Preparation and melting of single strand circular DNA loops.

A method for preparation of single strand DNA circles of almost arbitrary sequence is described. By ligating two sticky ended hairpins together a linear duplex is formed, closed at both ends by single stranded loops. The melting characteristics of such loops are investigated using optical absorbance and NMR. It is shown by comparison with the corresponding linear sequence (closed circle minus the end loops) that the effects of end fraying and the strand concentration dependence of the melting temperature are eliminated in the circular form. Over the concentration range examined (0.5 to 2.0 micromolar strands), the circular DNA has a monophasic melting curve, while the linear duplex is biphasic, probably due to hairpin formation. Since effects of duplex to single strands dissociation do not contribute to melting of the circular molecules (dumbells), these DNAs present a realistic experimental model for examining local thermal stability in DNA.     

Influence of base-pair changes and cooperativity parameters on the melting curves of short DNAs

Theoretical melting curves were calculated for four DNA restriction fragments, 157–257 base pairs (bp), and a series of hypothetical block DNAs with sequences d(C_2xA_xC_2x). d(C_2xT_xG_2x), 5 ? x ? 40. These DNAs provided a mixture of A·T/G·C sequence distributions with which to investigate the effects of parameters and base-pair changes on the melting of short DNAs. The sensitivity of DNA melting curves to changes in internal loop melting parameters σ and κ was examined. As Expected, theoretical melting curves of short DNAs with a quasirandom base-pair sequence vary little with changes in internal loop parameters. End melting dominates the transition behaviour of these moleucles. This was also observed for the block DNAs up to x = 22. Beyond this length, melting curves are highly sensitive to the internal loop parameters. Sensitivity is also predicted for a 157-bp fragment with a block distribution of A·T and G·C pairs. These results indicate that accurate evaluation of internal loop parameters is possible with short DNAs (100–200 bp) containing a G·C/A·T/G·C block distribution with at least 22 bp in each block. Duplex-to-single-strands dissociation parameters were reevaluated form experimental melting curve data of eight DNA fragments using a least squares fit approach. This analysis confirmed parameter values previously found with a simplified dissociation model. A Priori predictions are made on the effects of base-pair changes on the melting curves of three characterized DNA restriction fragments. Single base-pair changes are predicted to induce small but measurable changes in the melting curves. The characteristics of the altered melting curves depend on the location of the base-pair change.     

Triple helix DNA oligomer melting measured by fluorescence polarization anisotropy

A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 mer single strand sequence onto the complementary purinic sequence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10-90 degrees C. The T(m) of the triplex, 37 degrees C, was well separated from the onset of duplex melting. When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the T(m) of the triplex was shifted to approximately 32 degrees C and overlapped the melting of the duplex. We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and triplex samples to determine the hydrodynamic parameters in the temperature range 10-40 degrees C. The fluorescence lifetime of EB in the samples of double and triple stranded DNA is the same (21.3 +/- 0.5 ns) at 20 degrees C, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal triplex, and nicked triplex samples were 10.7 +/- 0.2, 12.2 +/- 0.2, and 12.0 +/- 0.2 A. FPA measurements on normal triplex DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the triplex carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal triplex sample. The torsional rigidity of the triplex forms was found to be higher than that of the duplex form.     

The four-state model of a linear lattice: application to ligand-controlled hybridization of short duplex DNAs

An algorithm for calculating the order-disorder transition of a four-state linear lattice is presented. Recursion schemes for the probabilities of each site along the lattice occupying one of the four possible states were derived following the work of D. Poland (Biopolymers, 1974, Vol. 13, pp. 1859-1871) and H. Tachibana and A. Wada (Biopolymers, 1982, Vol. 21, pp. 1873-1885). The algorithm was parameterized to consider melting of short duplex DNA in the presence of duplex and single-strand binding ligands. Model calculations were performed for two 31 base-pair duplex DNAs with very different percent guanine-cytosine base pairs, and thus very different thermodynamic stabilities. In the absence of ligands, calculated melting curves of the two DNAs under identical solvent conditions and identical concentrations were separated by over 15 degrees C. In the presence of either duplex or single-strand binding ligands, if a sequence dependence to ligand binding is assumed, the melting curves of the two DNAs can be made to coalesce, i.e., stability of the two DNAs can be normalized! This example demonstrates the feasibility of controlling hybridization of short DNAs by sequence specific ligand binding.     

