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Analysis of a DNA simulation model through hairpin melting experiments
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10.1063/1.3480685
/content/aip/journal/jcp/133/12/10.1063/1.3480685
http://aip.metastore.ingenta.com/content/aip/journal/jcp/133/12/10.1063/1.3480685

Figures

Image of FIG. 1.
FIG. 1.

Example of a two-bead model of the single-stranded sequence ; this chain is used for illustration purposes and is not a sequence in the study. The chain is comprised of a series of contiguous backbone beads and a series of base beads. The chain is initialized in either a linear or square U configuration. The Watson–Crick base pairing stem and noninteracting loop sections are labeled.

Image of FIG. 2.
FIG. 2.

Postprocessing of the experimental data for the sequence. (a) Average background fluorescence signal, . (b) Average background corrected unbound SYBR Green I temperature dependence, . (c) An example well’s melting curve, ; the temperature dependence of the bound SYBR Green I signal is fit from the closed hairpin state, . (d) Normalized and averaged replicates for the sequence. The temperature variation range is reported at selected normalized intensity values.

Image of FIG. 3.
FIG. 3.

The three metrics used to determine the “closed” state in the simulation data. Metrics 1 and 2 enumerate all stem possible bonding pairs (1) and aligned stem possible bonding pairs (2). Metric 3 only considers the hairpin system to be closed when all of the possible pairs in the aligned stems are bonded. The simulation data are presented for each of the three metrics with nondimensional temperature and normalized intensity. A sigmoidal curve is fit to the data from each metric.

Image of FIG. 4.
FIG. 4.

Plot of the normalized fluorescence intensity as a function of temperature for different DNA and dye concentrations with the sequence . The (red) dashed line in the center plot was chosen as the experimental condition for all subsequent studies: DNA solution and 2X SYBR Green I dye. This sets the ratio of dye molecules to stem base pairs at 2X: stem base pairs.

Image of FIG. 5.
FIG. 5.

Plot of the three metrics with the experimental data overlaid for the base case sequence. Metric 2 best captures the slope in the transition regime of the experimental data.

Image of FIG. 6.
FIG. 6.

Comparison of the metric 2 simulation data and experimental sigmoidal fit curve with a factor. Each of the seven investigated sequences is depicted and sorted into their original classes. The values of these fits are reported in Table IV.

Image of FIG. 7.
FIG. 7.

Comparison of the metric 2 simulation data and experimental sigmoidal fit curve for the sequence with , which is the conversion required to match the melting-point temperature. The value is 0.707.

Tables

Generic image for table
Table I.

List of single-stranded DNA sequences. The classes refer to the change in stem length (1), loop (2), or sequence (3) when compared to the base case . The index j will be used throughout to denote the DNA hairpin type. The mfold predicted melting-point temperatures were found for monovalent sodium, similar to buffer A, and are given for the aligned stem bonding configuration (Ref. 30).

Generic image for table
Table II.

List of well types randomly loaded on each 96 well plate. One of the seven DNA hairpins under study was loaded into each experimental signal well. The number of replicates of each well type per plate is the number of well numbers, k, corresponding to that experimental condition.

Generic image for table
Table III.

Summary of the and values for each of the values and metrics examined in the study. Row 2 is depicted graphically in Fig. 5 and the center, bolded cell contains the optimal values utilized for the remainder of the investigation.

Generic image for table
Table IV.

Summary of values for for each of the sequences examined.

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/content/aip/journal/jcp/133/12/10.1063/1.3480685
2010-09-22
2014-04-18
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Analysis of a DNA simulation model through hairpin melting experiments
http://aip.metastore.ingenta.com/content/aip/journal/jcp/133/12/10.1063/1.3480685
10.1063/1.3480685
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