Single-base extension

Single-base extension (SBE) is a molecular biology technique used in genotyping, DNA sequencing, and molecular diagnostics. The principle of SBE involves the extension of a primer by a single nucleotide, the identity of which is determined by the template DNA. This technique is particularly useful for the detection of single nucleotide polymorphisms (SNPs), which are the most common type of genetic variation among people.

Principle

The SBE reaction is initiated by annealing a primer to a complementary DNA template adjacent to the SNP site. A DNA polymerase is then used to extend the primer by adding a single, labeled nucleotide that is complementary to the nucleotide at the SNP site on the template strand. The identity of the incorporated nucleotide reveals the genotype at the SNP site. The labeled nucleotides can be detected through various methods, including fluorescence or mass spectrometry, allowing for the determination of the specific nucleotide present at the SNP site.

Applications

SBE has a wide range of applications in genetic research and diagnostics:

• Genotyping: SBE is commonly used in genotyping assays to identify specific alleles or mutations in genes. This is crucial for genetic studies, disease association studies, and pharmacogenomics.
• Molecular Diagnostics: In the field of molecular diagnostics, SBE is used to detect mutations that may cause genetic disorders or influence drug metabolism.
• DNA Sequencing: Although not as common as other sequencing methods, SBE can be used for sequencing short DNA fragments. It is particularly useful in targeted sequencing applications, where specific regions of the genome are of interest.

Advantages and Limitations

Advantages:

• High specificity and accuracy in SNP detection.
• Flexibility in the choice of detection methods (fluorescence, mass spectrometry, etc.).
• Suitable for high-throughput genotyping.

Limitations:

• Limited to the analysis of SNPs or very short DNA sequences.
• Requires prior knowledge of the DNA sequence surrounding the SNP for primer design.
• The cost and complexity of the detection systems may be prohibitive for some applications.

Technological Advances

Recent advances in SBE technology include the development of multiplexing strategies, allowing for the simultaneous detection of multiple SNPs in a single reaction. This increases throughput and reduces costs, making SBE an even more attractive option for large-scale genotyping studies.

Conclusion

Single-base extension is a powerful tool in the field of molecular biology, offering high specificity and flexibility for SNP detection and genotyping. Despite its limitations, ongoing technological improvements continue to expand its applications in genetic research and molecular diagnostics.