Development of an automated SNP analysis method using a paramagnetic beads handling robot

Biological and medical importance of the single nucleotide polymorphism (SNP) has led to development of a wide variety of methods for SNP typing. Aiming for establishing highly reliable and fully automated SNP typing, we have developed the adapter ligation method in combination with the paramagnetic beads handling technology, Magtration(R). The method utilizes sequence specific ligation between the fluorescently labeled adapter and the sample DNAs at the cohesive end produced by a type IIS restriction enzyme. Evaluation of the method using human genomic DNA showed clear discrimination of the three genotypes without ambiguity using the same reaction condition for any SNPs examined. The operations following PCR amplification were automatically performed by the Magtration(R)-based robot that we have previously developed. Multiplex typing of two SNPs in a single reaction by using four fluorescent dyes was successfully preformed at the almost same sensitivity and reliability as the single typing. These results demonstrate that the automated paramagnetic beads handling technology, Magtration(R), is highly adaptable to the automated SNP analysis and that our method best fits to an automated in-house SNP typing for laboratory and medical uses.     

Sequence-dependent effects on DNA stability resulting from guanosine replacements by inosine.

The effect of replacing a G.C base-pair with an I.C base-pair on DNA stability was investigated for a related set of 14-mers. DNA melting analysis of the 14-mers was used to determine delta Hzero, delta Szero and delta G(zero)37 of the double to single stranded transition. All 14mers were shown to have B-DNA character by circular dichroism analysis. 14mers substituted with a single inosine in place of guanosine at different positions showed that consequences on DNA stability are sequence-dependent. Large changes in delta Hzero and delta Szero result when inosine is substituted within the trinucleotide sequence d(TCG).d(CGA) while substitution within d(TCC).d(GGA) causes minor changes in enthalpy and entropy. Moreover, some 14-mers with two inosine substitutions five base-pairs apart showed non-additive free energy changes for the double to single stranded transition. These results clearly indicate that the structural consequences of replacing a single guanosine with an inosine are transmitted over a significant distance.     

A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants

High-resolution melting (HRM) analysis relies on the use of fluorescent dyes, such as LCGreen,ResoLight, and SYTO9, which bind in a saturated manner to the double-stranded DNAs. These dyes are expensive in use and may not be affordable when dealing with a large quantity of samples. EvaGreen is a much cheaper DNA helix intercalating dye and has been used in quantitative real-time polymerase chain reaction (PCR) and post-PCR DNA melt curve analysis. Here we report on the development of an EvaGreen-based HRM analysis and its performance, in comparison with the popular LCGreen-based HRM analysis, in detection of DNA polymorphism in plants. We found that various polymorphisms ranged from single nucleotide polymorphisms (SNPs) to Indels were equally detected by using EvaGreen- or LCGreen-based HRM. EvaGreen dye was sensitive enough in discovery of SNPs in fivefold pooled samples.Using this economical dye we successfully identified multiple novel mutant alleles of Gln1-3 gene,which produces a cytosolic glutamine synthetase isoenzyme (GS1), in a maize ethyl methanesulfonate (EMS)-mutagenized library, and genotyped rice mapping populations with SNP markers. The current results suggest that EvaGreen is a promising dye for HRM analysis for its ease to use and cost effectiveness.     

Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures

Melting temperatures, T(m), were systematically studied for a set of 92 DNA duplex oligomers in a variety of sodium ion concentrations ranging from 69 mM to 1.02 M. The relationship between T(m) and ln [Na(+)] was nonlinear over this range of sodium ion concentrations, and the observed melting temperatures were poorly predicted by existing algorithms. A new empirical relationship was derived from UV melting data that employs a quadratic function, which better models the melting temperatures of DNA duplex oligomers as sodium ion concentration is varied. Statistical analysis shows that this improved salt correction is significantly more accurate than previously suggested algorithms and predicts salt-corrected melting temperatures with an average error of only 1.6 degrees C when tested against an independent validation set of T(m) measurements obtained from the literature. Differential scanning calorimetry studies demonstrate that this T(m) salt correction is insensitive to DNA concentration. The T(m) salt correction function was found to be sequence-dependent and varied with the fraction of G.C base pairs, in agreement with previous studies of genomic and polymeric DNAs. The salt correction function is independent of oligomer length, suggesting that end-fraying and other end effects have little influence on the amount of sodium counterions released during duplex melting. The results are discussed in the context of counterion condensation theory.     

SNP genotyping through the melting analysis of unlabelled oligonucleotide applied on dilute PCR amplicon

Adaptation of DNA melting analysis for polymorphic single nucleotides (SNPs) genotyping using an unlabeled oligonucleotide probe for polymorphic DNAs under the presence of fluorescent DNA binding dye necessitates a reaction condition where the probe efficiently associates with a target strand that is PCR amplified. We present experimental evidence that application of an unlabeled probe to a dilute PCR amplicon provides a condition such that the fluorescent signals gained subsequently by probe melting are sufficient to discriminate allelic identities. This approach is best exploited by adapting the multiplexing PCR technique in order to cover multiple SNPs for given samples. 3′-end modification of the probe is unnecessary as the amplicon dilution step provides a way of inactivating the polymerase through divalent cation chelation. With the use of low-cost reagents and ordinary laboratory equipment, this method offers a rapid, simple and cost-efficient way of SNP genotyping.     

Inverted repeat sequences can influence the melting transitions of linear DNAs

The influence of inverted repeat sequences on the melting transitions of linear DNAs has been examined. Derivative melting curves (DMC) of a 514 base pair (bp) DNA, seven subfragments of this DNA, and four other DNAs have been compared to predictions of DNA melting theory. The 514-bp DNA contains three inverted repeat sequences that can form cruciform structures in supercoiled DNA. We refer to these sequences as c-inverted repeats. Previous work showed that the DMC of this DNA, unlike a number of other DNAs, is not accurately predicted by DNA melting theory. Since the theoretical model does not include hairpin-like structures, it was suggested that hairpin or cruciform formation in these inverted repeats may be responsible for this discrepancy. Our results support this hypothesis. Predicted DMCs are in good agreement with DNAs with no inverted repeats, or inverted repeats not evident in supercoiled DNA. Differences between the theoretical and experimental Tm's are less than or equal to 0.3 degrees C. DNA molecules that contain one or more of the three c-inverted repeats are not as accurately predicted. Experimental Tm values are lower than predicted values by 0.7-3.8 degrees C. It is concluded that some inverted repeat sequences can form hairpin-like structures during the melting of linear DNAs. These structures appear to lower overall DNA stability.     

Evaluation of DHPLC in the analysis of hemophilia A

The manifestation of hemophilia A, a common hereditary bleeding disorder in humans, is caused by abnormalities in the factor VIII (FVIII) gene. A wide range of different mutations has been identified and provides the genetic basis for the extensive variability observed in the clinical phenotype. The knowledge of a specific mutation is of great interest as this may facilitate genetic counseling and prediction of the risk of anti-FVIII antibody development, the most serious complication in hemophilia A treatment to date. Due to its considerable size (7.2 kb of the coding sequence, represented by 26 exons), mutation detection in this gene represents a challenge that is only partially met by conventional screening methods such as denaturing gradient gel electrophoresis (DGGE) or single stranded conformational polymorphism (SSCP). These techniques are time consuming, require specific expertise and are limited to detection rates of 70-85%. In contrast, the recently introduced denaturing high performance liquid chromatography (dHPLC) offers a promising new method for a fast and sensitive analysis of PCR-amplified DNA fragments. To test the applicability of dHPLC in the molecular diagnosis of hemophilia A, we first assessed a cohort of 156 patients with previously identified mutations in the FVIII gene. Applying empirically determined exon-specific melting profiles, a total of 150 mutations (96.2%) were readily detected. Five mutations (3.2%) could be identified after temperatures were optimized for the specific nucleotide change. One mutation (0.6%) failed to produce a detectable heteroduplex signal. In a second series, we analyzed 27 hemophiliacs in whom the mutation was not identified after extensive DGGE and chemical mismatch cleavage (CMC) analysis. In 19 of these patients (70.4%), dHPLC facilitated the detection of the disease-associated nucleotide alterations. From these findings we conclude that the dHPLC technology is a highly sensitive method well suited to the molecular analysis of hemophilia A.     

