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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.


Measurements of the melting temperature, Tm, for sequences divided by abasic sites into groups with size M for the same experimental conditions used to obtain the data in Fig 1.(a) M = 4 results where i is the same as Fig 1D, ii is the L result for the same system, and iii is the R result for the system shown in main text Fig 1A-iii where the abasic sites are shifted by two with respect to the system shown in i and ii. (b) Analogous results for a system with M = 6. For the M = 6 system that is shifted, some single mismatches fall on an abasic site. Curves corresponding to mismatches on abasic sites are shown as magenta dashed lines. The results are in very good agreement with the perfect match. (c) Analogous results for a system with M = 8. (d) Analogous result for an undivided probe.
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pone.0130875.g002: Measurements of the melting temperature, Tm, for sequences divided by abasic sites into groups with size M for the same experimental conditions used to obtain the data in Fig 1.(a) M = 4 results where i is the same as Fig 1D, ii is the L result for the same system, and iii is the R result for the system shown in main text Fig 1A-iii where the abasic sites are shifted by two with respect to the system shown in i and ii. (b) Analogous results for a system with M = 6. For the M = 6 system that is shifted, some single mismatches fall on an abasic site. Curves corresponding to mismatches on abasic sites are shown as magenta dashed lines. The results are in very good agreement with the perfect match. (c) Analogous results for a system with M = 8. (d) Analogous result for an undivided probe.

Mentions: Though the data in Fig 1 indicates that including abasic sites has a strong effect on the melting behavior of dsDNA, it is not clear whether the result is due to the mere presence of abasic sites, or whether the grouping of the abasic sites also plays an important role. In Fig 1, the sequences are divided into groups of M = 4, separated by abasic sites. Fig 2 shows melting curves for larger M values. The results in Fig 2 clearly indicate that the melting temperatures increase as the group size increases. This is not surprising since the presence of abasic sites must alter stacking interactions. Interestingly, the melting curves for matched base pairings (magenta) are indistinguishable from the melting curves for pairings in which the “mismatches” fall on abasic sites (dashed magenta curves). In addition, the melting curves for the two M = 4 systems are very similar even though the position and number of the abasic sites are different for the two cases. Thus, positions of individual mismatched bases could be detected by sliding the position of the abasic sites while maintaining group size.


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

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

Measurements of the melting temperature, Tm, for sequences divided by abasic sites into groups with size M for the same experimental conditions used to obtain the data in Fig 1.(a) M = 4 results where i is the same as Fig 1D, ii is the L result for the same system, and iii is the R result for the system shown in main text Fig 1A-iii where the abasic sites are shifted by two with respect to the system shown in i and ii. (b) Analogous results for a system with M = 6. For the M = 6 system that is shifted, some single mismatches fall on an abasic site. Curves corresponding to mismatches on abasic sites are shown as magenta dashed lines. The results are in very good agreement with the perfect match. (c) Analogous results for a system with M = 8. (d) Analogous result for an undivided probe.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482597&req=5

pone.0130875.g002: Measurements of the melting temperature, Tm, for sequences divided by abasic sites into groups with size M for the same experimental conditions used to obtain the data in Fig 1.(a) M = 4 results where i is the same as Fig 1D, ii is the L result for the same system, and iii is the R result for the system shown in main text Fig 1A-iii where the abasic sites are shifted by two with respect to the system shown in i and ii. (b) Analogous results for a system with M = 6. For the M = 6 system that is shifted, some single mismatches fall on an abasic site. Curves corresponding to mismatches on abasic sites are shown as magenta dashed lines. The results are in very good agreement with the perfect match. (c) Analogous results for a system with M = 8. (d) Analogous result for an undivided probe.
Mentions: Though the data in Fig 1 indicates that including abasic sites has a strong effect on the melting behavior of dsDNA, it is not clear whether the result is due to the mere presence of abasic sites, or whether the grouping of the abasic sites also plays an important role. In Fig 1, the sequences are divided into groups of M = 4, separated by abasic sites. Fig 2 shows melting curves for larger M values. The results in Fig 2 clearly indicate that the melting temperatures increase as the group size increases. This is not surprising since the presence of abasic sites must alter stacking interactions. Interestingly, the melting curves for matched base pairings (magenta) are indistinguishable from the melting curves for pairings in which the “mismatches” fall on abasic sites (dashed magenta curves). In addition, the melting curves for the two M = 4 systems are very similar even though the position and number of the abasic sites are different for the two cases. Thus, positions of individual mismatched bases could be detected by sliding the position of the abasic sites while maintaining group size.

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.