Sanger Sequencing: Concept and Applications

Sanger sequencing, a pioneering molecular biology technology, has been instrumental in revealing the genetic code’s riddles. This technology, developed by Frederick Sanger in the late 1970s, revolutionized DNA sequencing and laid the groundwork for the Human Genome Project. Sanger sequencing, which allows scientists to decipher the precise order of nucleotides within a DNA strand, has become a crucial technique for understanding the genetic basis of life. In this article, we will look at the concepts, and applications of Sanger sequencing, as well as how it is still shaping the landscape of genomics and contributing to advances in various scientific disciplines.

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Prerequisites for Sanger Sequencing

To understand the technique of Sanger Sequencing, one must understand the importance of each element used in the technique. Here are the key prerequisites for Sanger Sequencing:

Sanger sequencing, while a powerful and widely used technique, does have certain prerequisites and requirements to ensure accurate and successful results. Here are some key prerequisites for Sanger sequencing:

1. High-Quality DNA Template:

Firstly, Sanger Sequencing requires a high-quality, purified DNA template. Contaminants or degraded DNA cannot be used because it can lead to unreliable results.

2. Primers:

Secondly, specific primers complementary to the regions of interest are necessary for initiating DNA synthesis during the sequencing reaction. A critical step in the process is designing appropriate primers.

3. DNA Polymerase:

A DNA polymerase enzyme is needed to carry out the replication of the DNA template. The polymerase should have proofreading activity to minimize errors in the synthesized DNA strands.

4. dNTPs (Deoxynucleotide Triphosphates):

Another prerequisite is regular deoxynucleotide triphosphates (dATP, dTTP, dCTP, dGTP) for DNA synthesis during the sequencing reaction.

5. Fluorescently labelled deoxynucleotide triphosphates (ddNTPs):

These are the most crucial components in this process of sequencing. Dideoxynucleotide triphosphates (ddATP, ddTTP, ddCTP, and ddGTP), each tagged with a distinct fluorescent dye, are required. They end DNA synthesis at specific places, resulting in several fragments. Their ability to do so is explained later in this article.

6. Thermal Cycler:

It is the instrument inside which the formation of further DNA fragments happens. During the sequencing reaction, a thermal cycler is employed to accomplish cycling reactions such as DNA denaturation, primer annealing, and DNA synthesis.

7. Capillary or gel electrophoresis equipment:

Either capillary electrophoresis or gel electrophoresis apparatus can separate the DNA fragments. These fragments are separated based on size, which allows for the determination of the sequence.

8. Analysis Software:

At last, specialized software is required to analyze the electropherogram data and determine the nucleotide sequence from the fluorescent signals.

How do ddNTPs work?

As we already know, DNA comprises four different nucleotides called deoxynucleotide triphosphates (dNTPs). To continue the chain during the replication process, DNA polymerase adds new bases (dATPs, dTTPs, dGTPs, dCTPs) to the DNA templates. The incoming dNTP’s phosphate group reacts with the bound dNTP’s ribose oxygen. This is how the chain continues.

However, the ddNTPs meaning “Dideoxynucleotide” have one less oxygen atom than dNTPs. They are missing the -OH (hydroxyl) group at 3′ carbon. This is where another nucleotide attaches to form a bond and continues a chain. Due to this absence of an -OH group, the next nucleotide cannot bind, resulting in the termination of the chain. Hence, these ddNTPs are also called chain-terminating Inhibitors. These ddNTPs are also labelled with a radioactive dye (as in Frederick Sanger’s experiment) or a fluorescent dye (used in modern-day sequencing).  

In the end, the result is several fragments of DNA with different lengths with their last nucleotide detectable by a fluorescence detecting software. 

The Basic Concept of Sanger Sequencing

The fundamental idea while sequencing is to create a complementary strand while chain-terminating dideoxynucleotides (ddNTPs) are present to ascertain a DNA strand’s nucleotide sequence.

