The Power Behind Genetic Science: What is Genome Sequencing?

Gene sequencing technologies are one of the most important tools in basic genetic research, and modern genetic studies have been able to make significant progress thanks to gene sequencing techniques. Gene sequencing is the process of determining the nucleotide sequence (A-T-G-C) of nucleic acids (DNA-RNA). This sequencing information is essential for a wide range of applications, including investigating the causes of genetic diseases, detecting mutations, identifying regions involved in protein synthesis, comparing genomes, predicting diseases early, pinpointing genetic control regions, and resolving forensic cases.

DNA Sequencing Technologies

The foundations of gene sequencing technologies were laid by Allan Maxam-Walter Gilbert, and Frederick Sanger in the 1970s. Over time, advancements led to the development of next-generation sequencing (NGS) and third-generation sequencing technologies.

Maxam-Gilber Sequencing and Sanger Sequencing

The Maxam-Gilbert method, also known as chemical DNA sequencing, was developed by Allan Maxam and Walter Gilbert, earning them the Nobel Prize in Chemistry in 1980. This method begins with the separation of the DNA chain and radioactive labeling of DNA fragments. The DNA fragments are then exposed to specific chemicals that cut the DNA at designated points in four separate tubes, resulting in DNA fragments of varying lengths in each tube. These fragments are separated by size through electrophoresis, and the sequence is determined by combining overlapping fragments using autoradiography. This method is less commonly used today due to its complexity and the use of hazardous chemicals.

Frederick Sanger’s sequencing technique (chain termination method) also won a Nobel Prize. This method is based on the DNA synthesis process with a slight modification. In this method, primers, nucleotides (dNTPs), DNA polymerase, and DNA sample -essentially all the materials required for the PCR reaction, are placed in four reaction tubes. Additionally, a different type of modified dNTPs, known as ddNTPs, is added to each tube. (for example, ddNTP of Adenine base to the first tube, ddNTP of Thymine to the second tube.). DNA elongation occurs at the 3'OH end, but ddNTPs have a 3'H end instead of a 3'OH end. As a result, when the DNA polymerase encounters a ddNTP during nucleotide insertion, the reaction cannot continue. Afterward, the resulting DNA fragments are separated by size using gel electrophoresis, and the gene sequence is determined by analyzing these fragments. Today, all reactions can be performed in a single tube with the aid of color labeling of the bases and advanced detection systems. Furthermore, with advancements in computer technology, manual gel electrophoresis is no longer necessary; instead, capillary electrophoresis connected to computers is used. This allows for automatic gene sequencing by detecting different wavelengths of bases as the sample passes through the tube. Sanger sequencing remains widely used for small-scale sequencing or for verifying results obtained with other technologies due to its high accuracy.

Next-Generation Sequencing (NGS)- Illumina and Ion Torrent

While the Sanger method significantly advanced sequencing, newer techniques were developed to shorten sequencing times and reduce costs. Despite their differences, these techniques share several common steps: sample preparation (including DNA isolation, fragmentation, and adapter addition), cluster creation, and result analysis.

In Illumina technology, the process begins with the separation of double DNA strands, followed by the attachment of small known sequences called adapters to the ends of the DNA chains. These prepared samples are then transferred to a chip, known as a flow cell, which has many wells on its surface. Each well contains short oligonucleotide sequences complementary to the adapter sequences. The sample is spread across the chip, where adapters bind to the complementary oligonucleotides, thus attaching the DNA to the surface. A PCR reaction is then performed on this chip. In addition to the standard components, modified ddNTPs are used in the PCR. These ddNTPs have their 3'OH group blocked and are labeled with fluorescent dyes. When DNA encounters a ddNTP, elongation stops, and fluorescence is emitted. By detecting this fluorescence, multiple sequences are obtained. Finally, a computer analyzes these sequences, combines overlapping sections, and determines the final sequence.

DNA Sequencing

In Ion Torrent technology, sequencing is performed by detecting the release of H+ ions during the sequencing reaction. The system uses a semiconductor chip with wells containing special beads. DNA is attached to these beads, and a PCR reaction is carried out. Nucleotides are added one by one in sequence. As the reaction proceeds, the chip measures voltage changes caused by the release of H+ ions.

Third Generation Sequencing- Nanopore and PacBio

Sequencing technologies are continually advancing, leading to the development of new methods known as third-generation sequencing. Currently, Nanopore and PacBio are among the most prominent technologies in this technology.

Nanopore technology relies on using a nanoscale pore to read changes in electrical current as DNA (or RNA) passes through the pore. The key advantage of this method over previous sequencing technologies is that it does not require a PCR reaction. The pore can be produced either biologically or synthetically, and features such as size, shape, and electrical conductivity are important factors for sequencing. Enzymes or electrophoresis may be used to guide the DNA fragment to the pore. Once the DNA is attached to the pore, its negative charge pulls it toward the positive side, allowing DNA bases to pass through one by one. Each base (A-T-G-C-U) induces a distinct voltage change due to its unique chemical structure. Detectors measure these voltage changes to determine the DNA sequence. One of the major advantages of this technology is its ability to perform real-time sequencing.

In PacBio sequencing technology, adapters are added to the ends of isolated DNA to form a ring-shaped structure, which is then transferred to a surface known as the SMRT cell. This SMRT cell contains a large number of specially designed wells, each with DNA polymerase attached. The DNA sample is immobilized in these wells, and nucleotides labeled with fluorescent dyes are introduced. As DNA is synthesized, the labeled nucleotides emit fluorescence, which is recorded to determine the DNA sequence.

This brief summary highlights the evolution and diversity of sequencing technologies, from the pioneering methods of Maxam-Gilbert and Sanger to the advanced capabilities of next-generation and third-generation sequencing. For more detailed information and videos, please refer to the provided links. Each technique has made unique contributions to the field, enhancing our ability to understand genetic information with increasing speed and accuracy. Ongoing advancements in sequencing technologies continue to drive significant progress in genetic research, leading to breakthroughs in disease understanding, personalized medicine, and beyond.

References

1)Maxam, A. M., & Gilbert, W. (1977). A new method for sequencing DNA. Proceedings of the National Academy of Sciences of the United States of America, 74(2), 560–564. https://doi.org/10.1073/pnas.74.2.560

2)Sanger, F., Nicklen, S., & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America, 74(12), 5463–5467. https://doi.org/10.1073/pnas.74.12.5463

3)An Introduction to Genetic Engineering Third Edition Desmond S. T. Nicholl

4)Clark, D. P., Pazdernik, N. J., & McGehee, M. R. (2019). Molecular biology (Third edition.). London ; San Diego, CA: Elsevier/AcademicPress.