment demonstrated that KOD, 9°Nm and Phusion DNA polymerases can efficiently accept α-L-LNA TTP as a substrate and afford the full-length extension products (lane 2). In particular, KOD is very quick at extending the primer to full length, however accompanied by some product degradation. Although HIV RT could also produce the fully extended product in low yield, the reaction did not progress to completion in the time the other three polymerases required.

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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