This is a detailed description of the Sanger sequencing procedure:

Denaturation of DNA: It is the process used to produce single-stranded DNA by heating a double-stranded DNA template and separating its two complementary strands.

Primer Annealing: The single-stranded DNA is annealed to a brief DNA primer that is complementary to the template strand. The primer acts as the template for the production of DNA.

DNA Synthesis: Normal deoxynucleotides (dNTPs) and trace amounts of chain-terminating dideoxynucleotides (ddNTPs) are combined with DNA polymerase to start DNA synthesis. As we already know, chain termination results from these ddNTPs’ absence of the 3′-OH group required to insert the subsequent nucleotide.

Fragment Separation: The synthesis reactions are carried out in separate tubes, each containing a different ddNTP (A, T, C, or G). Different-length DNA strands are created as the polymerase incorporates the ddNTPs. These strands are also amplified in what is called “cycle sequencing” using PCR with Taq polymerase. 

Gel Electrophoresis (PAGE): Next, the artificial DNA fragments are sorted based on size using this technique. Smaller pieces can pass through the gel matrix more quickly thanks to the gel, creating a definite ladder-like structure of bands.

Detection: Autoradiography or fluorescence is commonly used to visualize the isolated DNA fragments. The location of the corresponding band on the gel indicates the identity of the terminating nucleotide (A, T, C, or G) at each position.

Reading the Sequence: Scientists can read the sequence by examining the band pattern on the gel.

Base Calling 

After the band pattern is obtained, how do scientists read the sequence? The term used for reading a DNA sequence is “base calling”. The DNA is read from 5′ to 3′ to call the bases. So we start with the shortest fragment first. In this case, it’s in the lane of the ddTTP, so the first nucleotide is a “T”. The next is in the ddGTP lane and thus is a “G”. You continue up the gel based on size to read the whole sequence. So, on this gel, it would read TGCATGCCA.

Why is Capillary Electrophoresis used in Modern-day Sequencing?

In capillary electrophoresis, a tiny quantity of gel is contained in a tiny tube. A laser at the other end detects the DNA after it is taken at one end and passed through the gel while an electric current is applied. Heat can escape through the fine tube used in capillary electrophoresis. Consequently, it is possible to apply a larger current without the gel overheating. Moreover, a greater current allows for a faster run time and improved resolution when running the gel. In 1989, Beckman Coulter introduced the first capillary electrophoresis device for sale. The ABI PRISM 310, a capillary-based Sanger sequencing machine, was made possible by this launch. With the introduction of this technique by Applied Biosystems in 1995, modern Sanger sequencing became possible.

Applications of Sanger Sequencing

For many years, Sanger sequencing has been a vital technique in molecular biology and genetics research. Among its most important uses are:

1. Sanger sequencing is mostly used to identify DNA fragments’ nucleotide sequences. Deciphering the genomes of different organisms, such as people, animals, plants, and microorganisms, has been made possible with its help.

2. Sanger sequencing is a widely used method for detecting mutations and changes in DNA sequences. It is especially helpful in the detection of genetic abnormalities, somatic mutations in cancer cells, and single nucleotide polymorphisms (SNPs) linked to disease susceptibility in clinical diagnostics.

3. It is also a tool that forensic scientists can use for DNA profiling and identification. Short tandem repeat (STR) loci and other genetic markers are sequenced using this method to produce DNA profiles that are utilized for paternity testing and criminal investigations.

4. The Sanger method is used to confirm the sequences of cloned DNA fragments or genes after gene cloning. In molecular cloning experiments, it guarantees that the appropriate DNA sequence has been introduced into a cloning vector or expression vector.

5. It is also a valuable tool for researching the genetic variety and evolution of viruses, including influenza, hepatitis, and HIV. This process is well-known as viral genotyping. All in all, it helps monitor the spread of viruses, comprehend treatment resistance mutations, and develop vaccinations.

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