**4. Method for detection of SNPs**

SNP genotyping strategies typically involve allele-discrimination and allele-detection. The other methods based on physical properties of DNA [29].

#### **4.1. Allele-discrimination**

#### *4.1.1. Primer extension*

These approaches involve allele-specific incorporation of nucleotides in primer extension reaction with a DNA template, utilizing enzyme specificity to accomplish allelic discrimination. In the reaction of a common primer extension (CPE) protocol, a designed primer is annealing with its 3′ end near a SNP site and nucleotides are added by polymerase enzyme [30]. The extended nucleotide is examined by either mass or fluorescence to verify SNP genotype. Because of the simplicity in primer and assay design, it can detect multiple SNPs at the same time, therefore, several SNP genotyping use CPE [29]. CPE-based methods that use MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) for the discrimination of alleles include PinPoint assay [31, 32], MassEXTEND™ [33, 34], SPC-single base extension (SBE) [35], and GOOD assay [36]. In the reaction of these methods, SNP-specific primers are simultaneously extended with numerous nucleotides using PCR products as a template yielding extended products of dissimilar masses. The genotypes of SNPs of the products are examined by mass analysis (**Figure 4a**). The PinPoint assay performs the simplest technique by using dideoxynucleotides (ddNTPs) for single base extension (SBE) of primer [31, 32]. The CPE approaches that use fluorescence-based detection involve SBE of primer with fluorescently labeled ddNTPs (**Figure 4b**). Specific primer extension (SPE) methods use two primers that have the same nucleotide sequences except specific allele/base at their 3′ end. The primers can extend if the nucleotide at their 3′ end of the primers perfectly binds with the SNP of complementary template, then allelic discrimination can be examined by mass different using gel electrophoresis (**Figure 4c**).

**MassEXTEND™** procedure uses a mixture of dNTPs and ddNTPs for primer extension and discriminate the allele by mass detection. It provides medium-to-high-throughput genotyping

**4. Method for detection of SNPs**

8 Genetic Diversity and Disease Susceptibility

**4.1. Allele-discrimination**

*4.1.1. Primer extension*

jpg).

other methods based on physical properties of DNA [29].

SNP genotyping strategies typically involve allele-discrimination and allele-detection. The

**Figure 3.** Genome-wide association studies (GWAS). (http://mmg-233-2014-genetics-genomics.wikia.com/wiki/File:GWAS.

These approaches involve allele-specific incorporation of nucleotides in primer extension reaction with a DNA template, utilizing enzyme specificity to accomplish allelic discrimination. In the reaction of a common primer extension (CPE) protocol, a designed primer is annealing with its 3′ end near a SNP site and nucleotides are added by polymerase enzyme [30]. The extended nucleotide is examined by either mass or fluorescence to verify SNP genotype. Because of the simplicity in primer and assay design, it can detect multiple SNPs at the same time, therefore, several SNP genotyping use CPE [29]. CPE-based methods that use MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) for the discrimination of alleles include PinPoint assay [31, 32], MassEXTEND™ [33, 34], SPC-single base extension (SBE) [35], and GOOD assay [36]. In the reaction of these methods, SNP-specific primers are simultaneously extended with numerous nucleotides using PCR products as a template yielding extended products of dissimilar masses. The genotypes of SNPs of the products are examined by mass analysis (**Figure 4a**). The PinPoint assay performs the simplest technique by using dideoxynucleotides (ddNTPs) for single base extension (SBE)

**Figure 4.** Primer extension approaches for SNP genotyping. (a) Mass spectrometry-based detection. This method uses a primer that anneals one base upstream of the SNP site followed by its extension with ddNTPs. Extension products are detected by mass spectrometry, and the difference between mass of extension product and primer identifies incorporated nucleotide(s) and therefore the SNP genotype. (b) Fluorescence detection using capillary electrophoresis. This technique uses a primer that anneals one base upstream of the SNP site after that its extension with ddNTPs that are labeled with different fluorescent tags. Products are detected by fluorescence after capillary electrophoresis and the color of dye indicates incorporated base(s). (c) Allele-specific primers with detection of PCR products. This method uses two allele-specific primers that anneal with their 3′ ends at the SNP site and a common reverse primer (not shown) for PCR reaction. The amplification occurs when the forward primer perfectly matches to the SNP at its 3′ end and the genotype can be determined by gel electrophoresis of PCR product [29].

that can be used to genotype a group of SNPs in candidate genes, SNPs for GWAS and SNPs in a large sample set [33, 34].

