Limits...
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.


ΔTm as a function of the position of single mismatches.The sequence is shown at the top of the figure The single base pair mismatch replacements are shown below in colors corresponding to the colors of the curves shown in Fig 1D and 1E. The cyan, black, and purple lines and symbols correspond to the systems shown in Fig 1i, ii, and iii, respectively. The solid triangles represent the L data. The hollow circles correspond to the R data. The solid lines connect the data points from R measurements, except for the Tm mismatch at position 30, which is derived from the L data. The arrows on the right (left) side of the graph indicate Tm values calculated from R (L) measurements for a mismatch at position 30(8). For the undivided probe, the fluorophore pair nearest the mismatch separates at a significantly lower temperature than the true Tm, but for the divided probe the ends melt at the true Tm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482597&req=5

pone.0130875.g003: ΔTm as a function of the position of single mismatches.The sequence is shown at the top of the figure The single base pair mismatch replacements are shown below in colors corresponding to the colors of the curves shown in Fig 1D and 1E. The cyan, black, and purple lines and symbols correspond to the systems shown in Fig 1i, ii, and iii, respectively. The solid triangles represent the L data. The hollow circles correspond to the R data. The solid lines connect the data points from R measurements, except for the Tm mismatch at position 30, which is derived from the L data. The arrows on the right (left) side of the graph indicate Tm values calculated from R (L) measurements for a mismatch at position 30(8). For the undivided probe, the fluorophore pair nearest the mismatch separates at a significantly lower temperature than the true Tm, but for the divided probe the ends melt at the true Tm.

Mentions: (a) Schematic of the pairing of the target ssDNA containing 35 nucleotides (purple) with the ssDNA probe (red), (i) probe with no abasic sites, (ii) and (iii) probes divided by abasic sites into groups with size M. i. M = 4 nt groups. The positions of the abasic sites are highlighted by the cyan rectangles. ii and iii correspond to the same M = 4 value, but have different numbers of abasic sites because they are shifted with respect to each other by 2 bases. (b) Schematic of the melting experiment with R constructs. (c) Schematic of the melting experiment with L constructs. The colored arrows indicate positions of different single isolated mismatches. The black and orange circles indicate the positions of the BHQ-1 and Texas Red fluorophores. Fluorescence is high if the fluorophores are widely separated, but low if they are close. (ii)-(iv) show partial melting and (v) shows full melting. (d) Texas Red fluorescence as a function of temperature in a buffer containing 150 mM NaCl for the system shown in a-i; perfectly matched sequence (magenta) and different single mismatches (green and gray as specified in Fig 3). (e) Texas Red fluorescence as a function of temperature in a buffer containing 150 mM NaCl for the system shown in a-ii and same color code as in (d).


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

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

ΔTm as a function of the position of single mismatches.The sequence is shown at the top of the figure The single base pair mismatch replacements are shown below in colors corresponding to the colors of the curves shown in Fig 1D and 1E. The cyan, black, and purple lines and symbols correspond to the systems shown in Fig 1i, ii, and iii, respectively. The solid triangles represent the L data. The hollow circles correspond to the R data. The solid lines connect the data points from R measurements, except for the Tm mismatch at position 30, which is derived from the L data. The arrows on the right (left) side of the graph indicate Tm values calculated from R (L) measurements for a mismatch at position 30(8). For the undivided probe, the fluorophore pair nearest the mismatch separates at a significantly lower temperature than the true Tm, but for the divided probe the ends melt at the true Tm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130875.g003: ΔTm as a function of the position of single mismatches.The sequence is shown at the top of the figure The single base pair mismatch replacements are shown below in colors corresponding to the colors of the curves shown in Fig 1D and 1E. The cyan, black, and purple lines and symbols correspond to the systems shown in Fig 1i, ii, and iii, respectively. The solid triangles represent the L data. The hollow circles correspond to the R data. The solid lines connect the data points from R measurements, except for the Tm mismatch at position 30, which is derived from the L data. The arrows on the right (left) side of the graph indicate Tm values calculated from R (L) measurements for a mismatch at position 30(8). For the undivided probe, the fluorophore pair nearest the mismatch separates at a significantly lower temperature than the true Tm, but for the divided probe the ends melt at the true Tm.
Mentions: (a) Schematic of the pairing of the target ssDNA containing 35 nucleotides (purple) with the ssDNA probe (red), (i) probe with no abasic sites, (ii) and (iii) probes divided by abasic sites into groups with size M. i. M = 4 nt groups. The positions of the abasic sites are highlighted by the cyan rectangles. ii and iii correspond to the same M = 4 value, but have different numbers of abasic sites because they are shifted with respect to each other by 2 bases. (b) Schematic of the melting experiment with R constructs. (c) Schematic of the melting experiment with L constructs. The colored arrows indicate positions of different single isolated mismatches. The black and orange circles indicate the positions of the BHQ-1 and Texas Red fluorophores. Fluorescence is high if the fluorophores are widely separated, but low if they are close. (ii)-(iv) show partial melting and (v) shows full melting. (d) Texas Red fluorescence as a function of temperature in a buffer containing 150 mM NaCl for the system shown in a-i; perfectly matched sequence (magenta) and different single mismatches (green and gray as specified in Fig 3). (e) Texas Red fluorescence as a function of temperature in a buffer containing 150 mM NaCl for the system shown in a-ii and same color code as in (d).

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.