Single-Strand Conformational Polymorphism (SSCP)
Single-strand conformational polymorphism (SSCP) is a technique for detecting single nucleotide variations in DNA fragments based on the difference in secondary structure of different DNA fragments under non-denaturing conditions. These structures have different migration rates in gel electrophoresis and, thus, can be used to distinguish normal and mutated DNA. SSCP has the characteristics of being fast, simple, sensitive, and economical and can be widely used in the diagnosis of genetic diseases, population genetics research, evolutionary biology analysis, and other fields.
Principles and Procedures of SSCP
SSCP is based on the principle that different DNA fragments form different secondary structures under non-denaturing conditions. These structures have different migration rates in gel electrophoresis and, thus, can be used to distinguish normal and mutated DNA. The principle of SSCP can be divided into several steps: DNA fragment selection, amplification, labeling, denaturation, renaturation, electrophoresis, and detection. Each step has its own role and influencing factors in the SSCP process. For example, the length and GC content of DNA fragments determine the complexity and stability of their secondary structures; the temperature, pH value, and salt concentration affect the denaturation and renaturation of DNA fragments; the type and concentration of gel and buffer affect the separation and resolution of DNA fragments; and the type and method of labeling affect the detection and visualization of DNA fragments.
Table 1. The experimental operation process of SSCP
| Step | Main Content | Purpose |
|---|---|---|
| DNA fragment selection | Design suitable primers, covering the region of interest and avoiding secondary structures. | Select the DNA fragment to be detected, determine its length, and determine its GC content. |
| Amplification | Amplify DNA fragments by the PCR method, making their quantity enough for SSCP analysis. | Increase the quantity and purity of DNA fragments, facilitating subsequent labeling and detection. |
| Labeling | Label PCR products with radioisotopes or fluorescent dyes, making them visible on the gel. | Increase the signal intensity of DNA fragments, facilitating subsequent separation and detection. |
| Denaturation | Denature labeled DNA fragments by high temperature or chemical reagents, making them from double-stranded to single-stranded | Break the complementary base pairing of DNA fragments, providing conditions for subsequent renaturation and secondary structure formation. |
| Renaturation | Renature denatured DNA fragments at a low temperature or in a buffer solution, making them form different secondary structures. | DNA fragments spontaneously form different secondary structures under non-denaturing conditions; these structures have different migration rates in gel electrophoresis. |
| Electrophoresis | Separate renatured DNA fragments by the non-denaturing gel electrophoresis method, making them form bands or spots on the gel according to different migration rates. | Separate the different secondary structures of DNA fragments and distinguish between normal and mutated DNA fragments. |
| Detection | Detect electrophoresed gel by radioautography or fluorescence imaging systems, making them show bands or spots on film or screen. | Detect the position and intensity of different secondary structures of DNA fragments on the gel and judge whether there are single nucleotide variations. |
Characteristics of SSCP
SSCP has both advantages and disadvantages as a technique for detecting single nucleotide variations in DNA fragments. The characteristics of SSCP can be evaluated in several aspects: sensitivity, specificity, reproducibility, resolution, reliability, and applicability. Each aspect reflects the performance and limitations of SSCP in different scenarios and is compared with other techniques. For example, SSCP has high sensitivity and specificity for detecting single nucleotide variations, but it may miss some mutations that do not affect the secondary structure of DNA fragments; SSCP has good reproducibility and resolution for separating different DNA fragments, but it is sensitive to experimental conditions such as temperature, pH value, and salt concentration; SSCP has high reliability and applicability for analyzing various DNA fragments, but it cannot determine the type and location of mutations without further sequencing or hybridization. In this part, we will summarize the advantages and disadvantages of SSCP from each aspect and compare and analyze it with other similar techniques such as restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE), single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), allele-specific oligonucleotide (ASO) hybridization, and direct sequencing.
Applications of SSCP
SSCP has a wide range of applications in various fields, such as the diagnosis of genetic diseases, population genetics research, evolutionary biology analysis, and others. SSCP can be used to detect common single nucleotide variations in genetic diseases, which are often responsible for the phenotypic differences and pathogenic mechanisms of these diseases. For example, SSCP can be used to detect mutations in the β-globin gene that cause β-thalassemia, mutations in the CFTR gene that cause cystic fibrosis, mutations in the NF1 gene that cause neurofibromatosis, and others. SSCP can also be used to analyze the distribution and variation of genotypes and allele frequencies in populations, which reflect the genetic diversity and structure of these populations. For example, SSCP can be used to analyze the polymorphism of mitochondrial DNA, microsatellites, HLA genes, and others in different human populations. SSCP can also be used to analyze the phylogenetic relationships and evolutionary history among and within species, which reveal the origin and divergence of these species. For example, SSCP can be used to analyze the variation of ribosomal RNA genes, chloroplast DNA, cytochrome b genes, and others in different plant and animal species.
References
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- Kakavas KV. Sensitivity and applications of the PCR Single-Strand Conformation Polymorphism method. Mol Biol Rep. 2021 Apr;48(4):3629-3635.
- Jordan D, et al. Resolving human DNA mixtures with F-108 polymer and capillary electrophoresis single-strand conformational polymorphism analysis. J Sep Sci. 2020 Aug;43(16):3327-3332.
- Gasser RB, et al. Applications of single-strand conformation polymorphism (SSCP) to taxonomy, diagnosis, population genetics and molecular evolution of parasitic nematodes. Vet Parasitol. 2001 Nov 22;101(3-4):201-13.
- Gasser RB, et al. Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation. Nat Protoc. 2006;1(6):3121-8.
- Schmalenberger A, et al. Profiling the diversity of microbial communities with single-strand conformation polymorphism (SSCP). Methods Mol Biol. 2014;1096:71-83.