The DNA fragment containing SNPs are amplified from genomic DNA. The PCR products are cleaved, tagged, and hybridized to the probe array under stringent conditions, then wash, and label with fluorescent. The genotypes of SNPs are examined from the fluorescence signal based on probe-target hybridization. Numerous probes that differ at a single base are used to confirm each SNP to increase genotyping accuracy. In this method, a single array contains millions of probes and be used for parallel genotyp-

Biosystems, CA) combines the procedure of hybridization and 5′ nuclease activity of polymerase coupled with fluorescence detection (**Figure 5b**). It employs four oligonucleotides containing two allele-specific oligonucleotide probes that have a single base mismatch and a pair of PCR primers neighboring the SNP covering region [29, 39].

Ligation method discriminates the allele by using specificity of ligase enzymes. When two oligonucleotides (allele-specific probes and ligation probes) hybridize to single-stranded template DNA with perfect complementarity, they are nearby to each other, then ligase enzymes join them together to form a single oligonucleotide. Normally, three oligonucleotide probes are used in ligation assays, two probes are allele specific that bind to the template at the SNP site and ligation probe [40]. Combinatorial fluorescence energy transfer (CFET) tags have been used with ligation for SNP genotyping [41]. CFET tags are composed of fluorescent dyes that can transfer energy when they are in close proximity. For SNP genotyping, two probes of allele specific are labeled with CFET tags and common probe is labeled with biotin (**Figure 6a**). After the ligation reaction, product is separated using the biotin-streptavidin interaction. Genotyping is carried out by using capillary array gel electrophoresis based on tag fluorescence. In Padlock technology, a linear oligonucleotide probe is used with its ends designed to mimic the allele-

The method is based on the capacity of enzymes to cleave DNA by recognition of specific sequences and structures. Difference between alleles can be discriminated when SNPs are

The ability of restriction enzymes has been used for detection of genetic variation by the method of restriction fragment length polymorphism (RFLP) [42]. These enzymes recognize specific sequences in double-stranded DNA and cleave both strands at a specific site in the sequence or near it to generate smaller DNA fragments (**Figure 7a**). For SNP genotyping, the PCR product containing the SNP is incubated with appropriate restriction enzyme and separated by gel electrophoresis. The SNP genotype is simply determined from sizes of the digested products. This method does not need any probes but it has limited amount and a number

The Invader® assay (Third Wave™ Technologies, WI) utilizes structure-specific cleavage by a flap endonuclease enzyme (**Figure 7b**). It uses three probes for genotyping a SNP, two allele-

specific probe and common probe for ligation at the SNP site (**Figure 6b**).

located in an enzyme recognition site and affect the enzyme activity.

specific probes and a third common probe (invader) [43].

SNPs [38]. The TaqMan® genotyping assay (Applied

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ing of 10<sup>4</sup>

*4.1.3. Ligation*

*4.1.4. Enzymatic cleavage*

of SNPs.

to approximately 10<sup>5</sup>

#### *4.1.2. Hybridization*

Hybridization methods use differences in thermal stability of double-stranded DNA to separate between perfectly matched and mismatched target-probe pairs for succeeding allelic discrimination. Hybridization methods have been applied on high-throughput platforms using microarrays (**Figure 5a**). In the GeneChip® array technology (Affymetrix, CA) protocol, probe array is synthesized in an ordered fashion on a solid surface using 25-bases oligonucleotides with specific allele by photolithography [37].

**Figure 5.** Hybridization-based approaches for SNP genotyping. (a) Target hybridization to a probe array. This method uses allele-specific probes attached to a solid surface for hybridization with tagged-targets containing SNPs. The surface is washed to remove mismatched targets, then the genotype of the perfectly matched target-probe pairs is detected by fluorescence. (b) TaqMan® assay. The assay uses two allele-specific probes with different dyes, reporter (R) and quencher (Q), at either end with a mismatch at the SNP site. The perfectly matched probe is cleaved during PCR amplification of the SNP-containing region, release its reporter, and the SNP genotype indicates by fluorescence analysis [29].

