What is Sanger Sequencing?
Sanger sequencing, also known as the chain-termination method, is a technique for determining the nucleotide sequence of DNA. It was developed by Frederick Sanger and his colleagues in 1977. This method involves the selective incorporation of chain-terminating dideoxynucleotides during DNA replication.
How is Sanger Sequencing Applied in Pediatrics?
In
pediatrics, Sanger sequencing is used to diagnose and understand a variety of genetic disorders. By analyzing a child's DNA, clinicians can identify mutations that may be responsible for conditions such as congenital abnormalities, metabolic disorders, and hereditary diseases.
DNA Extraction: DNA is extracted from the child's cells, typically from a blood sample.
Amplification: The region of interest in the DNA is amplified using
Polymerase Chain Reaction (PCR).
Sequencing Reaction: The amplified DNA is subjected to the sequencing reaction, where dideoxynucleotides terminate the DNA synthesis at specific points.
Electrophoresis: The resulting DNA fragments are separated by size using capillary electrophoresis.
Data Analysis: The sequence data is analyzed to identify any mutations or variations.
Cost and Time: It is more expensive and time-consuming compared to other methods like NGS.
Scale: It is less suitable for large-scale genomic studies.
Detection Limits: It may not detect low-frequency mutations or large structural variations.
High Accuracy: It provides highly accurate results, making it ideal for confirmatory testing.
Simplicity: The technique is well-established and straightforward to perform.
Reliability: It is less prone to errors compared to some high-throughput methods.
NGS can be used for initial screening, with Sanger sequencing employed for validation.
Microarray analysis can identify larger genomic changes, while Sanger sequencing can pinpoint specific mutations.
What is the Future of Sanger Sequencing in Pediatrics?
Although newer technologies are emerging, Sanger sequencing will continue to play a crucial role in pediatric genetic testing due to its reliability and accuracy. Ongoing advancements may further reduce costs and improve efficiency, ensuring its continued relevance in clinical practice.
