SSCP analysis

The SSCP method follows the steps of DNA extraction, PCR amplification of the target fragment and finally denaturation of the double-stranded PCR product by heat and formamide and electrophoresis on a non-denaturing polyacrylamide gel. During electrophoresis, single-stranded DNAs are converted to stable conformations (there is none theory can predict the exact folded structure of ssDNA) depending on their major sequence. The SSCP method is based on the ability of single-stranded DNAs to fall into unique conformations depending on their primary sequence whose structures are stabilized by intramolecular interactions, under non-denaturing conditions. As a result, even a base change can lead to a modulation change, which can be detected by the altered mobility of the single-stranded DNA molecule in the SSCP method. Therefore, for each ssDNA fragment, the number of constant configurations that create bands of different mobility during SSCP electrophoresis must be determined experimentally under strictly controlled conditions. Several parameters have been found empirically to affect the sensitivity of SSCP analysis [1,2,3,4,5]. Among them is (i) a type of mutation (base substitutions, small insertions, deletions and rearrangements); (ii) DNA fragment size (no longer than 300 bp); (iii) G and C fragment; (iv) polyacrylamide (use of low percentage of crosslinker) or other gel matrix composition (addition of glycerol enhances the sensitivity); (v) gel size and potential; (vi) electrophoresis gel temperature (ssDNA have more compact conformation at lower temperatures); (vii) DNA concentration; (viii) buffer composition, including ionic strength and pH (low pH buffer can improve the sensitivity) and (ix) buffer additives. All these parameters are described in detail by Kakavas et al. [6].

SSCP vs RFLP

A detailed comparison of these methods is made in the work of Hashim et al. [7]. Both of them have advantages and disadvantages. On the one hand PCR-SSCP can detect unknown mutations and is rapid, inexpensive and convenient on the other hand PCR-RLFP is fast and can detect only known SNPs. We can’t claim that one method is better than the other.

Medical diagnosis applications

Detection of somatic mutations in cancer, genes responsible for hereditary diseases, polymorphisms and other applications are described in detail at Kakavas et al. [8,9,10]. Recent researches in the last 4 years shows dispersion of SSCP method in the above applications: on cancer prognosis [11, 12], on asthma disease [13], on blood groups [14], on gilbert’s syndrome [15], on diabetes disease [16], on respiratory distress syndrome [17], on infertile men with varicocele [18], on gastric mucosa [19], on traditional Chinese medicine [20], detection of bacterial DNA [21], identification of trichomonas vaginalis [22].

Other applications

Animals

PCR-SSCP has been used for several traits of animals such as wool characteristics [23], milk production traits [24], profile of sheep fatty acids [25], detection of Escherichia coli in the black swan [26], growth and carcass traits in lambs [27], reproductive traits in cattle [28], egg production traits in hens [29], body weight traits on chicken [30,31,32], gene study for fat-tailed and nonfat-tailed sheep [33], serotyping dichelobacter nodosus [34], mitochondrial diversity role in the productivity of quails [35], miR-9 gene affects litter size in goats [36], mutations in porcine [37], prolactin gene and association with egg production in ducks [38], wool fibre diameter in ewes [39], lactoferrin gene affects milk content in buffaloes [40], milk traits in yaks [41], association between MyoG gene and egg quality [42].

Birds

Genes identification in ostrich populations [43] and mtDNA identification of Alectoris rufa populations [44].

Fishes

Identification of shrimp species [45], SNPs and white spot syndrome virus resistance in black tiger shrimp [46].

Amphibians

Caretta-caretta mtDNA polymorphisms detection [47].

Plants

PCR-SSCP could be used for the authentication of snapper species by SSCP [48], the population genetic structure of zeugodacus tau species complex [49], for the genetic diversity of citrus trees [50], identification of phytophthora genes polymorphism of potato [51], identification of genetic diversity of lemon and mandarin varieties [52], detection of the genetic variation of alfalfa gene [53], prediction of the chloroplast gene polymorphism in barley varieties [54] and bacteria identification in wood tick [55].

Microbial organisms

Molecular epidemiological studies on the exposure of farm children to bacteria in environmental dust [56], analysis of the fungal flora in environmental dust samples [57], development of methods to analyze bacterial species diversity in freshwater and soil ecosystems [58], impact of Fe oxides mineralogy on iron solubilization and associated microbial communities [59], difference in some biological properties of soils [60], gene comparison from culturable denitrifying bacteria [61], food waste composting and microbial community structure profiling [62].

Food

Microbial communities identification in cheese [63] and for food authenticity [64].

Applications in forensic medicine and biochemistry

Usually, a big number of biological samples used, are found in a crime because SSCP uses mtDNA for it΄s different variations [65, 66].

An application with coronavirus

A very interesting research work concerned the cat infection with coronaviruses by Battilani et al. [67]. This study investigated the biological importance of the mutations detected in the genome of feline coronaviruses from naturally infected cats.

All the applications of SSCP analyzed above are shown in Table 1 and Fig. 1.

Table 1 Applications of SSCP
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Fig. 1
figure 1

Number of SSCP applications on deferent living beings

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An exhaustive search in the literature determined that the SSCP method was first used in 1989, it reached its peak in the year 1999 and by 2021 demonstrated a downward trend with spread to applications of environmental, veterinary and forensic medicine interest. The total of the research works amounts to 9.944 in a depth of 32 years (Fig. 2). This may encourage us to use SSCP in routine analyses in clinical, environmental, veterinary, microbiological, and forensic medicine laboratories.

Fig. 2
figure 2

Number of articles on the subject of SSCP over the past 32 years

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Conclusions

The PCR-SSCP method is widely used in the detection of mutations in both basic and applied biological and environmental science. The separation of mutated sequences could be further improved by using a gel matrix rather than a polyacrylamide one [68] or adding agents that can affect the folding of single-stranded DNA. Thus, PCR-SSCP is considered still modern as a method not only to screen sequence variations but also to identify new mutations. Among the various methods used for the detection of mutations, SSCP is one of the simplest and most sensitive methods, thus making it attractive for use in the clinical diagnostic, veterinary, environmental, microbiological, food and forensic medicine laboratories. Everyone’s expectation for the future application of SSCP technique in genotyping experiments is very high especially with the detection of different and new coronavirus (SARS-CoV-2) strains.