The DNA fragment containing SNPs are amplified from genomic DNA. The PCR products are cleaved, tagged, and hybridized to the probe array under stringent conditions, then wash, and label with fluorescent. The genotypes of SNPs are examined from the fluorescence signal based on probe-target hybridization. Numerous probes that differ at a single base are used to confirm each SNP to increase genotyping accuracy. In this method, a single array contains millions of probes and be used for parallel genotyping of 10<sup>4</sup> to approximately 10<sup>5</sup> SNPs [38]. The TaqMan® genotyping assay (Applied Biosystems, CA) combines the procedure of hybridization and 5′ nuclease activity of polymerase coupled with fluorescence detection (**Figure 5b**). It employs four oligonucleotides containing two allele-specific oligonucleotide probes that have a single base mismatch and a pair of PCR primers neighboring the SNP covering region [29, 39].

#### *4.1.3. Ligation*

that can be used to genotype a group of SNPs in candidate genes, SNPs for GWAS and SNPs

Hybridization methods use differences in thermal stability of double-stranded DNA to separate between perfectly matched and mismatched target-probe pairs for succeeding allelic discrimination. Hybridization methods have been applied on high-throughput platforms using microarrays (**Figure 5a**). In the GeneChip® array technology (Affymetrix, CA) protocol, probe array is synthesized in an ordered fashion on a solid surface using 25-bases oligonucleotides with specific allele by photolithography [37].

**Figure 5.** Hybridization-based approaches for SNP genotyping. (a) Target hybridization to a probe array. This method uses allele-specific probes attached to a solid surface for hybridization with tagged-targets containing SNPs. The surface is washed to remove mismatched targets, then the genotype of the perfectly matched target-probe pairs is detected by fluorescence. (b) TaqMan® assay. The assay uses two allele-specific probes with different dyes, reporter (R) and quencher (Q), at either end with a mismatch at the SNP site. The perfectly matched probe is cleaved during PCR amplification of

the SNP-containing region, release its reporter, and the SNP genotype indicates by fluorescence analysis [29].

in a large sample set [33, 34].

10 Genetic Diversity and Disease Susceptibility

*4.1.2. Hybridization*

Ligation method discriminates the allele by using specificity of ligase enzymes. When two oligonucleotides (allele-specific probes and ligation probes) hybridize to single-stranded template DNA with perfect complementarity, they are nearby to each other, then ligase enzymes join them together to form a single oligonucleotide. Normally, three oligonucleotide probes are used in ligation assays, two probes are allele specific that bind to the template at the SNP site and ligation probe [40]. Combinatorial fluorescence energy transfer (CFET) tags have been used with ligation for SNP genotyping [41]. CFET tags are composed of fluorescent dyes that can transfer energy when they are in close proximity. For SNP genotyping, two probes of allele specific are labeled with CFET tags and common probe is labeled with biotin (**Figure 6a**). After the ligation reaction, product is separated using the biotin-streptavidin interaction. Genotyping is carried out by using capillary array gel electrophoresis based on tag fluorescence. In Padlock technology, a linear oligonucleotide probe is used with its ends designed to mimic the allelespecific probe and common probe for ligation at the SNP site (**Figure 6b**).

#### *4.1.4. Enzymatic cleavage*

The method is based on the capacity of enzymes to cleave DNA by recognition of specific sequences and structures. Difference between alleles can be discriminated when SNPs are located in an enzyme recognition site and affect the enzyme activity.

The ability of restriction enzymes has been used for detection of genetic variation by the method of restriction fragment length polymorphism (RFLP) [42]. These enzymes recognize specific sequences in double-stranded DNA and cleave both strands at a specific site in the sequence or near it to generate smaller DNA fragments (**Figure 7a**). For SNP genotyping, the PCR product containing the SNP is incubated with appropriate restriction enzyme and separated by gel electrophoresis. The SNP genotype is simply determined from sizes of the digested products. This method does not need any probes but it has limited amount and a number of SNPs.

The Invader® assay (Third Wave™ Technologies, WI) utilizes structure-specific cleavage by a flap endonuclease enzyme (**Figure 7b**). It uses three probes for genotyping a SNP, two allelespecific probes and a third common probe (invader) [43].

