Classification of DNA sequences based on thermal melting profiles

A new classification scheme based on the melting profile of DNA sequences simulated thermal melting profiles. This method was applied in the classification of (a) several species of mammalian - β globin and (b) α-chain class II MHC genes. Comparison of the thermal melting profile with the molecular phylogenetic trees constructed using the sequences shows that the melting temperature based approach is able to reproduce most of the major features of the sequence based evolutionary tree. Melting profile method takes into account the inherent structure and dynamics of the DNA molecule, does not require sequence alignment prior to tree construction, and provides a means to verify the results experimentally. Therefore our results show that melting profile based classification of DNA sequences could be a useful tool for sequence analysis.     

High‐resolution calorimetric and optical melting profiles of DNA plasmids: Resolving contributions from intrinsic melting domains and specifically designed inserts

We demonstrate that differential scanning calorimetry (DSC) can be used to yield high‐resolution melting profiles for DNA plasmids that agree in all major features with the corresponding plasmid melting profiles derived using more traditional optical techniques. We further demonstrate that by combining information derived from both calorimetric and optical melting profiles one can glean insights that are unavailable from either melting curve alone. By using both optical and calorimetric observables, we show how one can resolve, identify, and measure the thermodynamic properties of particular sequences/domains of interest within a plasmid. We also show that complementary DSC and optical melting studies on plasmids with and without specifically designed inserts can provide fundamental advantages over the corresponding melting studies on other model system constructs for thermodynamically characterizing nucleic acid sequences/structures. © 1999 John Wiley & Sons, Inc. Biopoly 50: 303–318, 1999     

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves

Many factors that change the temperature position and interval of the DNA helix–coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, T_m, and temperature melting width, ΔT, of the entire DNA transition. Changes in T_m and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining T_m and ΔT. Indeed, some of the methods give unreasonable “jagged” T_m and ΔT dependences on varying relative concentration of DNA chemical modifications (r_b), [Na⁺], and GC content. At the same time, T_m determined as the helix–coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of T_m and ΔT on r_b, [Na⁺], and GC content. For multi-peak DMCs, T_m value determined in this way is the closest to the thermodynamic melting temperature (the helix–coil transition enthalpy/entropy ratio).     

高分辨率熔解曲线法的非标记探针基因分型技术

PCR反应中利用荧光检测技术对已知位点进行基因分型时常采用荧光标记的寡核苷酸做探针。近年来新兴起的高分辨率熔解曲线技术可以采用非标记的探针对已知位点的SNP(single nucleotide polymorphism)或突变进行基因分型研究。采用非标记探针法对已知位点的基因分型研究具有廉价、快速、简便等特点,因此被大量应用在和疾病、形状等相关的一些多肽位点的研究中。本文较详细地介绍该技术的基本原理和实验中的注意事项。     

Predicting sequence-dependent melting stability of short duplex DNA oligomers

Many important applications of DNA sequence-dependent hybridization reactions have recently emerged. This has sparked a renewed interest in analytical calculations of sequence-dependent melting stability of duplex DNA. In particular, for many applications it is often desirable to accurately predict the transition temperature, or t_m, of short duplex DNA oligomers (∼ 20 base pairs or less) from their sequence and concentration. The thermodynamic analytical method underlying these predictive calculations is based on the nearest-neighbor model. At least 11 sets of nearest-neighbor sequence-dependent thermodynamic parameters for DNA have been published. These sets are compared. Use of the nearest-neighbor sets in predicting t_m from the DNA sequence is demonstrated, and the ability of the nearest-neighbor parameters to provide accurate predictions of experimental t_m's of short duplex DNA oligomers is assessed. © 1998 John Wiley & Sons, Inc. Biopoly 44: 217–239, 1997     

一种用于优化PCR引物设计的短链DNA熔点分析方法

目前,PCR引物设计主要依赖于软件对引物熔点的模拟计算,而PCR退火条件的优化需进行不同条件下的扩增实验。为开发一种可高效、精确评价引物和确定退火条件的方法,本研究采用高分辨率熔解曲线（high resolution melting,HRM）测定技术直接分析短链DNA的熔点,用于引物优劣性的评价,并为退火条件的优化提供参考。本文用HRM法直接测定了非完全互补的双链DNA以及DNA发卡结构的熔点,结果显示：（1）与完全互补的双链DNA相比,较为稳定的单碱基错配A?G、G?G和T?G的熔点只降低2℃ ~ 3℃,部分双碱基错配的熔点只降低4℃ ~ 6℃,单碱基突出熔点只降低4℃~ 5℃。因此,如果采用的退火温度不当,部分错配的非目的模板可能会被扩增。（2）即使发卡结构的茎干区只有6 bp,当其环区碱基少于10 nt时,其熔点也可达到60℃以上。此外,环区的长度对发卡熔点也有较大影响。根据本研究结果发现,引物设计时应尽量避免模板引物结合区同其邻近的30 nt碱基有6 bp以上的互补部分。综上所述,本研究证明HRM熔点法是一种高效评价引物及确定退火温度的方法。     

