High Resolution Melt analysis is a powerful technique in molecular biology

Molecular biology

Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 for the detection of mutation

Mutation

In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...

s, polymorphisms

Polymorphism (biology)

Polymorphism in biology occurs when two or more clearly different phenotypes exist in the same population of a species — in other words, the occurrence of more than one form or morph...

 and epigenetic differences in double-stranded DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 samples. It was discovered and developed by Idaho Technology and the University of Utah. It has advantages over other genotyping

Genotyping

Genotyping is the process of determining differences in the genetic make-up of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their...

 technologies, namely:

• It is cost effective vs. other genotyping
  Genotyping
  Genotyping is the process of determining differences in the genetic make-up of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their...
  
   technologies such as sequencing
  DNA sequencing
  DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
  
   and Taqman SNP typing. This makes it ideal for large scale genotyping projects.
• It is fast and powerful thus able to accurately genotype many samples rapidly.
• It is simple. With a good quality HRM assay, powerful genotyping can be performed by non-geneticists in any laboratory with access to an HRM capable real-time PCR machine.

Method

HRM analysis is performed on double stranded DNA samples. Typically the user will use polymerase chain reaction

Polymerase chain reaction

The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....

 (PCR) prior to HRM analysis to amplify the DNA region in which their mutation of interest lies. Essentially the PCR process turns a tiny amount of your region of DNA of interest in to a large amount, so you have quantities large enough for better analysis. In the tube there are now many of copies of your region of DNA of interest. This region that is amplified is known as the amplicon.
After the PCR process the HRM analysis begins. The process is simply a precise warming of the amplicon DNA from around 50˚C up to around 95˚C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or “melt” apart.
The secret of HRM is to monitor this process happening in real-time. This is achieved by using a fluorescent dye. The dyes that are used for HRM are known as intercalating dyes and have a unique property. They bind specifically to double-stranded DNA and when they are bound they fluoresce brightly. In the absence of double stranded DNA they have nothing to bind to and they only fluoresce at a low level.
At the beginning of the HRM analysis there is a high level of fluorescence in the sample because of the billions of copies of the amplicon. But as the sample is heated up and the two strands of the DNA melt apart, presence of double stranded DNA decreases and thus fluorescence is reduced. The HRM machine has a camera that watches this process by measuring the fluorescence. The machine then simply plots this data as a graph known as a melt curve, showing the level of fluorescence vs the temperature:

Spot the difference

The melting temperature of the amplicon at which the two DNA strands come apart is entirely predictable. It is dependent on the sequence of the DNA bases. If you are comparing two samples from two different people, they should give exactly the same shaped melt curve. However if one of the people has a mutation in the DNA region you have amplified, then this will alter the temperature at which the DNA strands melt apart. So now the two melt curves appear different. The difference may only be tiny, perhaps a fraction of a degree, but because the HRM machine has the ability to monitor this process in “high resolution”, it is possible to accurately document these changes and therefore identify if a mutation is present or not.

Wild type, heterozygote or homozygote?

Things become slightly more complicated than this because organisms contain two (or more

Polyploidy

Polyploid is a term used to describe cells and organisms containing more than two paired sets of chromosomes. Most eukaryotic species are diploid, meaning they have two sets of chromosomes — one set inherited from each parent. However polyploidy is found in some organisms and is especially common...

) copies of each gene, known as the two allele

Allele

An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...

s. So, if a sample is taken from a patient and amplified using PCR both copies of the region of DNA (alleles) of interest are amplified. So if we are looking for mutation there are now three possibilities:

1. Neither allele contains a mutation
2. One or other allele contains a mutation
3. Both alleles contain a mutation.

These three scenarios are known as “Wild –type”, “Heterozygote” or “Homozygote” respectively. Each gives a melt curve that is slightly different. With a high quality HRM assay it is possible to distinguish between all three of these scenarios.

SNP typing/Point mutation detection

Conventional SNP typing methods are typically time consuming and expensive, requiring several probe based assays to be multiplexed together or the use of DNA microarrays. HRM is more cost effective and reduces the need to design multiple pairs of primers and the need to purchase expensive probes. The HRM method has been successfully used to detect a single G to A substitution in the gene Vssc (Voltage Sensitive Sodium Channel) which confers resistance to the acaricide permethrin

Permethrin

Permethrin is a common synthetic chemical, widely used as an insecticide, acaricide, and insect repellent. It belongs to the family of synthetic chemicals called pyrethroids and functions as a neurotoxin, affecting neuron membranes by prolonging sodium channel activation. It is not known to...

 in Scabies mite. This mutation results in a coding change in the protein (G1535D). The analysis of scabies mites collected from suspected permethrin susceptible and tolerant populations by HRM showed distinct melting profiles. The amplicons

Amplicons

thumb|75px|PCR ThermocyclerAn amplicon is a piece of DNA formed as the product of natural or artificial amplification events. For example, it can be formed via polymerase chain reactions or ligase chain reactions , as well as by natural gene duplication.Artificial amplification can be used to...

 from the sensitive mites were observed to have a higher melting temperature relative to the tolerant mites, as expected from the higher thermostability of the GC base pair

Base pair

In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...

