Frederick Sanger, the English biochemist, and his colleagues created Sanger sequencing in 1977. They designed it to determine nucleotide bases’ sequence in DNA pieces. Frederick and his team consider Sanger sequencing with about 99.9% base accuracy as a ‘gold standard’ that helps to validate sequences of DNA, including those that are sequenced through NGS (next-generation sequencing).
They also applied Sanger sequencing in the Human Genome Project to know the sequences of human DNA’s small fragments, which were then used in assembling bigger DNA and the entire chromosomes.
Why Sanger Sequencing?
The sequencing is a gold standard procedure that accurately detects small deletions/insertions and single variants of nucleotide. Compared to NGS, it’s also more flexible when it comes to testing specific familial variants.
In addition to that, the sequencing is cost-effective, especially when you need to urgently test single samples to ensure they don’t batch up in prenatal testing and parental carrier tests during pregnancy.
Sequencing Steps
- Mainly, there are six Sanger sequencing steps. These steps include the following:
- Denaturing dsDNA into ssDNA.
- Attaching primers that correspond to the sequence’s one end.
- Adding one type of ddNTP, four dNTPs, and four solutions of polymerase.
- Reaction of DNA synthesis starts and the chain extends until the termination of nucleotides are incorporated randomly.
- Denaturing the resulting fragments of DNA into ssDNA.
- Determining sequencing and separating denatured fragments through gel electrophoresis.
Applications
Researchers widely use Sanger sequencing for research purposes, such as detecting mutation and pharmacogenomics. In mutation detection, Sanger sequencing often plays an important role in detecting mutations that cause genetic diseases. It helps researchers and clinicians pinpoint mutations, which underlie various disorders, such as different types of cancer, sickle cell anemia, and cystic fibrosis. By detecting these changes, clinicians will understand disease mechanisms and develop targeted therapies. On the other hand, pharmacogenomics explores how the makeup genetic of people determines how they react to drugs. Through the procedure, clinicians can identify genetic variations, which affect adverse reactions, efficacy, and metabolism of drugs. Other applications include validating results from NGS studies, sequencing variable regions, and genotyping microsatellite markers.
NGS and Sanger Sequencing Working Together
Sanger sequencing complements next-generation sequencing in various ways. For instance, it can fill NGS data gaps, especially in hard-to-sequence places where the depth of coverage is low. It can also re-sequence and validate new approaches of NGS by analyzing samples with both techniques. By re-sequencing, clinicians can confirm the results of NGS in small but important genome sections.
Choosing Between NGS and Sanger Sequencing
With more than 99% accuracy, Sanger sequencing remains a ‘gold standard’ technique for both clinical and basic research applications. Many clinical labs depend on the method to validate variants of genes. However many recent studies support NGS accuracy for identifying variants and questioning redundant use of costly process.
In conclusion, researchers and clinicians should use both NGS and Sanger sequencing as complementary, and not rival technologies. It will be waste of resources to use next-generation sequencing for needs, which Sanger sequencing can meet and satisfy especially in situations where low throughput is still enough to get genomic data required to make necessary clinical decisions.
The post by Special to SouthFloridaReporter.com appears on South Florida Reporter.