A Beginner’s Guide To Sanger Sequencing

In 1977, an English biochemist Frederick Sanger with his colleagues invented another proficient method of DNA sequencing called Sanger sequencing. The goal of this method of gene sequencing is to determine the sequence of the nucleotide bases in a DNA strand. The length of the DNA strands that are the target in these types of bimolecular reactions is about 1,000 bp. Sanger sequencing is known to deliver 99.99% base accuracy that is crucial for optimum validation in the field of genetics. It is considered the “gold standard” when the job is to understand how the genes carry out information (The Genomic Services Company, 2020).

How does Sanger Sequencing work?

There are two ways you can carry out sanger sequencing. Either manually or by using a sequencing machine. Performing sanger sequencing via automated equipment is more common than the manual approach.

The following are the steps that take place in typical Sanger sequencing.

Step # 1 DNA sequence for chain termination PCR

The first and foremost step in automated Sanger sequencing is the production of a DNA template for a distinct type of PCR, known as chain termination PCR. The working of chain termination PCR is similar to standard PCR, but with only one substantial difference; the use of di deoxyribonucleotides (ddNTPs) that are a type of modified nucleotides (dNTPs). The dNTPs addition to a growing DNA strand by the DNA polymerase catalyzes the formation of a phosphodiester bond between the adjacent nucleotides.

In a basic chain termination PCR, the mixing of a lower ratio of chain-terminating ddNTPs and a higher concentration of normal dNTPs occurs. For the formation of a phosphodiester bond, the presence of a 3’OH- group is necessary, but ddNTPs lack them. Therefore, when a DNA polymerase incorporates a ddNTP into the mix, extension ceases. The result of a chain termination PCR is the formation of billion copies of single-stranded nucleotide copies of the template DNA sequence.

Manual set-up: In a manual set-up, four PCR reactions are arranged. Each one has only a single type of ddNTP in the mix. The choices include; ddGTP, ddATP, ddTTP, and ddCTP.

Automated set-up: In an automated set-up, all types of ddNTPs are incorporated in a single reaction. However, each ddNTP carries a distinct fluorescent label.

Step # 2 Size grading via gel electrophoresis

The second step of sanger sequencing revolves around the separation of chain-terminated oligonucleotides via gel electrophoresis based on size. In this step, the DNA samples are loaded into one end of a gel matrix. The mixture is subjected to an electric current. Since DNA carries a negative charge, the DNA samples will be attracted to the opposite end. As these gene molecules have the same unit per mass, the only way to gauge their size is to analyze the speed with which they move. Molecules that are smaller in size will face less friction when they are on the move. Thus, the DNA fragments that move faster and smoother of all in the gel, the smaller will be their size. With this technique, the oligonucleotides can be arranged from smallest to largest by reading the gel from top to bottom.

Manual set-up: The speed of oligonucleotides is analyzed in all the four PCR reacting by making them run on a gel matrix in four separate lanes. This step is vital to know which oligonucleotides correspond to which ddNTP.

Automated set-up: Since all ddNTPs are mixed in a single reaction, all oligonucleotides are made to run on a single gel electrophoresis machine.

Step # 3 Gel reading and analysis of DNA sequence

In the last step, the only job left is the analysis of the gel for the determination of resultant DNA. DNA polymerase can only synthesize DNA in the 5’ and 3’ direction, starting from the region where the primer gets incorporated. Hence, each ddNTP will correspond to the complementary base that was present in the original DNA sequence. Therefore, if the user reads the gel band from smallest to largest, the determination of the sequence becomes easier.

Manual set-up: The user analyses the lanes of all four reaction mixes by reading the gel band from top to bottom.

Automated set-up: The computer reads the band itself and pinpoints the sequence of the DNA. (Sanger, 1977, 1563-1567)

Advantages of Sanger Sequencing

Even with the development of modern DNA sequencing technologies like next-generation DNA sequencing (NGS) and third-generation sequencing, Sanger sequencing is still used extensively in laboratories due to many advantages. Those are:

1. Cost-effective.
2. 99.99% accuracy, which is vital for cloning operations and mutagenesis.
3. Target several small genomic regions belonging to a large number of samples.
4.  Carry out the sequencing of a variety of regions.
5. Validation and confirmation of the accuracy of results taken from next-generation DNA sequencing.
6. Part of HLA typing.
7. Takes part in the genotyping of microsatellite markers.
8. Identification of single disease-causing viruses. (The Genomic Services Company, 2020).

Sanger Sequencing VS Next Generation DNA Sequencing

The field of genetics is a discipline that is the most important for the conservation of life. The more knowledge we have in hands about how our cells work, the better we can tackle issues like epidemics, cure of viral diseases, and extinction of living forms. However, the development in the field of DNA technology technologies has taken new heights in the department of genomic research. Even with low input DNA, NGS can simultaneously read and copy 100 genes and entire genomes smoothly.

Nevertheless, next-generation DNA sequencing is now considered a competitor technique in the field of genetics. It is preferred when the objectives of the sequencing are:

1. Analysis of experimental samples with low input DNA.
2. Cost-efficient.
3. Interrogation of more than a hundred genes at one time.
4. Isolation of distinct and commercially crucial variants by elevating the number of target regions per run.
5. Sequencing of entire genomes, especially microbial genomes that plays a substantial role in studying the microbial activity of many bacterias.

The bottom line is that both of these DNA sequencing approaches are vital for advanced genetic studies and play individual roles that contribute to making understanding living species easier. (Sikkema-Raddatz, 2013).

References

The Genomic Services Company. (2020, February 21). Sanger Sequencing: Introduction, Principle, and Protocol. CD Genomics. https://www.cd-genomics.com/blog/sanger-sequencing-introduction-principle-and-protocol/Sanger, F. (1977, December). DNA sequencing with chain terminator inhibitors. Proceedings of the National Academy of Sciences of Sciences of the United States of America, 74(USA), 5463-5467.Sikkema-Raddatz, B. (2013, April 8th). Targeted Next‐Generation Sequencing can Replace Sanger Sequencing in Clinical Diagnostics. Human Mutation, 34.7(Netherlands).