DNA Sequencing

What is DNA?

DNA, entwined in a double helix structure resembling a spiral ladder, serves as the fundamental genetic material in all living organisms. Comprising four chemical letters—adenine (A), thymine (T), guanine (G), and cytosine (C)—this molecular script weaves together 6 billion letters (3 billion bases), encapsulating the entirety of life's information.

Deoxyribonucleic acid (DNA) stands as a crucial nucleic acid, carrying the genetic instructions vital for RNA and protein synthesis within living cells. It emerges as a biomolecule indispensable for the development and proper functioning of organisms. The sequence of bases in DNA's nucleotides forms the basis of genetic information. This information undergoes transcription to create RNA, and subsequently, the mRNA within it undergoes translation to generate polypeptides, constituting proteins.

DNA replication, a process preceding cell division, ensures the preservation of genetic information, averting its loss across generations. In eukaryotes, DNA resides in structures termed chromosomes within the nucleus. In organisms lacking a nucleus, DNA is present in chromosomes or other tissues (bacteria feature single-looped double-stranded DNA molecules, while viruses harbor DNA or RNA genomes). Chromatin proteins like histones, cohesins, and cohesins play a pivotal role in maintaining the ordered structure of DNA within chromosomes. These structures orchestrate the interplay between the genetic code and proteins responsible for transcription, exerting control over gene transcription.

Why Engage in DNA Sequencing?

• DNA sequencing serves the purpose of unraveling the intricacies of life, exploring the distinctions among organisms, and delving into the evolutionary and developmental histories of diverse life forms.
• This scientific technique plays a pivotal role in guiding the examination of recombinant DNA.
• Moreover, DNA sequencing holds significant practical importance in disease diagnosis and genotyping.

What is DNA Sequencing?

DNA sequencing technology stands at the forefront of genomics and ranks among the most extensively employed and crucial technologies in contemporary life sciences.

In 1953, Watson and Crick not only unveiled the double helix structure of DNA but, more significantly, underscored the significance of sequencing for deciphering the sequence of bases in DNA molecules.

The evolution of sequencer technology has traversed four distinct stages and achieved four breakthroughs, progressing from "pre-direct reading" to "direct reading," from manual processes to automation, from plate-based methods to capillary gel electrophoresis (marking the initial realization of large-scale sequencing), and eventually advancing to massively parallel sequencing.

4 Milestones in DNA Sequencing Technology

First Breakthrough: Direct Reading

• Pre-Direct Reading: The original sequencing method predating direct reading involved techniques such as molecular cloning, PAGE, and radiographic autoradiography.
• SBC Method: A chemical degradation reaction using specific reagents to directly degrade DNA molecules. Gilbert received the Nobel Prize in Chemistry in 1980 for inventing the SBC method.
• SBS Method: Based on enzymatic synthesis reaction and DNA synthesis, also known as the Sanger method or double deoxyribonucleic acid terminal termination method. This method offers advantages such as non-toxic reagents, ease of operation, stable results, high accuracy, and excellent reproducibility.

Second Breakthrough: Automation

• Pioneers of Automated Sequencing: Akiyoshi Wada, Wilhelm Ansorge.
• The key breakthrough in automating the SBS method came with Leroy Hood's invention of the automated four-color fluorescent-labeled SBS sequencer. This innovation played a crucial role in supporting and launching the Human Genome Project (HGP).

Third Breakthrough: Scale-Up

• The emergence and enhancement of capillary electrophoresis sequencing technology facilitated scale-up.
• In 1998, ABI introduced the ABIPrism 3700 capillary sequencer (ABI3700), enabling direct scaling up of sequencing technology. The subsequent introduction of the MegaBACE series of sequencers significantly contributed to the early completion of the HGP.

Fourth Breakthrough: Massively Parallel Sequencing

This represents a substantial leap forward, characterized by the ability to sequence DNA in a massively parallel manner, resulting in a rapid and simultaneous analysis of multiple fragments. One of the key features of sequencing is its significant contribution to the precipitous drop in sequencing costs.