**Figure 6.** Ligation-based SNP genotyping approaches using ligase enzymes. (a) Detection using CFET labels. This protocol uses two allele-specific probes that have different CFET labels and a biotin-tagged common probe that hybridizes adjacent to them. Allele-specific probe and common probe bind to the template containing SNP. If the sequences are perfectly matches at SNP site, the two probes are ligated. The ligated product is proved by isolation using the biotin tag on the common probe and the fluorescence signal from the CFET label to indicate SNP genotype. (b) Ligation of padlock probe. This method uses two allele-specific probes that hybridize to target DNA with their ends aligned end-to-end at the SNP site. One end of the probe is allele-specific that is ligated to the other end only when there is a perfect match at the SNP site, leading to circularization of the probe. After rolling circle amplification using a specific prime, the product is analyzed by gel electrophoresis [29].

#### **4.2. Allele detection methods**

#### *4.2.1. Mass-based detection*

For mass analysis of oligonucleotides, MALDI-TOF MS is a widely used method. It involves the use of a small organic molecule termed matrix that absorbs energy from a laser source of certain wavelength for ionization. When analytes are mixed and cocrystallized with matrix, they are ionized in the form of intact molecules owing to transfer of energy from the matrix molecules [44, 45]. **Figure 8** demonstrates a multiplex SNP genotyping by primer extension and MALDI-TOF MS detection.

*4.2.2. Fluorescence signal-based detection*

fluorescence analysis [29].

Monitoring fluorescence signal is widely used in genotyping technologies because its operation is simple and detection is fast with high sensitivity. Fluorescence detection is used for direct sequencing (DS) using capillary array electrophoresis. Fluorescence polarization (FP)-based detection uses the change in polarization of plane polarized light by a fluorescent dye molecule owing to change in its molecular weight under conditions of constant temperature and solvent viscosity [46]. FP has been coupled with other SNP genotyping techniques including TaqMan® and Invader® [47]. TaqMan® assay is a single-step assay that uses fluorescence-based detection.

**Figure 7.** Enzymatic cleavage for SNP genotyping. (a) Digested with specific restriction enzyme. This method uses a restriction enzyme that cleaves only one of the alleles. The digested products are run on agarose gel electrophoresis, and the SNP genotype is determined based on the size and number of DNA fragments. (b) Invader® assay. This assay uses two allele-specific probes with different dyes, reporter (R) and quencher (Q), at either end or one common invader probe. The allele-specific probe and invader probe hybridize with target DNA to form a three-dimensional structure at the SNP site which recognized by cleavase enzyme. The allele-specific probe that complementary to the SNP is cleaved by the enzyme and releases its reporter dye in which SNP genotype can be discriminated by

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**Figure 7.** Enzymatic cleavage for SNP genotyping. (a) Digested with specific restriction enzyme. This method uses a restriction enzyme that cleaves only one of the alleles. The digested products are run on agarose gel electrophoresis, and the SNP genotype is determined based on the size and number of DNA fragments. (b) Invader® assay. This assay uses two allele-specific probes with different dyes, reporter (R) and quencher (Q), at either end or one common invader probe. The allele-specific probe and invader probe hybridize with target DNA to form a three-dimensional structure at the SNP site which recognized by cleavase enzyme. The allele-specific probe that complementary to the SNP is cleaved by the enzyme and releases its reporter dye in which SNP genotype can be discriminated by fluorescence analysis [29].

#### *4.2.2. Fluorescence signal-based detection*

**4.2. Allele detection methods**

12 Genetic Diversity and Disease Susceptibility

and MALDI-TOF MS detection.

For mass analysis of oligonucleotides, MALDI-TOF MS is a widely used method. It involves the use of a small organic molecule termed matrix that absorbs energy from a laser source of certain wavelength for ionization. When analytes are mixed and cocrystallized with matrix, they are ionized in the form of intact molecules owing to transfer of energy from the matrix molecules [44, 45]. **Figure 8** demonstrates a multiplex SNP genotyping by primer extension

**Figure 6.** Ligation-based SNP genotyping approaches using ligase enzymes. (a) Detection using CFET labels. This protocol uses two allele-specific probes that have different CFET labels and a biotin-tagged common probe that hybridizes adjacent to them. Allele-specific probe and common probe bind to the template containing SNP. If the sequences are perfectly matches at SNP site, the two probes are ligated. The ligated product is proved by isolation using the biotin tag on the common probe and the fluorescence signal from the CFET label to indicate SNP genotype. (b) Ligation of padlock probe. This method uses two allele-specific probes that hybridize to target DNA with their ends aligned end-to-end at the SNP site. One end of the probe is allele-specific that is ligated to the other end only when there is a perfect match at the SNP site, leading to circularization of the probe. After rolling circle amplification using a specific prime, the product is analyzed by gel electrophoresis [29].