Formation of joint DNA molecules by two eukaryotic strand exchange proteins does not require melting of a DNA duplex

We have examined whether DNA strand exchange activities from nuclear extracts of HeLa cells or Drosophila melanogaster embryos have detectable helicase or melting activities. The partially purified recombinases have been shown to recognize homologous single strand and double strand DNA molecules and form joint molecules in a DNA strand exchange reaction. The joint molecule product consists of a linear duplex joined at one end by a region of DNA heteroduplex to a homologous single strand circular DNA. Using two different partially duplex helicase substrates, we are unable to detect any melting of duplex regions under conditions that promote joint molecule formation. One substrate consists of a 32P-labeled oligonucleotide 20 or 30 bases long annealed to M13mp18 circular single strand DNA. The second substrate consists of a linear single strand region flanked at each end by short duplex regions. We observe that even in the presence of excess recombinase protein or after prolonged incubation no helicase activity is apparent. Control experiments rule out the possibility that a helicase is masked by reannealing of displaced single strand fragments. Based on these findings and other data, we conclude that the human and D. melanogaster recombinases recognize and pair homologous sequences without significant melting of duplex DNA prior to strand exchange.     

Effect of sulphur crosslinking on the stability and transition of triple helical DNA

In continuation to our work on order-order and order-disorder transition in triple stranded DNA when it is bounded to netropsin, we report in this communication the stabilizing/destabilizing effect of disulphide linkage on the phase dynamics of the triplex using the amended Zimm-Bragg theory. It is observed that in contrast to the sequential triplex-->duplex -->single strand melting of the uncrosslinked triplex, crosslinking causes the triplex state to melt directly to the single stranded state, with no apparent intermediary of a duplex state. Since there is no overall difference in the enthalpy of crosslinked and uncrosslinked triplexes, the transition is entropy driven.     

A cooperative self-consistent microscopic theory of thermally induced melting of a repeat sequence DNA polymer

We attempt to extend the modified self-consistent phonon theory to describe thermal fluctuational base-pair opening of repeat sequence DNA polymers in the helix–coil transition region as well as in the premelting region. A microscopic base-pair open state is introduced and the effect of this open state is taken into account self-consistently in a mean field system that models the DNA polymer. Our analysis indicates the structure of this open state changes with temperature in such a manner that on average a base pair opens and unstacks with its neighbors more completely as temperature increases. We apply this theory to a homopolymer—poly(dG) · poly(dC) to evaluate the base-pair opening probability in a temperature range from 273 to 366.5 K. At 366.5 K the system undergoes cooperative melting. Our calculated base-pair opening probabilities are in general agreement with several experimental estimates at room temperature. The calculated probabilities show typical melting curve behavior at temperatures close to the observed melting temperature. The cooperative modified self-consistent phonon approximation approach becomes a viable microscopic theory of melting. © 1993 John Wiley & Sons, Inc.     

