DNA Sequencing- Definition, Principle, Steps, Types, Uses

The genome of all living organisms, which is made up of long strands of deoxyribonucleic acids (DNA), is encoded with the basic information required for the development and function of the organism. The order of the nucleotide bases arranged in our DNA contains the biological instructions and makes us what we are. Therefore, it is essential to determine the sequence of a gene or the genome.

DNA sequencing can be defined as the method that allows the identification of the exact order or sequence of the nucleotide bases – adenine (A), guanine (G), cytosine (C), and thymine (T) in the DNA molecule.

DNA Sequencing Applications

There are various applications of DNA sequencing in different fields, including:

DNA sequencing has helped to obtain detailed information about the genetic makeup of living organisms. It has also helped to discover new genes and understand their function.
It can be used as a diagnostic tool to identify genetic variations associated with diseases, which helps in the development of new therapeutic tools.
It can also be used in evolutionary studies to study the relationship between different species and understand evolutionary processes.
It can be used in environmental studies to study the diversity of life which aids in better understanding and protecting biodiversity.
It can also be used in criminal investigations to analyze DNA evidence and identify suspects.
It is a fundamental aspect of biological research and is utilized in diverse fields such as biotechnology, virology, forensic biology, and medical diagnostics.

DNA Sequencing Types and Methods

On the basis of their timeline of development, characteristics, and outputs, DNA sequencing technologies can be classified into several generations.

However, it should be noted that the boundaries between the different generations of sequencing are not always well-defined, and there can be significant overlap between them. With new advancements and innovations, these boundaries may change.

First Generation Sequencing

Sanger Sequencing

Sanger sequencing, also known as enzymatic DNA sequencing or dideoxy sequencing, was developed by Frederick Sanger and his team in 1977. This sequencing method is based on chain termination by dideoxynucleotides (ddNTPs). The ddNTPs are a third form of ribose in which both hydroxyl group is missing.

Sanger Sequencing Working Principle

At first, the double-stranded DNA (dsDNA) fragment of interest is isolated and denatured into single strands (ssDNA). Then, a labeled primer complementary to the template DNA is attached.
The template DNA annealed with primer is added to four separate sequencing reactions that also contain DNA polymerase, the four deoxynucleotides (A, T, C, and G), and a different dideoxynucleotide (ddNTP) in each reaction.
Whenever a ddNTP is added, the chain terminates as no phosphodiester bond can be formed by DNA polymerase. This generates DNA fragments of different lengths that end in a chain-terminating base.
Then, the reaction products are loaded into four lanes of a single gel, and gel electrophoresis is performed. The DNA fragments are separated according to their sizes.

An automated version of the classical Sanger sequencing method includes using fluorescently labeled bases and detection using capillary electrophoresis. Sequencing has become significantly faster with this improvement.

Advantages of Sanger sequencing

Sanger sequencing is a well-established technology as it has been widely used for several decades.
Data from sanger sequencing is relatively easy to analyze.
It is less reliant on computational tools.
It is fast and cost-effective for low numbers of targets

Limitations of Sanger sequencing

It is time-consuming.
It is not cost-effective when compared to newer sequencing technologies.
It is unsuitable for high-throughput applications.
It requires high-quality DNA.
It requires higher amounts of sample input.

Maxam-gilbert Sequencing

The Maxam-Gilbert method, also known as the chemical degradation DNA sequencing method, uses chemicals to generate base-specific cleavages of the DNA to be sequenced. It was developed by Allan Maxam and Walter Gilbert in 1977. Like Sanger sequencing, it was one of the earliest methods for sequencing DNA. However, due to the use of toxic chemicals and a labor-intensive method, it was less often used.

Maxam-gilbert Sequencing Working Principle

It works by breaking the DNA strands at specific positions using a series of chemical reactions.
At first, the DNA to be sequenced is purified and labeled with a radioactive (³²P) or fluorescent marker at one end.
Next, the single-stranded labeled DNA template is separated and used for base-specific reactions.
Four base-specific reactions are set up: G, G+A, C, and C+T. Specific chemicals are added to each tube.
Then, these reactions are run in sequencing gel, and the DNA sequence is determined from the autoradiogram.

Advantages of Maxam-Gilbert sequencing

Template DNA can be used for sequencing directly.
It is useful in studying methylation of DNA and genetic imprinting,
It can sequence longer stretches of DNA compared to Sanger sequencing.

Limitations of Maxam-Gilbert sequencing

It uses hazardous chemicals.
It is a time-consuming and labor-intensive method.
It cannot be easily automated.

Second Generation Sequencing

Next-Generation Sequencing (NGS)

Second-generation sequencing methods, also known as Next-Generation Sequencing (NGS), are a group of automated DNA sequencing technologies used for rapid DNA sequencing. The key characteristic of NGS is the rapid and high-throughput sequencing of large quantities of DNA.

