DNA is the building block of life. It consists of a recurring sequence of four chemical bases. The arrangement of these bases encodes vital information necessary for various biological processes. Each of the basespairs exclusively with its respective “base pair”, adenine (A) pairs with thymine (T), while cytosine (C) pairs with guanine (G), forming stable bonds. This complementarity ensures the accuracy of DNA replication and transcription. In the human genome, approximately 3 billion base pairs are arranged, containing the instructions, which are essential for constructing and upholding a human organism.
Understanding the genetic diversity within populations enables healthcare professionals to develop diagnostic protocols that can be personalised. This approach aligns healthcare treatment with the genetic characteristics of each person, ensuring that they receive equitable and effective care. To achieve these results, it is essential to integrate these ideas with comprehensive scientific approaches.
One such actionable strategy is personalised medicine through genomic sequencing, allowing for customised drug therapies based on genetic profiles. DNA sequencing technology is a key method for personalised and equitable healthcare. It allows clinicians to identify genetic variations that influence disease susceptibility and treatment response. This enables targeted interventions based on an individual’s genetic profile, potentially minimising adverse reactions. Additionally, DNA sequencing aids in the early detection of genetic diseases, enabling the ability to proactively mitigate their impact.
British biochemist and Nobel laureate Frederick Sanger pioneered a groundbreaking DNA sequencing method using dideoxynucleotide triphosphates (ddNTPs). These special molecules stop DNA strands from growing, allowing scientists to pinpoint the order of nucleotides. By mixing ddNTPs with regular nucleotides and analysing the results through gel electrophoresis, researchers can determine the exact DNA sequence, with each lane on the gel revealing where the DNA synthesis was halted.
Sanger's DNA sequencing method relied on several key materials, each carefully selected for their specific properties and functionalities. One crucial component was the dideoxynucleotide triphosphates (ddNTPs), which were the chain-terminating nucleotides used to halt DNA synthesis. These ddNTPs were chemically modified versions of regular nucleotides, lacking a 3' hydroxyl group, which is essential for DNA polymerase to continue extending the DNA strand. In the late 1970s and early 1980s, the templates used for sequencing were single-stranded DNA (ssDNA) typically derived from fragments cloned into filamentous phage vectors. However, with the advent of PCR, the process evolved: PCR amplified products, generated from genomic DNA, became the new templates for sequencing. This shift simplified the template preparation process and allowed for more efficient sequencing.
Following Sanger's work, the next generation of sequencing developed, with a key player being Illumina, whose working principle was a ‘bridge amplification’. In this process, a single DNA fragment is attached to the surface of a silicon chip, initiating the bridge amplification process. Through this process, the DNA fragment is replicated into a cluster of many copies, forming a bridge-like structure with multiple copies extending outward from the chip surface. As sequencing begins, fluorescently labeled nucleotides are introduced one at a time, each labeled with a different fluorescent dye corresponding to one of the four DNA bases. These nucleotides are incorporated into the growing DNA strands, and the fluorescence signal emitted by each nucleotide is detected by a camera. This sequential addition of nucleotides allows for the determination of the DNA sequence base by base.
First and second-generation sequencing technologies made significant progress in the field of DNA sequencing with high accuracy. However, both Sanger’s sequencing and Next-Generation Sequencing(NGS) simplification failed to provide act quickly leading to slower rates of sequencing. Additionally, these technologies could not read longer sequences of entire DNA strands. This led to the development of third generation technologies.
The idea of single-molecule DNA sequencing evolved in the 1990s to address these challenges. Oxford Nanopore Technologies (ONT) developed one such method of nanopore sequencing that could read ultra‐long, single-stranded DNA and RNA molecules at a lower cost. This application provided a more portable and efficient method of sequencing DNA.
Nanopore sequencing decodes the DNA sequence using a very small, nanoscale protein pore that acts as a biosensor. This pore is a tiny hole embedded in an electrically resistant polymer membrane and immersed in a solution that conducts electricity. Each DNA molecule is made of different combinations of building blocks, called nucleotides which are negatively charged. Due to this charge, in the presence of electric current, the DNA molecules move through the pore from the negative side towards the positive side. The nanopore contains a motor protein that controls how fast the DNA strand moves through the pore. As the DNA strand passes through the nanopore, each nucleotide changes the electric current differently. These changes in current act as a fingerprint to uniquely identify the nucleotide present in the DNA strand one molecule at a time. Computer programs are further used to combine these readings and decode the overall genetic sequence in real-time.
One significant contribution of DNA sequencing to the medical industry is its role in cancer treatment. DNA sequencing enables identification of specific mutations within a cancer patient's tumour DNA, allowing for the selection of targeted therapies. For example, certain mutations in genes like EGFR or BRAF are associated with specific types of cancer and may respond better to particular drugs. Without DNA sequencing, identifying these mutations and prescribing the most effective treatment would be challenging. This personalized approach to cancer treatment has led to improved patient outcomes, with higher response rates.
Nanopore sequencing emerged as a crucial tool in combating the COVID-19 pandemic. Its real-time capabilities enabled rapid detection of genomic mutations in the virus and understanding spread patterns. Due to its ability to perform ultra-long reads, nanopore sequencing reduced the risk of errors in detecting the presence of the virus. Unlike traditional diagnosis methods for COVID-19 such as RT-PCR, this technology provided higher accuracy of results with a smaller number of false positives and negatives. The portability, high accuracy, and low cost of nanopore sequencing make it particularly well suited for monitoring the spread and mutations of the COVID‐19 in different countries and regions.
DNA sequencing, particularly through innovative methods like nanopore technology, has revolutionised the field of disease diagnostics and medical research. It offers promising personalised healthcare solutions to health challenges like the COVID-19 pandemic and cancer treatment. DNA sequencing evolved from Sanger’s sequencing, to the NGS bridge amplification technique and finally nanopore sequencing to address major challenges such as the need for high throughput, longer sequence reads, and portability. By enabling ultra-long reads and real-time analysis, nanopore sequencing has overcome limitations of previous generation sequencing technologies.
Nanopore technology promises versatility, lower cost, and efficient performance. However, methods to slow down DNA translocation through the nanopore for better control need to be researched and implemented. Overcoming this challenge will make nanopore sequencing a pivotal technology for personalised disease diagnosis and precision medicine.
Jia Bhargava and Tanay Srinivasa are BTech students at Plaksha University, Mohali.