Types of DNA Sequencing Technologies

• Sanger Sequencing

The initial era of DNA sequencing technology relied on either the strand termination method, introduced by Sanger and Coulson in 1975, or the chemical method involving strand degradation, pioneered by Maxam and Gilbert in 1976-1977. These techniques are commonly grouped together as the Sanger method for simplicity. A pivotal moment occurred in 1977 when Sanger successfully determined the first genome sequence—specifically, that of phage phiX-174, consisting of a mere 5,375 bases.

Please read our article:

Sanger Sequencing: Introduction, Workflow, and Applications.

Sanger Sequencing Q&A

• Next Generation Sequencing (NGS)

With the continual evolution and enhancement of genome projects, the first-generation sequencing faced limitations in throughput, speed, and cost, making it unsuitable for large-scale sequencing demands like deep sequencing and repeat sequencing. To address these challenges, the emergence of next-generation sequencing marked a transformative milestone. Over more than a decade of technological evolution and market dynamics, pioneering technologies such as Roche454 and ABI Solid, which once shone brightly, have gradually phased out.

Please read our article: What is Next Generation Sequencing (NGS)?

Presently, the landscape is dominated by mainstream NGS equipment manufacturers, including Illumina's Nextseq and MiniSeq series (recognized as the global mainstream NGS platform) and UWM's MGISEQ series and DNBSEQ sequencers. Notably, UWM's NGS platform continues to hold a competitive position.

The advancement and widespread adoption of these technologies have significantly expedited research endeavors in genome resequencing, large-scale genome sequencing, DNA methylation sequencing, transcriptome sequencing, targeted genomic region sequencing, detection of gene epigenetic modifications, microbial identification, and more. Notably, these innovations have effectively addressed the challenges posed by the high cost and low throughput associated with first-generation sequencing technologies. Their broad application extends to pathogen diagnosis, precision medicine, clinical tests, and molecular pathology, marking a transformative impact on diverse fields.

• Long-Read Sequencing

In recent years, researchers have pioneered the development of the third generation of sequencing technology—single molecule real time sequencing. This innovative approach aims to mine DNA sequence information with greater accuracy and efficiency. Unlike its predecessors, this technology eliminates the need for PCR amplification and relies on detecting electrical or chemical signals from individual DNA molecules, enabling the sequencing of each DNA molecule independently.

The current frontrunners in long-read sequencing platforms are Oxford Nanopore and PacBio.

• Nanopore Sequencing Technology

Oxford Nanopore Technologies has introduced single-molecule nanopore DNA sequencing, centered on electrical signal sequencing. The principle involves the passage of a single base or DNA molecule through a nanopore channel, causing discernible changes in the channel's electrical properties. The distinct chemical properties of the bases A, T, C, and G lead to varying electrical parameter changes during their passage through the nanopore. Detection of these changes enables the determination of the corresponding base type, ultimately revealing the DNA strand sequence. Notably, nanopore technology excels in directly detecting a larger number of bases, including methylation sites and different amino acids.

• PacBio Single-Molecule Real-Time (SMRT) Sequencing Technology

PacBio RS sequencing technology, a widely utilized core technology in long-read sequencing, is based on fluorescent labeling during synthesis. The principle involves capturing the DNA template with a polymerase, where four distinct fluorescently labeled deoxyribonucleoside triphosphates (dNTP) randomly enter the detection area and combine with the polymerase through Brownian motion. Bases matching the DNA template form chemical bonds, resulting in a longer duration of interaction compared to non-matching bases. This extended interaction facilitates the excitation of fluorescently labeled dNTP and allows for the detection of corresponding fluorescent signals. By identifying the emitted fluorescent groups at different wavelengths, the species of the extended bases can be determined, culminating in the sequencing of the DNA template after information processing.