Melting studies of short DNA hairpins: Influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were performed. These hairpins form by intramolecular folding of 16 base self‐complementary DNA oligomer sequences. Sequence design dictated that the hairpin structures have a six base pair duplex linked by a four base loop and that the first five base pairs in the stem are the same in every molecule. Only loop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried out to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained from their temperature‐induced melting transitions in buffered 115 mM Na⁺ solvent, monitored by ultraviolet absorbance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5°C and are concentration independent over strand concentrations of 0.5 to 260 μM; thus, as expected for hairpins, the melting transitions are apparently unimolecular. Model independent thermodynamic transition parameters, ΔH_cal, ΔS_cal, and ΔG_cal, were determined from DSC measurements. Model dependent transition parameters, ΔH_vH, ΔS_vH, and ΔG_vH were estimated from a van't Hoff (two‐state) analysis of optical melting transitions. Results of these studies reveal a significant sequence dependence to DNA hairpin stability. Thermodynamic parameters evaluated by either procedure reveal the transition enthalpy, ΔH_cal (ΔH_vH) can differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy ΔS_cal (ΔS_vH) can differ by as much as 60 cal/Kmol (eu) for different molecules. Differences in free energies ΔG_cal (ΔG_vH) are as large as 4 kcal/mol for hairpins with different sequences. Comparisons between the model independent calorimetric values and the thermodynamic parameters evaluated assuming a two‐state model reveal that 10 of the 28 hairpins display non‐two‐state melting behavior. The database of sequence‐dependent melting free energies obtained for the hairpins was employed to extract a set of n‐n (nearest‐neighbor) sequence dependent loop parameters that were able to reproduce the input data within error (with only two exceptions). Surprisingly, this suggests that the thermodynamic stability of the DNA hairpins can in large part be reasonably represented in terms of sums of appropriate nearest‐neighbor loop sequence parameters. © 1999 John Wiley & Sons, Inc. Biopoly 50: 425–442, 1999     

Application of high-resolution melting for genotyping bovine mitochondrial DNA

Recent studies have demonstrated that mitochondrial DNA (mtDNA) haplotype has a significant impact on the efficiency of bovine somatic cell nuclear transfer. Conventional methods for detecting mtDNA variations and haplotypes, such as restriction fragment length polymorphism (RFLP), temporal temperature gradient gel electrophoresis, dHPLC and sequencing, are labor intensive or expensive and have low sensitivity. High-resolution melting (HRM) analysis is a new technique for mutation detection and has the advantages of speed, cost, and accuracy. Here, we describe the genotyping of bovine mtDNA using HRM analysis. DNA samples containing mtDNA were extracted from 75 Holstein cows and subjected to rapid-cycle (<20 min) PCR of small amplicons (<120 bp) using specific primer sets. Capillaries containing the PCR products were then subjected to HRM analysis; data were acquired in 2 min and analyzed using the instrument's software. Five common bovine mtDNA single nucleotide polymorphisms were identified: 9602 G>A, 169 A>G, 166A>G with 173A>G, and 363C>G. These results agree with both sequencing and RFLP analysis. In addition, a very small amount of heteroplasmic variants (<5%) was sufficiently to be distinguished by HRM analysis that would be very useful to differentiate heteroplasmy vs. homoplasmy. HRM analysis thus provides a new approach to genotyping bovine mtDNA sequence variations and has many advantages over other methods, including speed of analysis, cost, and accuracy. We believe this will be a valuable technique for determining the efficiency of nuclear transfer in cloned embryos and for studying maternal effects on nuclear-cytoplasm interactions.     

