Sanger sequencing, commonly known as the chain-termination method, transformed the field of genetics by providing a quick and dependable method for determining DNA sequence. Frederick Sanger invented this approach in 1977, which cleared the path for modern genetic research, such as the Human Genome Project and personalized treatment.
What is Sanger Sequencing?
Sanger sequencing is a method for decoding the nucleotide sequence of DNA. It employs a strategy based on the selective inclusion of chain-terminating dideoxynucleotides (ddNTPs) during DNA replication. This technique produces DNA fragments of varied lengths, which are then sorted and examined to determine the DNA sequence.
The Principle of Sanger Sequencing
Sanger sequencing depends on the selective inclusion of chain-terminating dideoxynucleotides (ddNTPs) during DNA synthesis. In this approach, DNA polymerase replicates a single-stranded DNA template in the presence of four standard deoxynucleotides (dNTPs) plus a minor amount of modified ddNTPs. These ddNTPs lack a hydroxyl group at the 3′ position, which prevents further nucleotide addition once they are incorporated into the developing DNA strand. This produces a succession of DNA pieces of varied lengths, each of which ends at a certain nucleotide. Labeling the ddNTPs with fluorescent or radioactive markers allows the fragments to be sorted and examined to identify the target DNA’s sequence.
The Working Mechanism of Sanger Sequencing
- The DNA sample to be sequenced is prepared and isolated, then converted into a single-stranded form.
- A short primer is added to bind to a specific starting point on the DNA template.
- A reaction mixture is set up, including DNA polymerase, standard nucleotides (dNTPs), and modified chain-terminating nucleotides (ddNTPs), each tagged with a fluorescent dye.
- DNA polymerase extends the primer by adding nucleotides to create a complementary DNA strand, but the incorporation of a ddNTP halts further extension.
- This process produces DNA fragments of varying lengths, each ending at a specific nucleotide due to random ddNTP incorporation.
- The DNA fragments are separated by size using capillary electrophoresis, with shorter fragments migrating faster than longer ones.
- As the fragments pass through a laser detector, the fluorescent tags on the ddNTPs emit light signals that are recorded.
The sequence is determined by analyzing the emitted signals and reconstructing the nucleotide order.
Illustration of Sanger Sequencing Through Examples
Sanger sequencing can be illustrated with a simple example. Suppose the target DNA template has the sequence 5’-ATCGTACGTA-3’. To sequence this, a primer complementary to the starting region of the template is added along with DNA polymerase, deoxynucleotides (dNTPs), and chain-terminating dideoxynucleotides (ddNTPs), each labeled with a unique fluorescent dye. During DNA synthesis, the polymerase adds dNTPs to extend the strand, but the random incorporation of a ddNTP halts the chain. This results in a collection of DNA fragments of varying lengths, each ending at a specific nucleotide. For instance, fragments might terminate after “A,” “T,” or “C,” creating a series of differently sized fragments. These fragments are separated by capillary electrophoresis, where a laser detects the fluorescently labeled ddNTPs. The resulting pattern of signals is analyzed to determine the sequence of the complementary strand, which reads as 5’-TACGTACGAT-3’.
Applications of Sanger Sequencing
Validation of NGS Results: Researchers often use Sanger sequencing to confirm findings from high-throughput sequencing experiments.
Small-Scale DNA Analysis: Ideal for sequencing single genes, small DNA fragments, or specific regions of interest.
Clinical Diagnostics: Used to identify genetic mutations linked to diseases, including cystic fibrosis and certain cancers.
Forensic Science: Helps in identifying individuals through DNA profiling.
Evolutionary Studies: Sanger sequencing aids in studying evolutionary relationships by comparing genetic material from different species.
Conclusion
Sanger sequencing exemplifies the power of innovative thinking in science. Its simplicity, accuracy, and versatility have made it an indispensable method for DNA analysis for decades. Whether you’re studying single genes or validating genomic data, Sanger sequencing continues to play a pivotal role in advancing our understanding of genetics.