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ssDNA Pairing Accuracy Increases When Abasic Sites Divide Nucleotides into Small Groups.

Peacock-Villada A, Coljee V, Danilowicz C, Prentiss M - PLoS ONE (2015)

Bottom Line: We demonstrate that appropriately grouping of 35 bases in ssDNA using abasic sites increases the difference between the melting temperature of correct bases and the melting temperature of mismatched base pairings.Importantly, in the presence of appropriately spaced abasic sites mismatches near one end of a long dsDNA destabilize the annealing at the other end much more effectively than in systems without the abasic sites, suggesting that the dsDNA melts more uniformly in the presence of appropriately spaced abasic sites.In sum, the presence of appropriately spaced abasic sites allows temperature to more accurately discriminate correct base pairings from incorrect ones.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Harvard University, 17 Oxford St., Cambridge, MA 02138, United States of America.

ABSTRACT
Accurate sequence dependent pairing of single-stranded DNA (ssDNA) molecules plays an important role in gene chips, DNA origami, and polymerase chain reactions. In many assays accurate pairing depends on mismatched sequences melting at lower temperatures than matched sequences; however, for sequences longer than ~10 nucleotides, single mismatches and correct matches have melting temperature differences of less than 3°C. We demonstrate that appropriately grouping of 35 bases in ssDNA using abasic sites increases the difference between the melting temperature of correct bases and the melting temperature of mismatched base pairings. Importantly, in the presence of appropriately spaced abasic sites mismatches near one end of a long dsDNA destabilize the annealing at the other end much more effectively than in systems without the abasic sites, suggesting that the dsDNA melts more uniformly in the presence of appropriately spaced abasic sites. In sum, the presence of appropriately spaced abasic sites allows temperature to more accurately discriminate correct base pairings from incorrect ones.

No MeSH data available.


Effect of M on melting curves: differences between the ΔTm for a perfectly matched 35 bp sequence with and without abasic sites and various single bp mismatches in 150 mM NaCl.The sequence is shown in black at the top of the figure. The mismatches are shown below the sequence. In all graphs, the purple squares correspond to a 35 bp sequence without abasic sites for the R data except for the mismatch at position 30 which was taken from the L data. (a) Results for 35 bp sequence with 5 abasic sites where M = 6 (aqua circles). (b) Results for another case where M = 6 shifted by two base pairs from the system shown in (a) (blue triangles). (c) Results for 35-bp sequences with 3 abasic sites where M = 8 (dark blue squares).
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pone.0130875.g004: Effect of M on melting curves: differences between the ΔTm for a perfectly matched 35 bp sequence with and without abasic sites and various single bp mismatches in 150 mM NaCl.The sequence is shown in black at the top of the figure. The mismatches are shown below the sequence. In all graphs, the purple squares correspond to a 35 bp sequence without abasic sites for the R data except for the mismatch at position 30 which was taken from the L data. (a) Results for 35 bp sequence with 5 abasic sites where M = 6 (aqua circles). (b) Results for another case where M = 6 shifted by two base pairs from the system shown in (a) (blue triangles). (c) Results for 35-bp sequences with 3 abasic sites where M = 8 (dark blue squares).

Mentions: Analogous results for probes divided into larger groups are shown in Fig 4. That figure shows that the insertion of abasic sites does not enhance mismatch detection if the spacing between the basic sites is too large. The top row corresponds to results for M = 6, where Fig 4A and 4B correspond to the melting curve data shown in Fig 2Bi and 2Biii, respectively. The results in Fig 4A show no significant improvement over results in the absence of abasic sites, and the results shown in Fig 4B are somewhat worse than the undivided result. The results in Fig 4C correspond to the M = 8 results shown in Fig 2C. Like the M = 6 results, the M = 8 results are not an improvement over the undivided system. For the M = 8 results the high ΔTm is an artifact due to partial melting near the fluorophore, indicating that by M = 8 the melting along the length of the dsDNA is no more uniform than it was for the case without abasic sites. Thus, the data shown in Fig 4 highlights the importance of dividing the nucleotides into small groups.


ssDNA Pairing Accuracy Increases When Abasic Sites Divide Nucleotides into Small Groups.