Several different methods of second-generation sequencing have been developed. Some are briefly explained below:

454 pyrosequencing

454 pyrosequencing is one of the first second-generation sequencing methods that is based on pyrosequencing. In this method, the DNA fragment of interest is attached with adaptors, and nucleotides are added to the growing DNA strand. This hybridization will release a pyrophosphate molecule that generates a light signal. This signal is detected by a camera and is used to determine the sequence of the DNA fragment.

Illumina sequencing

Illumina sequencing is the most widely used second-generation sequencing technology. In this method, DNA fragments are first bound to a solid surface and amplified using PCR. Next, fluorescently labeled nucleotides are added and the sequence of the DNA fragments is determined by analyzing the location of the labeled nucleotides.

Ion Torrent sequencing

Ion Torrent sequencing is the method of sequencing that detects the hydrogen ions released during DNA synthesis. Unlike the other two methods, this method does not use light signals. It measures the direct release of H+ ions when individual bases are added by DNA polymerase. The order of hydrogen ion release determines the sequence of the DNA fragment.

Advantages of NGS

It has high sensitivity.
It can process millions of sequencing reactions in parallel.
It has significantly reduced the cost of sequencing.

Limitations of NGS

The high-throughput nature can result in a higher error rate.
Although the cost of NGS has reduced considerably over time, it is still comparatively costly.
It produces a large amount of data which is difficult to analyze and interpret.

Third Generation Sequencing

Third-generation sequencing (TGS), also known as large fragment single molecule sequencing, is a real-time sequencing method used to directly sequence DNA molecules without the need for template amplification. It produces longer sequencing reads than earlier generations of sequencing and hence is also known as long-read sequencing.

SMRT (Single Molecule, Real-Time) sequencing

SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences (PacBio) is the most widely used example of third-generation sequencing.

SMRT working principle

SMRT sequencing reads the sequence of a DNA fragment using the SMRT chip containing thousands of zero-mode waveguides (ZMW). A ZMW is a tiny chamber that helps to focus light energy into a really small area.
A single DNA polymerase is attached to the base of ZMW along with the target DNA and the four nucleotides labeled with specific fluorescent dyes.
When a nucleotide is added to the growing DNA strand, it generates a fluorescent signal.
This signal is analyzed by the SMRT cell and thus can determine the DNA sequence.

Fourth Generation Sequencing

Fourth-generation sequencing methods are emerging sequencing technologies that are able to read repeated or difficult-to-sequence regions of the genome, such as highly repetitive DNA, centromeres, and telomeres. They are based on new ways of preparing, handling, and reading DNA.

Nanopore sequencing

Examples of fourth-generation sequencing technologies include synthetic nanopore sequencing, molecular inversion probes (MIPs), and in situ sequencing.

Nanopore sequencing working principle

Nanopore sequencing, developed by Oxford Nanopore Technologies (ONT), is a portable sequencer that can generate sequencing data in real time.
This method uses flow cells that contain tiny nanopores embedded in a membrane that detect changes in electrical current when any molecule passes through it.
The sample DNA fragments are passed through the nanopore which creates fluctuations in the electric current.
The electrical signals are converted to base calls and thus the DNA sequence is determined.

Some of the famous DNA Sequencers

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References

DNA Sequencing Fact Sheet (genome.gov)
DNA Sequencing: Definition, Importance, Methods and More (praxilabs.com)
DNA Sequencing: Definition, Methods, and Applications | CD Genomics Blog (cd-genomics.com)
Griffiths, University Anthony J F, Wessler, University Susan R, Carroll, D. S. B., & Doebley, J. (2015). Introduction to genetic analysis (11th ed.). W.H. Freeman.
How nanopore sequencing works (nanoporetech.com)
Hoy, M. A. (2013). DNA Sequencing and the Evolution of the “-Omics.” Insect Molecular Genetics, 251–305. doi:10.1016/b978-0-12-415874-0.00007-x
McGinn, S., & Gut, I. G. (2013). DNA sequencing – spanning the generations. New Biotechnology, 30(4), 366–372. doi:10.1016/j.nbt.2012.11.012
Morey, M., Fernández-Marmiesse, A., Castiñeiras, D., Fraga, J. M., Couce, M. L., & Cocho, J. A. (2013). A glimpse into past, present, and future DNA sequencing. Molecular Genetics and Metabolism, 110(1-2), 3–24. doi:10.1016/j.ymgme.2013.04.024
Nanopore DNA Sequencing (genome.gov)
Saraswathy, N., & Ramalingam, P. (2011). DNA sequencing methods. Concepts and Techniques in Genomics and Proteomics, 57–76. doi:10.1533/9781908818058.57
Slatko, B. E., Gardner, A. F., & Ausubel, F. M. (2018). Overview of Next Generation Sequencing Technologies. Current protocols in molecular biology, 122(1), e59. https://doi.org/10.1002/cpmb.59
What is Next Generation DNA Sequencing? | Functional genomics II (ebi.ac.uk)
What is Next-Generation Sequencing? | Thermo Fisher Scientific – NP