Table 5.

Gene scanning methods for unknown mutations and their mechanisms.

Method	The Mechanism/Application	Ref.
Single-Strand Conformation Polymorphism (SSCP)	single-strand nucleotide sequence under the non-denaturing conditions forms a unique conformation based on its primary DNA sequence, altered strand takes on a different conformation than a non-altered strand and shows different electrophoretic mobility, even a single nucleotide change can be detected, steps: amplification of targeted fragment, double-strand denaturation, non-denaturing polyacrylamide gel electrophoresis, and the analysis of the results	[80,81]
Denaturing Gradient Gel Electrophoresis (DGGE)	the different melting points of altered and unaltered double-stranded DNA at different concentrations of chemical denaturants steps: amplification of the targeted fragment through PCR, polyacrylamide gel electrophoresis with a rising concentration of the denaturing agent, and migration of double-stranded PCR products until a concentration of denaturing agent is equal to their melting point (Tm)	[80,81]
Denaturing High-Pressure Liquid Chromatography (DHPLC)	one of the chromatography methods for detection of single nucleotide substitution and small insertions/deletions separation of hetero- and homoduplexes on a chromatographic column under partial denaturation temperature conditions, using buffer gradients with TEAA and acetonitrile steps: DNA isolation and amplification of targeted sequence (approximately 150–600 bp) via PCR reaction; formation of hereto- and homoduplexes through DNA strand denaturation and reannealing caused by slow temperature reduction; elution (heteroduplexes are less stable and show less affinity for the column, and as a consequence are eluted earlier); monitoring of separation by measuring absorbance at 260 nm wavelength; detection and results presented as the chromatograms (homozygous sample—1 peak, the heterozygous—up to 4 peaks)	[82,83]
Conformation-sensitive Gel Electrophoresis (CSGE)	heteroduplex analysis for screening of mismatches in large multi-exon genes based on different electrophoresis mobility of hetero- and homoduplexes on a modified polyacrylamide gel	[84]
High-Resolution Melting (HRM)	detection of point mutations, small deletions/insertions, and large genomic rearrangements (qPCR-HRM) gene scanning technique performed on double-stranded DNA samples; the separation of the double-strand is monitored in real-time by a progressive change in fluorescence (due to the release of fluorescence dye from double-strand DNA denatured under temperature) the differences in the melting points distinguish altered and normal (wild type) alleles	[85]