What the Most Common Applications of Sanger Sequencing?
Sanger sequencing is a DNA sequencing technique that hinges on the incorporation of chain-terminating dideoxynucleotides during in vitro DNA replication, often guided by DNA polymerase action. Frederick Sanger as well as his team pioneered the technique in 1977, which is now important in DNA polymerase reaction.
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How It Works
In order to determine which nucleotides are incorporated in the nucleotide chain, four ddNTPs often labeled with different fluorescent dye are utilized to terminate the reaction process of synthesis. Compared to dNTP, ddNTP has an oxygen atom, which is removed from a ribonucleotide. Following the synthesis process, the products’ reaction are usually loaded into four different lanes of a gel, based on different chain-terminating nucleotides and get subjected to the process of gel electrophoresis.
Ingredients
The technique involves making numerous copies of target DNA regions. Ingredients used in Sanger sequencing are the same as those required for replication of DNA in organisms or for PCR (polymerase chain reaction) that copies DNA in vitro. Some of these ingredients are not limited to the following:
- DNA templates to be sequenced
- Four nucleotides of DNA, including dGTP, dCTP, dTTP, and dATP
- Primers, which are short pieces of a single-strand DNA binds to DNA templates as well as serves as the starter for polymerase.
- Chain-terminating or dideoxy versions of nucleotides (ddGTP, ddCTP, ddTTP, and ddATP), all labeled with different dye colors.
Applications
The technique remains an accurate method of sequencing. It is widely used, especially in the clinical labs for various applications, including testing for specific familial sequence variants and diagnostic sequencing of genes. In addition, it can also be used to fill NGS data’s gaps and confirm variants that the NGS has identified. Other applications include the following:
1. Pharmacogenomics
Well, pharmacogenomics explore how genetic makeup of individual influences drugs’ response. Sanger sequencing helps to identify genetic variations, which affect metabolism, adverse reactions, and efficacy of drugs. This data guides personalized plans for treatment and minimizes the risks associated with drug events.
2. Genetic Diversity Study
The technique greatly contributes to the study of genetic diversity in the population. It often helps scientists to analyze chloroplast and mitochondrial DNA to infer evolutionary relationships, migration patterns, and population histories.
3. Forensic Analysis
The method helps in forensic investigation by analyzing samples of DNA collected from a crime scene. It helps to identify people through DNA fingerprinting that involves sequencing of very specific genome regions with a high variability, like STRs (short tandem repeats).
4. Mutation Detection
Sanger plays an important role in identifying all the mutations liable for genetic illnesses. It allows researchers and clinicians to pinpoint mutations, which underlie disorders, such as sickle cell anemia, cystic fibrosis, and different types of cancer.
In conclusion, Sanger sequencing remains a gold standard in the technology of sequencing. It doesn’t just guarantee long-read abilities. It is also offers a high-degree of accuracy and flexibility to support different applications in research fields. These applications include detecting mutations, conducting forensic investigations, doing genetic diversity studies, and determining genetic variations, to name a few.
