Unravelling the Secrets of DNA: The Sanger Sequencing Method
Sanger sequencing, also known as dideoxy sequencing, is a method of DNA sequencing that was developed by Frederick Sanger in 1977. It is based on the principle of chain termination, in which DNA synthesis is interrupted at specific nucleotides by the incorporation of modified nucleotides, known as dideoxy nucleotides.
The Sanger sequencing method is based on the concept of DNA replication, in which a template strand of DNA is used to synthesize a complementary strand. The process involves using a specific type of DNA polymerase enzyme to synthesize a new strand of DNA from a template strand.
The process of Sanger sequencing can be broken down into several key steps:
- Sample preparation:
The first step in Sanger sequencing is to prepare the DNA sample for analysis. This involves isolating the DNA of interest from other cellular components and then performing a polymerase chain reaction (PCR) to amplify the DNA of interest.
2. Primer extension:
The next step is primer extension, which is the process of adding new nucleotides to the 3' end of a primer sequence. This is done using a DNA polymerase, such as Taq polymerase, and one of four modified nucleotides (ddATP, ddCTP, ddGTP, or ddTTP) known as dideoxy nucleotides.
3. Chain termination:
The incorporation of a dideoxy nucleotide causes the chain termination as the nucleotides lacks the 3'OH required for polymerase to add more nucleotides. This causes the formation of several DNA fragments of different lengths, corresponding to the different locations where the dideoxy nucleotides are incorporated.
4. Size separation:
The DNA fragments that are generated during chain termination are then separated by size through gel electrophoresis, which separates the DNA based on its charge and size, where the smaller fragments travel faster through the gel than the larger fragment.
5. Detection:
After size separation, the fragments are visualized by staining with ethidium bromide and UV illumination. The resulting pattern of bands, which corresponds to the sequence of the DNA, can then be read to determine the exact order of nucleotides in the original DNA molecule.
6.Data analysis:
The final step of the Sanger sequencing process is data analysis, in which the raw data generated by the sequencing reactions is analyzed to determine the exact sequence of the DNA. This involves using computer software to align the sequences and identify any variations or mutations in the sample.
One of the key applications of Sanger sequencing is in determining the exact order of nucleotides in a piece of DNA. This is important in many areas of biology, including genetics, evolutionary biology and biotechnology. Sanger sequencing can be used to identify mutations in genes associated with genetic disorders, to study the evolution of different species, and to analyze the genome of microorganisms for biotechnology applications.
Another important application of Sanger sequencing is in the field of gene expression analysis. This technique, known as quantitative real-time PCR (qRT-PCR), and allows to quantify the expression of specific genes in different samples by determining the number of copies of a particular segment of DNA in the sample. In addition, Sanger sequencing can also be used to determine the sequence of small sections of DNA, making it useful for applications such as DNA fingerprinting, genetic testing, and the detection of genetic disorders.
The main disadvantage of Sanger sequencing is that it is relatively slow and labor-intensive compared to newer sequencing methods, such as next-generation sequencing (NGS). Another disadvantage is that it is limited in the amount of DNA it can sequence at one time, making it less useful for large-scale sequencing projects.
Overall, Sanger sequencing is a powerful tool that allows researchers to determine the exact order of nucleotides in a DNA molecule with high accuracy. Despite its relatively low throughput, it is still considered a go-to method for sequencing of small region of DNA. It continues to be widely used and is constantly being improved for the better sensitivity and specific identification of genetic variations.