 

In a field more relevant to clinical diagnostics, HRM has been shown to be suitable in principle for the detection of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. More than 400 mutations have been identified in these genes.
The sequencing of genes is the gold standard for identifying mutations. Sequencing is time consuming and labour intensive and is often preceded by techniques used to identify heteroduplex DNA, which then further amplify these issues. HRM offers a faster and more convenient closed-tube method of assessing the presence of mutations and gives a result which can be further investigated if it is of interest. In a study carried out by Scott et al. in 2006, 3 cell lines harbouring different BRCA mutations were used to assess the HRM methodology. It was found that the melting profiles of the resulting PCR products could be used to distinguish the presence or absence of a mutation in the amplicon. Similarly in 2007 Krypuy et al. showed that the careful design of HRM assays (with regards to primer placement) could be successfully employed to detect mutations in the TP53 gene, which encodes the tumour suppressor protein p53 in clinical samples of breast and ovarian cancer. Both these studies highlighted that fact that changes in the melting profile can be in the form of a shift in the melting temperature or an obvious difference in the shape of the melt curve. Both of these parameters are a function of the amplicon sequence.
The overall consensus is that HRM is a cost efficient method that can be employed as an initial screen for samples suspected of harbouring polymorphisms or mutations. This would reduce the number of samples which need to be investigated further using more conventional methods.

Zygosity testing

Currently there are many methods used to determine the zygosity

Zygosity

Zygosity refers to the similarity of alleles for a trait in an organism. If both alleles are the same, the organism is homozygous for the trait. If both alleles are different, the organism is heterozygous for that trait...

 status of a gene at a particular locus. These methods include the use of PCR with specifically designed probes to detect the variants of the genes (SNP typing is the simplest case). In cases where longer stretches of variation is implicated post PCR analysis of the amplicons may be required. Changes in enzyme restriction, electrophoretic and chromatographic profiles can be measured. These methods are usually more time consuming and increase the risk of amplicon contamination in the laboratory, due to the need to work with high concentrations of amplicons in the lab post-PCR. The use of HRM reduces the time required for analysis and the risk of contamination. HRM is a more cost effective solution and the high resolution element not only allows the determination of homo and heterozygosity, it also resolves information about the type of homo and heterozygosity, with different gene variants giving rise to differing melt curve shapes. A study by Gundry et al. 2003, showed that fluorescent labelling

Fluorescent labelling

Fluorescent labelling is the process of covalently attaching a fluorophore to another molecule, such as a protein or nucleic acid. This is generally accomplished using a reactive derivative of the fluorophore that selectively binds to a functional group contained in the target molecule...

 of one primer (in the pair) has been shown to be favourable over using an intercalating dye such as SYBR green I

SYBR Green

SYBR Green I is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light and emits green light . The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower...

. However, progress has been made in the development and use of improved intercalating dyes which reduce the issue of PCR inhibition and concerns over non-saturating intercalation of the dye.

Epigenetics

The HRM methodology has also been exploited to provide a reliable analysis of the methylation

DNA methylation

DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...

 status of DNA. This is of significance since changes to the methylation status of tumour suppressor genes, genes that regulate apoptosis

Apoptosis

Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

 and DNA repair, are characteristics of cancers and also have implications for responses to chemotherapy. For example, cancer patients can be more sensitive to treatment with DNA alkylating agents

Alkylating antineoplastic agent

An alkylating antineoplastic agent is an alkylating agent used in cancer treatment that attaches an alkyl group to DNA.The alkyl group is attached to the guanine base of DNA, at the number 7 nitrogen atom of the purine ring....

 if the promoter of the DNA repair gene MGMT

O-6-methylguanine-DNA methyltransferase

Methylated-DNA-protein-cysteine methyltransferase is an enzyme that in humans is encoded by the MGMT gene.- Function :O-alkyl-guanine is the major carcinogenic lesion in DNA induced by alkylating mutagens. This DNA adduct is removed by the repair protein, O-methylguanine-DNA methyltransferase...

 of the patient is methylated. In a study which tested the methylation status of the MGMT promoter on 19 colorectal samples, 8 samples were found to be methylated.

Methylated DNA can be treated by bi-sulphite modification, which converts non-methylated cytosines to uracil. Therefore, PCR products resulting from a template that was originally unmethylated will have a lower melting point than those derived from a methylated template. HRM also offers the possibility of determining the proportion of methylation in a given sample, by comparing it to a standard curve which is generated by mixing different ratios of methylated and non-methylated DNA together. This can offer information regarding the degree of methylation that a tumour may have and thus give an indication of the character of the tumour and how far it deviates from what is “normal”.

HRM also is practically advantageous for use in diagnostics, due to its capacity to be adapted to high throughput screening testing, and again it minimises the possibility of amplicon spread and contamination within a laboratory, owing to its closed-tube format.

Intercalating dyes

To follow the transition of dsDNA (double-stranded) to ssDNA (single-stranded), intercalating dyes are employed. These dyes show differential fluorescence emission dependent on their association with double-stranded or single-stranded DNA. SYBR Green I

SYBR Green

SYBR Green I is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light and emits green light . The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower...

is a first generation dye for HRM. It fluoresces when intercalated into dsDNA and not ssDNA. Because it may inhibit PCR at high concentrations, it is used at sub-saturating concentrations. Recently, some researchers have discouraged the use of SYBR Green I for HRM, claiming that substantial protocol modifications are required. This is because it is suggested that the lack of accuracy may result from “dye jumping”, where dye from a melted duplex may get reincorporated into regions of dsDNA which had not yet melted. New saturating dyes such as LC Green and LC Green Plus , ResoLight, EvaGreen, Chromofy and SYTO 9 are available on the market and have been used successfully for HRM. However, some groups have successfully used SYBR Green I for HRM with the Corbett Rotorgene instruments and advocate the use of SYBR Green I for HRM applications.

Further information

High Resolution Melting analysis information at gene-quantification.info

University of Southampton spinout company specialising in High Resolution Melting analysis
HRM Technology