Melting curve analysis is an assessment of the dissociation characteristics of double-stranded

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity, hyperchromicity. The temperature at which 50% of DNA is denatured is known as the melting temperature. Measurement of melting temperature can help us predict species by just studying the melting temperature. This is because every organism has a specific melting curve. The information gathered can be used to infer the presence and identity of

single-nucleotide polymorphisms In genetics and bioinformatics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in ...

(SNP). This is because G-C base pairing have 3

hydrogen bonds In chemistry, a hydrogen bond (H-bond) is a specific type of molecular interaction that exhibits partial covalent character and cannot be described as a purely electrostatic force. It occurs when a hydrogen (H) atom, covalently bonded to a mo ...

between them while A-T base pairs have only 2. DNA with mutations from either A or T to either C or G will create a higher melting temperature. The information also gives vital clues to a molecule's mode of interaction with DNA. Molecules such as

intercalator Intercalation is the reversible inclusion or insertion of a molecule (or ion) into layered materials with layered structures. Examples are found in graphite and transition metal dichalcogenides. : Examples Graphite One famous intercalation hos ...

s slot in between base pairs and interact through pi stacking. This has a stabilizing effect on DNA's structure which leads to a raise in its melting temperature. Likewise, increasing salt concentrations helps diffuse negative repulsions between the phosphates in the DNA's backbone. This also leads to a rise in the DNA's melting temperature. Conversely, pH can have a negative effect on DNA's stability which may lead to a lowering of its melting temperature.

Implementation

The energy required to break the base-base hydrogen bonding between two strands of DNA is dependent on their length, GC content and their complementarity. By heating a reaction-mixture that contains double-stranded DNA sequences and measuring dissociation against temperature, these attributes can be inferred. Originally, strand dissociation was observed using UV absorbance measurements, but techniques based on fluorescence measurements are now the most common approach. The temperature-dependent dissociation between two DNA-strands can be measured using a DNA-intercalating

fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...

such as SYBR green, EvaGreen or fluorophore-labelled DNA probes. In the case of SYBR green (which fluoresces 1000-fold more intensely while intercalated in the minor groove of two strands of DNA), the dissociation of the DNA during heating is measurable by the large reduction in fluorescence that results. Alternatively, juxtapositioned probes (one featuring a fluorophore and the other, a suitable quencher) can be used to determine the complementarity of the probe to the target sequence. The graph of the negative

first derivative First most commonly refers to: * First, the ordinal form of the number 1 First or 1st may also refer to: Acronyms * Faint Images of the Radio Sky at Twenty-Centimeters, an astronomical survey carried out by the Very Large Array * Far Infrared a ...

of the melting-curve may make it easier to pin-point the temperature of dissociation (defined as 50% dissociation), by virtue of the peaks thus formed. SYBR Green enabled

product differentiation In economics and marketing, product differentiation (or simply differentiation) is the process of distinguishing a product or service from others to make it more attractive to a particular target market. This involves differentiating it from c ...

in the LightCycler in 1997. Hybridization probes (or FRET probes) were also demonstrated to provide very specific melting curves from the single-stranded (ss) probe-to-amplicon hybrid. Idaho Technology and Roche have done much to popularize this use on the LightCycler instrument.

Applications

Since the late 1990s product analysis via SYBR Green, other double-strand specific dyes, or probe-based melting curve analysis has become nearly ubiquitous. The probe-based technique is sensitive enough to detect single-nucleotide polymorphisms (SNP) and can distinguish between

homozygous Zygosity (the noun, zygote, is from the Greek "yoked," from "yoke") () is the degree to which both copies of a chromosome or gene have the same genetic sequence. In other words, it is the degree of similarity of the alleles in an organism. Mos ...

wildtype The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...

heterozygous Zygosity (the noun, zygote, is from the Greek "yoked," from "yoke") () is the degree to which both copies of a chromosome or gene have the same genetic sequence. In other words, it is the degree of similarity of the alleles in an organism. Mos ...

and homozygous

mutant In biology, and especially in genetics, a mutant is an organism or a new genetic character arising or resulting from an instance of mutation, which is generally an alteration of the DNA sequence of the genome or chromosome of an organism. It i ...

alleles by virtue of the dissociation patterns produced. Without probes, amplicon melting (melting and analysis of the entire PCR product) was not generally successful at finding single base variants through melting profiles. With higher resolution instruments and advanced dyes, amplicon melting analysis of one base variants is now possible with several commercially available instruments. For example: Applied Biosystems 7500 Fast System and the 7900HT Fast Real-Time PCR System, Idaho Technology's LightScanner (the first plate-based high resolution melting device), Qiagen's Rotor-Gene instruments, and Roche's LightCycler 480 instruments. Many research and clinical examples exist in the literature that show the use of melting curve analysis to obviate or complement sequencing efforts, and thus reduce costs. While most

quantitative PCR A real-time polymerase chain reaction (real-time PCR, or qPCR when used quantitatively) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule duri ...

machines have the option of melting curve generation and analysis, the level of analysis and software support varies. High Resolution Melt (known as either Hi-Res Melting, or HRM) is the advancement of this general technology and has begun to offer higher sensitivity for SNP detection within an entire dye-stained amplicon. It is less expensive and simpler in design to develop probeless melting curve systems. However, for genotyping applications, where large volumes of samples must be processed, the cost of development may be less important than the total throughput and ease of interpretation, thus favoring probe-based genotyping methods. Digital High Resolution Melting (dHRM) is also used in conjunction with digital PCR (dPCR) to improve quantitative power by providing additional information on the melting behavior of the amplified DNA, which can help in distinguishing between different genetic variants and in ensuring the accuracy of the quantification. dHRM is enabled by the use of sensitive DNA-binding dyes and digital PCR instrumentation, which allows for the collection of high-density data points to generate detailed melt profiles. These profiles can be used to identify even subtle differences in nucleic acid sequences, making dHRM a powerful tool for genotyping, mutation scanning, and methylation analysis dHRM is an advanced molecular technique used for the analysis of genetic variations, such as single nucleotide polymorphisms (SNPs), mutations, and methylations, by monitoring the melting behavior of double-stranded DNA. It is a post-PCR method that involves the gradual heating of PCR-amplified DNA in the presence of intercalating dyes that fluoresce when bound to double-stranded DNA. As the DNA melts, the fluorescence decreases, and the changes in fluorescence are monitored in real-time with digital PCR system. The resulting melting curves are then analyzed to detect genetic differences based on the melting temperatures of the DNA fragments. The technique has been further advanced by its application on digital microfluidics platforms, which can facilitate the analysis of single-nucleotide polymorphisms (SNPs) with high accuracy and sensitivity. Additionally, massively parallel dHRM has been developed to enable rapid and absolutely quantitative sequence profiling, which can be particularly useful in clinical and industrial settings where accurate quantification of nucleic acids is critical.

References

External links

* * {{DEFAULTSORT:Melting Curve Analysis Biochemistry

Implementation

Applications

See also

References

External links