Quantum Method for Fluorescence Background Removal in DNA Melting Analysis

Sanford, Lindsay N.; Kent, Jana; Wittwer, Carl T.

doi:10.1021/ac4024928

Cited by 9 publications

(4 citation statements)

References 21 publications

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“…The melting temperature of WT and DAP-substituted sequences was determined from fluorescently detected melting curves (44,45). Three 155, 147, and 156 basepair-long portions of the pBR322 plasmid were selected containing 40, 54, and 65% GC content, respectively.…”

Section: Melting Temperature Characterizationmentioning

confidence: 99%

“…S4) (46). Fluorescence curves were normalized to account for temperature Nanomechanics of DAP-DNA dependence of the fluorescent dye (44,45); details are included in the Supporting Materials and Methods. The Syto-84 findings were confirmed by roughly equivalent melting temperatures measured via Sybr-Green I (Fig.…”

Section: Dap Substitution Increases Melting Temperature But Preservesmentioning

confidence: 99%

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Nanomechanics of Diaminopurine-Substituted DNA

Cristofalo

Kovari

Corti

et al. 2019

Biophysical Journal

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2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.

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Section: Melting Temperature Characterizationmentioning

confidence: 99%

Section: Dap Substitution Increases Melting Temperature But Preservesmentioning

confidence: 99%

Nanomechanics of Diaminopurine-Substituted DNA

Cristofalo

Kovari

Corti

et al. 2019

Biophysical Journal

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“…Consecutive anneals were compared to ensure consistency. Using a python script replicate samples were averaged after normalized as per Sanford et al [31]. Fluorescence spectroscopy was gathered for 5 nM circle and bundle at 2.5×, 5×, 7.5×, 10×, 15×, 20×, and 40× staples.…”

Section: Fluorescence Spectroscopy Via Rt-pcr Equipmentmentioning

confidence: 99%

Competitive annealing of multiple DNA origami: formation of chimeric origami

2016

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Scaffolded DNA origami are a robust tool for building discrete nanoscale objects at high yield. This strategy ensures, in the design process, that the desired nanostructure is the minimum free energy state for the designed set of DNA sequences. Despite aiming for the minimum free energy structure, the folding process which leads to that conformation is difficult to characterize, although it has been the subject of much research. In order to shed light on the molecular folding pathways, this study intentionally frustrates the folding process of these systems by simultaneously annealing the staple pools for multiple target or parent origami structures, forcing competition. A surprising result of these competitive, simultaneous anneals is the formation of chimeric DNA origami which inherit structural regions from both parent origami. By comparing the regions inherited from the parent origami, relative stability of substructures were compared. This allowed examination of the folding process with typical characterization techniques and materials. Anneal curves were then used as a means to rapidly generate a phase diagram of anticipated behavior as a function of staple excess and parent staple ratio. This initial study shows that competitive anneals provide an exciting way to create diverse new nanostructures and may be used to examine the relative stability of various structural motifs.The commercial development of inexpensive and quickly produced DNA of arbitrary nucleobase sequence has fueled the growth of DNA nanotechnology as a field [1]. DNA nanotechnology has made promising advances toward light harvesting [2], computation [3], cancer treatement [4,5], and assembly of nanoelectronics [6]. While many DNA self-assembly strategies exist [7,8], scaffolded DNA origami has received significant attention as a convenient way to design and create discrete nanoscale objects [9]. Scaffolded DNA origami consist of single strand DNA (ssDNA) of two types, synthetic oligomers and circular viral genomes; the viral scaffold is forced to route through the designed structure by the binding of complementary subsequences on the synthetic oligomers, or staples. Staples are added in excess, often 10× relative to the scaffold concentration, strongly driving the viral ssDNA scaffold to fold into the target structure. Although 10× is a common staple excess anneals have been successfully performed as low as 2.5× staple excess. As this study involves multiple staple pools, staple excess will always refer to the total staple concentration relative to the scaffold, while the parent staple ratio will refer to how much of that total corresponds to the staple pool for each target origami. High production yield is achieved by thermal annealing and slow cooling of the system. Such annealing has also been performed chemically, isothermally, and mechanically [10][11][12].The hybridization of double strand DNA (dsDNA) has been well studied, particularly under physiological conditions [13,14]. The formation of dsDNA from ssDNA is driven by base stack...

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“…S5 show melting curves after normalization. Several methods have been described for normalization, including proportional linear baseline removal (3), exponential background subtraction (15), and quantum calculations (21) Exponential background removal is most robust, although the most appropriate method may depend on the DNA‐binding dye and the number of transitions in the melting curve. The success of background removal should be assessed by how horizontal the normalized curves are outside of the melting transitions (equivalent to how close to 0 the derivative curves baselines are).…”

Section: Discussionmentioning

confidence: 99%

Removal of artifact bias from qPCR results using DNA melting curve analysis

et al. 2019

Self Cite

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Quantitative PCR (qPCR) allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, analyses using validated Sybr Green I-based assays regularly amplify both the correct product and an artifact. Amplification of more than 1 product can be recognized when melting curve analysis is performed after the qPCR. Currently, such reactions need to be excluded from further analysis because the quantification result is considered meaningless. However, when the fraction of the fluorescence associated with the correct product can be determined, the quantitative result of the qPCR analysis can be corrected. The main assumptions of this correction model are: 1) the melting peak of the correct product can be identified, 2) the PCR efficiencies of all amplified products are similar, 3) the relative size of the melting peaks reflects the relative concentrations of the products, and 4) the relative concentrations do not change as the reaction reaches plateau. These assumptions were validated in a series of model experiments. The results show that the quantitative results can be corrected. Implementation of a correction for the presence of artifact amplification in the analysis of qPCR data leads to more reliable quantitative results in qPCR experiments.-Ruijter,

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Quantum Method for Fluorescence Background Removal in DNA Melting Analysis

Cited by 9 publications

References 21 publications

Nanomechanics of Diaminopurine-Substituted DNA

Nanomechanics of Diaminopurine-Substituted DNA

Competitive annealing of multiple DNA origami: formation of chimeric origami

Removal of artifact bias from qPCR results using DNA melting curve analysis

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