*4.2.1. Mass-based detection*

Monitoring fluorescence signal is widely used in genotyping technologies because its operation is simple and detection is fast with high sensitivity. Fluorescence detection is used for direct sequencing (DS) using capillary array electrophoresis. Fluorescence polarization (FP)-based detection uses the change in polarization of plane polarized light by a fluorescent dye molecule owing to change in its molecular weight under conditions of constant temperature and solvent viscosity [46]. FP has been coupled with other SNP genotyping techniques including TaqMan® and Invader® [47]. TaqMan® assay is a single-step assay that uses fluorescence-based detection.

of enzyme reactions [48]. Pyrosequencing™ is an approach that combines sequencing-bysynthesis with chemiluminescence detection. In SNP genotype, it provides sequence informa-

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SSCP discriminates the allele by using secondary structure of single-stranded DNA. The singlestranded DNA molecules which differ at a single base run on a nondenaturing gel electrophoresis

**Figure 9.** Single-strand conformation polymorphism (SSCP) (https://media.nature.com/m685/nature-assets/nprot/journal/

display different mobility based on their native conformations (**Figure 9**) [49].

tion on the region surrounding the SNP site.

*4.3.1. Single-strand conformation polymorphism (SSCP)*

**4.3. The other methods**

v1/n6/images/nprot.2006.485-F1.jpg).

**Figure 8.** Multiplex SNP genotyping by primer extension and MALDI-TOF MS [29]. First, genomic DNA is amplified in multiplex PCR reaction, and the PCR products are used for a primer extension reaction with a collection of primers specific to each SNP site. The mass of the primer indicates SNP, and the mass difference between primer and its extension product(s) tells identity of incorporated nucleotide(s) and the genotype. (a) MassEXTENDtm method in which primers are prolonged with a specific mixture of dNTPs and ddNTPs by one or more bases, specific to each allele. (b) Modified PinPoint method using mass-tagged ddNTPs for primer extension. This increases mass difference between extension products of alleles of a SNP, when compared with regular ddNTPs, increasing the accuracy of genotyping. (c) SPC-SBE method relating the production of primer extension products with biotinylated ddNTPs for isolation on a solid surface. This excludes excess primers from the mass spectrum, permitting the analysis of more products at the same time [29].

It is well suited for low-to-medium throughput genotyping applications but is currently limited to genotyping of one SNP per assay.

#### *4.2.3. Chemiluminescence*

Chemiluminescence has several advantages as a detection technique, such as high signalto-noise ratio, rapid detection, and feasibility for automation. Pyrosequencing™ (Biotage, Sweden) employs chemiluminescence-based detection for SNP genotyping using a cascade of enzyme reactions [48]. Pyrosequencing™ is an approach that combines sequencing-bysynthesis with chemiluminescence detection. In SNP genotype, it provides sequence information on the region surrounding the SNP site.

#### **4.3. The other methods**

It is well suited for low-to-medium throughput genotyping applications but is currently limited

**Figure 8.** Multiplex SNP genotyping by primer extension and MALDI-TOF MS [29]. First, genomic DNA is amplified in multiplex PCR reaction, and the PCR products are used for a primer extension reaction with a collection of primers specific to each SNP site. The mass of the primer indicates SNP, and the mass difference between primer and its extension product(s) tells identity of incorporated nucleotide(s) and the genotype. (a) MassEXTENDtm method in which primers are prolonged with a specific mixture of dNTPs and ddNTPs by one or more bases, specific to each allele. (b) Modified PinPoint method using mass-tagged ddNTPs for primer extension. This increases mass difference between extension products of alleles of a SNP, when compared with regular ddNTPs, increasing the accuracy of genotyping. (c) SPC-SBE method relating the production of primer extension products with biotinylated ddNTPs for isolation on a solid surface. This excludes excess primers from the mass spectrum, permitting the analysis of more products at the same time [29].