Target concentration dependence of DNA melting temperature on oligonucleotide microarrays

The design of microarrays is currently based on studies focusing on DNA hybridization reaction in bulk solution. However, the presence of a surface to which the probe strand is attached can make the solution‐based approximations invalid, resulting in sub‐optimum hybridization conditions. To determine the effect of surfaces on DNA duplex formation, the authors studied the dependence of DNA melting temperature (T_m) on target concentration. An automated system was developed to capture the melting profiles of a 25‐mer perfect‐match probe–target pair initially hybridized at 23°C. Target concentrations ranged from 0.0165 to 15 nM with different probe amounts (0.03–0.82 pmol on a surface area of 10¹⁸ Ų), a constant probe density (5 × 10¹² molecules/cm²) and spacer length (15 dT). The authors found that T_m for duplexes anchored to a surface is lower than in‐solution, and this difference increases with increasing target concentration. In a representative set, a target concentration increase from 0.5 to 15 nM with 0.82 pmol of probe on the surface resulted in a T_m decrease of 6°C when compared with a 4°C increase in solution. At very low target concentrations, a multi‐melting process was observed in low temperature domains of the curves. This was attributed to the presence of truncated or mismatch probes. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Immobilized DNA hairpins for assay of sequential breaking and joining of DNA backbones

Immobilized DNA hairpins are exploited in a novel approach to assay DNA ligases and nucleases. A fundamental characteristic of the assay is that a fluorophore at the remote terminus of the hairpin reports on the integrity of the DNA backbone. The functionality of the protocol is confirmed using ATP- and NAD+-dependent DNA ligases and the nicking enzyme N.BbvCIA. The assay format is amenable to high-throughput analysis and quantitation of enzyme activity, and it is shown to be in excellent agreement with the more laborious electrophoretic approaches that are widely used for such analyses. Significantly, the assay is used to demonstrate sequential breaking and rejoining of a specific nucleic acid. Thus, a simple platform for biochemically innovative studies of pathways in cellular nucleic acid metabolism is demonstrated.     

The effect of hydrostatic pressure on the thermal stability of DNA hairpins

DNA hairpins consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that loop and stem sequences can significantly affect the thermodynamic stability of short DNA hairpins. In this work we present the effect of hydrostatic pressure on the helix-coil transition temperature (T_M) for 11 16-base, hairpin-forming DNA oligonucleotides. All of the samples form a hairpin with a 6-base pair stem and a four-base loop. In addition, the four base pairs at the end of the stem distal from the loop are the same for every molecule. We have varied loop sequence and identity of the two duplex base pairs adjacent to the loop. Using the change in UV absorption to monitor the conformational state of the oligonucleotide the hairpin-coil transition temperature of these molecules was studied as a function of sodium ion concentration and pressure. From these data we calculated the volume change accompanying the transition. Model-dependent (van't Hoff) transition parameters such as ΔH_vH and transition volume (ΔV) were estimated from the analysis of conformational transitions. Experiments revealed that the ΔV for denaturation of these molecules range from − 2.35 to + 6.74 cm³ mol⁻¹. The expansibility (ΔΔV/ΔT) and the pressure dependence of cation release are also presented. The difference in the volume change for this transition is related to the differences in the hydration of these molecules.     

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.     

A rapid real-time PCR/DNA resolution melting method to identify Prototheca species

Aim: The study describes the development of a simple and rapid tool to identify yeast‐like microalgae belonging to the genus Prototheca. Methods and Results: The method, based on two‐step Real Time PCR reaction followed by DNA Resolution Melting Analysis (qPCR/RMA), has been developed using reference strains belonging to both pathogenic (P. zopfii genotype 2, P. wickerhamii and P. blaschkeae) and non‐pathogenic species (P. zopfii genotype 1, P. stagnora and P. ulmea). In order to validate the method, seventy recently isolated Prototheca strains were thus tested in parallel with both the first qPCR/RMA and the conventional genotype‐specific PCR assay: they were classified as P. zopfii genotype 1, P. zopfii genotype 2 and P. blaschkeae, with a perfect accordance between the two above methodologies. Furthermore, we used the second qPCR/RMA to identify the other species (P. stagnora, P. ulmea and P. wickerhamii), which cannot be discriminated by conventional PCR assay. Conclusions: The assay two‐step Real Time PCR is accurate, robust, cost‐effective and faster than auxonographical, biochemical or conventional molecular biology methods. Significance and Impact of the Study: the rapid and high throughout two‐step qPCR/RMA tool can be usefully used for the identification of clinical and environmental Prototheca species into the framework of the diagnosis of animal (e.g. bovine mastitis) or human protothecosis.     