Rapid genotyping of APOA5 -1131T>C polymorphism using high resolution melting analysis with unlabeled probes

The APOA5 -1131 T/C polymorphism (rs662799) exhibits a very strong association with elevated TG levels in different racial groups. High resolution melting (HRM) analysis with the use of unlabeled probes has shown to be a convenient and reliable tool to genotyping, but not yet been used for detecting rs662799 polymorphism. We applied the unlabeled probe HRM analysis and direct DNA sequencing to assay the -1131T>C SNP in 130 cases DNA samples blindly. This HRM analysis can be completed in <3 min for each sample. The two melting peaks were displayed at 66.1±0.4°C for CC homozygote and 68.7±0.2°C for TT homozygote; TC heterozygote showed the both melting peaks. The genotyping results by HRM method were completely concordant with direct DNA sequencing. The distribution of CC, TC, and TT genotypes for the -1131T>C SNP was 9.2, 49.2, and 41.5%, respectively. This assay was sensitive enough to detect C allele down to 20% and 10% for T allele. The limit of detection for C and T allele was 6.2 and 2.5 ng/μL DNA, respectively. The developed unlabeled probe HRM method provides an alternative mean to detect ApoA5 -1131T>C SNP rapidly and accurately.     

Applications of the method of high resolution melting analysis for diagnosis of Leber's disease and the three primary mutation spectrum of LHON in the Han Chinese population

Current screening methods, such as single strand conformational polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC) and direct DNA sequencing that are used for detecting mutation in Leber's hereditary optic neuropathy (LHON) subjects are time consuming and costly. Here we tested high-resolution melt (HRM) analysis for mtDNA primary mutations in LHON patients. In this study, we applied the high resolution melting (HRM) technology to screen mtDNA primary mutations in 50 LHON patients from their peripheral blood. In order to evaluate the reliability of this technique, we compared the results obtained by HRM and direct mtDNA sequencing. We also investigated the spectrum of three most common mtDNA mutations implicated in LHON in the Han Chinese population. The results showed HRM analysis differentiated all of the mtDNA primary mutations and identified 4 additional mtDNA mutations from 50 patients in the blind study. The prevalence of three primary mutations were 11778G>A (87.9%), 14484T>C (6.5%) and 3460G>A (1.7%) in the Han Chinese population. In conclusion, HRM analysis is a rapid, reliable, and low-cost tool for detecting mtDNA primary mutations and has practical applications in molecular genetics.     

High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.)

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs.     

Entropy and heat capacity of DNA melting from temperature dependence of single molecule stretching

When a single molecule of double-stranded DNA is stretched beyond its B-form contour length, the measured force shows a highly cooperative overstretching transition. We have measured the force at which this transition occurs as a function of temperature. To do this, single molecules of DNA were captured between two polystyrene beads in an optical tweezers apparatus. As the temperature of the solution surrounding a captured molecule was increased from 11 degrees C to 52 degrees C in 500 mM NaCl, the overstretching transition force decreased from 69 pN to 50 pN. This reduction is attributed to a decrease in the stability of the DNA double helix with increasing temperature. These results quantitatively agree with a model that asserts that DNA melting occurs during the overstretching transition. With this model, the data may be analyzed to obtain the change in the melting entropy DeltaS of DNA with temperature. The observed nonlinear temperature dependence of DeltaS is a result of the positive change in heat capacity of DNA upon melting, which we determine from our stretching measurements to be DeltaC(p) = 60 +/- 10 cal/mol K bp, in agreement with calorimetric measurements.     

Single-molecule atomic force spectroscopy reveals that DnaD forms scaffolds and enhances duplex melting

The Bacillus subtilis DnaD is an essential DNA-binding protein implicated in replication and DNA remodeling. Using single-molecule atomic force spectroscopy, we have studied the interaction of DnaD and its domains with DNA. Our data reveal that binding of DnaD to immobilized single molecules of duplex DNA causes a marked reduction in the ‘end-to-end’ distance of the DNA in a concentration-dependent manner, consistent with previously reported DnaD-induced looping by scaffold formation. Native DnaD enhances partial melting of the DNA strands. The C-terminal domain (Cd) of DnaD binds to DNA and enhances partial duplex melting but does not cause DNA looping. The Cd-mediated melting is not as efficient as that caused by native DnaD. The N-terminal domain (Nd) does not affect significantly the DNA. A mixture of Nd and Cd fails to recreate the DNA looping effect of native DnaD but produces exactly the same effects as Cd on its own, consistent with the previously reported failure of the separated domains to form DNA-interacting scaffolds.