Chemiluminescence has several advantages as a detection technique, such as high signalto-noise ratio, rapid detection, and feasibility for automation. Pyrosequencing™ (Biotage, Sweden) employs chemiluminescence-based detection for SNP genotyping using a cascade

to genotyping of one SNP per assay.

14 Genetic Diversity and Disease Susceptibility

*4.2.3. Chemiluminescence*

#### *4.3.1. Single-strand conformation polymorphism (SSCP)*

SSCP discriminates the allele by using secondary structure of single-stranded DNA. The singlestranded DNA molecules which differ at a single base run on a nondenaturing gel electrophoresis display different mobility based on their native conformations (**Figure 9**) [49].

**Figure 9.** Single-strand conformation polymorphism (SSCP) (https://media.nature.com/m685/nature-assets/nprot/journal/ v1/n6/images/nprot.2006.485-F1.jpg).

#### *4.3.2. High resolution melting analysis (HRM)*

This method, the fragment cover SNP is amplified by real-time PCR and followed by HRM. The HRM is a technique for the detection mutations and SNPs, which based on analysis of melting curve when double-strand DNA (dsDNA) separate into single-strand DNA (ssDNA) during increased temperature 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. This can visualize the melting behavior of the product through a fluorescent dye [50]. The fluorescent dye binds to double-strand DNA during the amplification resulting in an increase of fluorescence (**Figure 10**). This method is simple, cost-effective, fast and able to accurately genotype many samples rapidly. It also reduces the need to design multiple pairs of primers or purchase expensive probes.

#### *4.3.3. Denaturing high performance liquid chromatography (DHPLC)*

It is a method for screening DNA samples for SNPs and inherited mutations. The analysis begins with a PCR amplification, followed by a step of denaturation-renaturation to create hetero-and homoduplexes from the two populations in the PCR. The heteroduplexes with mismatch pairing and homoduplex can be detected on reversed-phase chromatography of denaturing high performance liquid chromatography (DHPLC). The heteroduplexes thermally that less stable than their corresponding homoduplexes will be resolved by chromatography when subjected to a sufficiently high temperature. This mismatch will decrease the interaction with the column and a reduced retention time compared to the homoduplexes (**Figure 11**).

**5. Application of SNPs**

mechanism [59].

**5.2. SNPs and drug development**

**5.1. SNPs as biological markers of human diseases**

tamop425/0011\_1A\_Molelkularis\_diagnoszitka\_en\_book/images/image047.png).

Most SNPs are not responsible for a disease state. They serve as biological markers for pinpointing a disease on the human genome map. SNPs occur on average once every 200 base pairs [18, 51–53] in the human genome. Common SNPs (a minor allele frequency range from 5% to *>*20%) can explain a proportion of common human disease. Most SNPs do not occur in the coding region of genes or even in genes [4]. Nonsynonymous SNPs that suspect in causing a human disease do not account for all SNPs that can cause disease or susceptibility to disease. Other functional SNPs associated with human disease or susceptibility to disease includes SNPs located in promoters [54, 55], introns [56], splice sites [57], and intragenic region [56, 58]. Even synonymous SNPs have been involved in functional consequences via unknown

**Figure 11.** Denaturing high performance liquid chromatography (DHPLC) (http://www.tankonyvtar.hu/en/tartalom/

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Variants of genes encoding drug metabolizing enzymes or drug targets have been studied in association with personal drug responses. SNPs are popular molecular markers in such pharmacogenomics studies. Using SNPs to study the genetics of drug response will help in the creation of personalized medicine or the most appropriate drug for an individual and could be determined in advance of treatment by analyzing a patient's SNP profile. SNPs may

**Figure 10.** High resolution melting (HRM) analysis (https://www.dna.utah.edu/Image/Hi\_Res%20Melting\_Normalized. JPG).

#### Polymorphisms http://dx.doi.org/10.5772/intechopen.76728 17

**Figure 11.** Denaturing high performance liquid chromatography (DHPLC) (http://www.tankonyvtar.hu/en/tartalom/ tamop425/0011\_1A\_Molelkularis\_diagnoszitka\_en\_book/images/image047.png).