Peacock-Villada A, Coljee V, Danilowicz C, Prentiss M - PLoS ONE (2015)

Effect of M on melting curves: differences between the ΔTm for a perfectly matched 35 bp sequence with and without abasic sites and various single bp mismatches in 150 mM NaCl.The sequence is shown in black at the top of the figure. The mismatches are shown below the sequence. In all graphs, the purple squares correspond to a 35 bp sequence without abasic sites for the R data except for the mismatch at position 30 which was taken from the L data. (a) Results for 35 bp sequence with 5 abasic sites where M = 6 (aqua circles). (b) Results for another case where M = 6 shifted by two base pairs from the system shown in (a) (blue triangles). (c) Results for 35-bp sequences with 3 abasic sites where M = 8 (dark blue squares).
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Related In: Results  -  Collection

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pone.0130875.g004: Effect of M on melting curves: differences between the ΔTm for a perfectly matched 35 bp sequence with and without abasic sites and various single bp mismatches in 150 mM NaCl.The sequence is shown in black at the top of the figure. The mismatches are shown below the sequence. In all graphs, the purple squares correspond to a 35 bp sequence without abasic sites for the R data except for the mismatch at position 30 which was taken from the L data. (a) Results for 35 bp sequence with 5 abasic sites where M = 6 (aqua circles). (b) Results for another case where M = 6 shifted by two base pairs from the system shown in (a) (blue triangles). (c) Results for 35-bp sequences with 3 abasic sites where M = 8 (dark blue squares).
Mentions: Analogous results for probes divided into larger groups are shown in Fig 4. That figure shows that the insertion of abasic sites does not enhance mismatch detection if the spacing between the basic sites is too large. The top row corresponds to results for M = 6, where Fig 4A and 4B correspond to the melting curve data shown in Fig 2Bi and 2Biii, respectively. The results in Fig 4A show no significant improvement over results in the absence of abasic sites, and the results shown in Fig 4B are somewhat worse than the undivided result. The results in Fig 4C correspond to the M = 8 results shown in Fig 2C. Like the M = 6 results, the M = 8 results are not an improvement over the undivided system. For the M = 8 results the high ΔTm is an artifact due to partial melting near the fluorophore, indicating that by M = 8 the melting along the length of the dsDNA is no more uniform than it was for the case without abasic sites. Thus, the data shown in Fig 4 highlights the importance of dividing the nucleotides into small groups.

Bottom Line: We demonstrate that appropriately grouping of 35 bases in ssDNA using abasic sites increases the difference between the melting temperature of correct bases and the melting temperature of mismatched base pairings.Importantly, in the presence of appropriately spaced abasic sites mismatches near one end of a long dsDNA destabilize the annealing at the other end much more effectively than in systems without the abasic sites, suggesting that the dsDNA melts more uniformly in the presence of appropriately spaced abasic sites.In sum, the presence of appropriately spaced abasic sites allows temperature to more accurately discriminate correct base pairings from incorrect ones.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Harvard University, 17 Oxford St., Cambridge, MA 02138, United States of America.

ABSTRACT
Accurate sequence dependent pairing of single-stranded DNA (ssDNA) molecules plays an important role in gene chips, DNA origami, and polymerase chain reactions. In many assays accurate pairing depends on mismatched sequences melting at lower temperatures than matched sequences; however, for sequences longer than ~10 nucleotides, single mismatches and correct matches have melting temperature differences of less than 3°C. We demonstrate that appropriately grouping of 35 bases in ssDNA using abasic sites increases the difference between the melting temperature of correct bases and the melting temperature of mismatched base pairings. Importantly, in the presence of appropriately spaced abasic sites mismatches near one end of a long dsDNA destabilize the annealing at the other end much more effectively than in systems without the abasic sites, suggesting that the dsDNA melts more uniformly in the presence of appropriately spaced abasic sites. In sum, the presence of appropriately spaced abasic sites allows temperature to more accurately discriminate correct base pairings from incorrect ones.

No MeSH data available.