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. 2004 Feb 18;32(3):e32.
doi: 10.1093/nar/gnh036.

Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays

Affiliations

Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays

Daniel A Di Giusto et al. Nucleic Acids Res. .

Abstract

The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3'-->5' exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance. The polymerase active sites of these enzymes also display a distinct preference. An L-1 LNA modification has modest effects upon poly merization, but an L-2 LNA group slows dTTP incorporation somewhat while virtually abolishing extension with ddTTP or acyTTP terminators, even with A488L Vent DNA polymerase engineered for terminator incorporation. These observations on active site preference have been utilized to demonstrate two novel assays: exonuclease-mediated single base extension (E-SBE) and proofreading allele-specific extension (PRASE). We show that a model PRASE genotyping reaction with L-2 LNA primers offers greater specificity than existing non-proofreading assays, whether or not the non-proofreading reaction employs LNA-modified primers.

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Figures

Figure 1
Figure 1
Exonucleolysis of natural and LNA-modified oligonucleotides with (A) Pfu DNA polymerase, (B) exo+ A488L Vent DNA polymerase, (C) exo+ Klenow DNA polymerase, (D) T7 DNA polymerase and (E) exo III. Oligonucleotides are PAI-L0 (squares), PAI-L1 (circles), PAI-L2 (triangles) and PAI-L1,2 (inverted triangles).
Figure 2
Figure 2
Exonucleolysis of LNA-modified primers before and after primer extension. Lanes: 1, PAI-L1 standard; 2, PAI-L2 standard; 3, PAI-T2a template standard; 4, exonuclease-III-treated PAI-L1 + PAI-T2a duplex; 5, exonuclease-III-treated PAI-L2 + PAI-T2a duplex; 6, PAI-L1 + PAI-T2a duplex extended with dTTP; 7, PAI-L2 + PAI-T2a duplex extended with dTTP; 8, exonuclease-III-treated PAI-L1 + PAI-T2a duplex following extension with dTTP; and 9, exonuclease-III-treated PAI-L2 + PAI-T2a duplex following extension with dTTP.
Figure 3
Figure 3
Polyacrylamide gel time course of LNA-modified primer extension by 0.2 units of exo A488L Vent DNA polymerase with aminoallyl-dUTP, followed by exo III digestion. (A) PAI-L1. (B) PAI-L1,2. Lanes: M, 25 bp ladder; 1, 0 min; 2, 15 min; 3, 30 min; 4, 60 min; 5, 120 min; 6, 240 min; 7, 960 min; and C, undigested oligonucleotide control.
Figure 4
Figure 4
Polymerase-mediated extension of LNA-modified primers with dTTP by (A) exo Klenow DNA polymerase and (B) exo A488L Vent DNA polymerase. Oligonucleotides are PAI-L1 (squares) and PAI-L1,2 (inverted triangles).
Figure 5
Figure 5
Exonuclease-mediated APEX of five replicated spots with (A) PAI-L0 and (B) PAI-L1. Using PAI-T2g as a template, primers were (1) extended with Cy3-dCTP, dATP, dTTP, dGTP, and then (2) treated with exo III to degrade susceptible nucleotides. Using PAI-T2a as a template, primers were (3) extended with Cy3-dCTP, dATP, dTTP, dGTP and then (4) treated with exo III.
Figure 6
Figure 6
Demonstration of PRASE PCR. (A) Forward allele-specific primers and template sequences. (B) PCR amplicons generated with exo Vent DNA polymerase. (C) PCR amplicons generated with exo+ Vent DNA polymerase. Lanes: M, 100 bp marker ladder and PCR amplicons generated with forward primers. Standard primers: 1, PAP-1g; 2, PAP-1a; 3, PAP-2a and 4, PAP-2g; L-1 LNA-modified primers: 5, PAP-1gL1; 6, PAP-1aL1; 7, PAP-2aL1; 8, PAP-2gL1; and L-2 LNA-modified primers: 9, PAP-1gL2; 10, PAP-1aL2; 11, PAP-2aL2; and 12, PAP-2gL2.

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