Single-strand conformation polymorphism

Single-strand conformation polymorphism (SSCP) is a molecular biology technique used for the detection of genetic variation or mutations in DNA. The method hinges on the principle that single-stranded DNA fragments of identical sequence but different conformations (shapes) can be separated by electrophoresis through a non-denaturing polyacrylamide gel. This separation occurs because the three-dimensional conformation of a single-stranded DNA molecule affects its mobility through the gel. Variations in DNA sequence can lead to changes in the conformation of the DNA strand, thus altering its electrophoretic mobility. SSCP is a sensitive method for detecting mutations that lead to changes in the primary structure of DNA, including point mutations, small deletions, and insertions.

Principle

The principle behind SSCP analysis is that under non-denaturing conditions, single-stranded DNA molecules fold into unique secondary structures. These structures are highly dependent on their nucleotide sequence. Even a single nucleotide change can significantly alter the conformation of the DNA strand, thereby affecting its mobility during electrophoresis. When a DNA fragment containing a mutation is run on a gel alongside a wild-type (mutation-free) fragment, the two may exhibit different mobilities, allowing for the detection of the mutation.

Methodology

The SSCP analysis involves several steps:

1. DNA Extraction: DNA is extracted from the sample of interest.
2. PCR Amplification: The region of DNA containing the potential mutation is amplified using Polymerase Chain Reaction (PCR).
3. Denaturation: The double-stranded PCR products are denatured to single strands by heating.
4. Electrophoresis: The denatured DNA fragments are then subjected to electrophoresis on a non-denaturing polyacrylamide gel. The gel concentration and electrophoresis conditions are optimized to maximize the resolution of DNA conformations.
5. Visualization: After electrophoresis, the DNA bands are visualized using a DNA staining method, such as silver staining or fluorescent dyes.

Applications

SSCP is widely used in genetic research and diagnostics to detect known and unknown mutations. Its applications include:

• Detection of genetic disorders caused by point mutations or small deletions/insertions.
• Identification of genetic variations in populations, useful in population genetics studies.
• Characterization of genetic diversity in non-human organisms, important for conservation genetics and evolutionary biology.

Advantages and Limitations

Advantages:

• SSCP is a relatively simple and cost-effective method for mutation detection.
• It does not require large amounts of DNA or sophisticated equipment.

Limitations:

• The sensitivity of SSCP can be affected by the size of the DNA fragment, with shorter fragments generally yielding better results.
• It may not detect all mutations, especially in larger DNA fragments, as not all sequence changes result in a conformational change that alters mobility.
• The method requires careful optimization of gel conditions for each DNA fragment analyzed.

See Also

• Polymerase Chain Reaction
• DNA Sequencing
• Molecular Genetics
• Genetic Variation