Influence of formamide on the thermal stability of DNA

The utility of formamide in the denaturation and renaturation of DNA has been examined. The melting temperature of duplex DNA is lowered by 0·6°C per per cent formamide. The depression of melting temperature is independent of the GC content. Formamide also increases the width of the thermal transition. Upto 30%, it does not affect the rate of DNA reassociation     

Spectral analysis for base composition of DNA undergoing melting

A microcomputer-controlled spectrophotometer is described for obtaining the base composition of melting domains in DNA from derivative melting curves. Values have been determined for the differential molar extinction coefficients for the A-T and G-C base pair at the three wavelengths most useful for spectral analysis of base composition, 260, 270 and 282 nm. The average RMS error for these values was 29 l(mol X cm)-1 for the melting of 14 DNA specimens ranging in base composition from 0-0.72 F(G + C). A precision of approximately 1% in base composition of domains is possible. Such analysis is useful for confirming or establishing assignments of domains to particular subtransitional features in high resolution melting curves.     

Effect of low-energy microwaves on DNA stability in solution

DNA isolated from liver of healthy and tumor-bearing (sarcoma 45) rats was irradiated in water-salt solution with weak microwaves (64.5 GHz, 50 μW/cm²). The heat stability of DNA increased with irradiation time (a raise of 1.5°C in T _m for “tumor” DNA after 90 min, without changes in ΔT), which may be associated with dehydration of the surrounding Na⁺ ions.     

Sensitive quantitative analysis of murine LINE1 DNA methylation using high resolution melt analysis

We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2’-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay design was also used to detect changes in methylation at other murine repeat elements (B1 and Intracisternal-A-particle Long-terminal Repeat elements). Pyrosequencing analysis revealed that L1 methylation changes were non-uniform across the CpGs within the L1-HRM target region, demonstrating that the L1 assay can detect small changes in CpG methylation among a large pool of heterogeneously methylated DNA templates. Application of the assay to various tissues from Balb/c and CBA mice, including previously unreported peripheral blood (PB), revealed a tissue hierarchy (from hypermethylated to hypomethylated) of PB > kidney > liver > prostate > spleen. CBA mice demonstrated overall greater methylation than Balb/c mice, and male mice demonstrated higher tissue methylation compared with female mice in both strains. Changes in DNA methylation have been reported to be an early and fundamental event in the pathogenesis of many human diseases, including cancer. Mouse studies designed to identify modulators of DNA methylation, the critical doses, relevant time points and the tissues affected are limited by the low throughput nature and exorbitant cost of many DNA methylation assays. The L1 assay provides a high throughput, inexpensive and sensitive screening tool for identifying and characterizing DNA methylation changes to L1 elements at multiple regions across the genome.     

The effect of ionic strength on melting of DNA modified by platinum(II) complexes

Thermal denaturation of calf thymus DNA modified by antitumor cis-diamminedichloroplatinum(II) (cis-DDP) and by two related Pt(II) compounds which had been shown to be clinically inefective, viz. trans-diamminedichloroplatinum(II) (trans-DDP) or monodentate diethylenetriaminechloroplatinum(II) chloride {[Pt(dien)Cl)]Cl}, was studied by monitoring changes of absorbance at 260 nm. The melting of DNA platinated to different levels was investigated in neutral media containing varying concentrations of Na⁺. It has been shown that the ionic strength has a strong influence on the character and magnitude of changes in the melting temperature of DNA (T_m) induced by the platination. The modification of DNA by either platinum complex used in this work results in an increase of T_m if DNA melting is measured in media containing low Na⁺ concentrations (ca. 1 mM). This effect is reversed at higher Na⁺ concentrations. The concentration of Na⁺ at which this reversal occurs is, however, markedly lower for DNA modified by cis-DDP than for DNA modified by the other two platinum complexes. These results have been iterpreted to mean that at least three factors affect the thermal stability of DNA modified by the platinum(II) complexes: stabilization effects of the positive charge on the platinum moiety and of interstrand cross-links, and a destabilization effect of conformational distortions in DNA. Thus, in order to compare and interpret the melting behavior of DNA modified by different compounds, a great attention has to be paid to the composition of the medium in which the melting experiments are carried out.     

On the thermal stability of DNA in solution of mixed solvents

The study of the changes in UV absorbance of DNA solutions in water/dioxane and water/ethylene glycol mixture at different concentrations shows that the thermal denaturation of DNA is sensitive to the electrical permittivity of the media and the water content. At relative low concentrations of co-solvent the dominant feature is the electrical permittivity. When water content is lower than a critical value, the electrical permittivity is no longer the determinant of the denaturation temperature but the partial volume fraction of water. The critical water content is about 0.69 partial volume fraction of water.