Melting curve analysis
Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA 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
The information also gives vital clues to a molecule's mode of interaction with DNA. Molecules such as
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,[1] but techniques based on fluorescence measurements[2] are now the most common approach.
The temperature-dependent dissociation between two DNA-strands can be measured using a
The graph of the negative
SYBR Green enabled product differentiation in the LightCycler in 1997.[4] 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
Many research and clinical examples[5] exist in the literature that show the use of melting curve analysis to obviate or complement sequencing efforts, and thus reduce costs.
While most
Digital High Resolution Melting (dHRM)
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.[9] 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.[10] 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.[11]
See also
- High Resolution Melt analysis
- Microscale thermophoresis, a method to determine the stability, the length, the conformation and the modifications of DNA and RNA[12]
- Nucleic acid thermodynamics
References
- PMID 1244898
- PMID 9056205
- ^ a b Hou, Shaw (2010). Biocatalysis and Biomolecular Engineering. John Wiley & Sons. pp. 314–317.
- ^ Ririe, 1997
- ^ Lay MJ, Wittwer CT. (1997) Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin Chem. 1997 Dec;43(12):2262-7
- ^ "Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples". academic.oup.com. Retrieved 2024-02-15.
- PMID 32295887.
- ^ "What is High Resolution Melting (HRM)? | Bio-Rad". www.bio-rad.com (in Korean). Retrieved 2024-02-15.
- ^ "An Introduction to High-Resolution Melting (also known as HRM, HRMA, melting curve analysis)". dna-utah.org. Retrieved 2024-02-15.
- ISSN 1473-0189.
- PMC 5296755.
- PMID 21297115.
External links
- Ririe, KM; Rasmussen, RP; Wittwer, CT (1997). "Product differentiation by analysis of DNA melting curves during the polymerase chain reaction". Anal Biochem. 245 (2): 154–160. PMID 9056205.
- Lo, Patcick C. H. (2014-10-21). "The Moments: Melting Curve Analysis". BioTechniques. Retrieved 2014-10-21.