How Does DNA Sequencing Work and Why Does It Matter?
The science of a specific gene and its interactions with one another and the surroundings is known as genomics. The functioning of genomes is sequenced, assembled, and analyzed using a mix of recombinant DNA, DNA sequencing techniques, and DNA sequence analysis. It evaluates an individual's full gene sequencing set rather than just one gene or gene output.
What is DNA Sequencing?
DNA sequencing is another term for genome sequencing. Let's have a closer glance at what genome sequencing entails.
The term "sequencing" simply refers to the process of determining the particular order in which the nucleotides sequencing in a strand of DNA is placed. Scientists do not need to record the two bases in a pair since they exist in pairs and the character of one of the bases in the pair determines the other person from the pair.
DNA polymerase (the enzyme in organisms that synthesizes DNA) is used to produce another strand of DNA using a thread of interest in the most extensively used type of decoding nowadays, termed sequencing by synthesis. The enzymes integrate a single nucleotide that was intentionally tagged with a fluorescence mark into the new DNA strand during the sequencing reaction. A light source excites the nucleotide sequencing, which causes a fluorescent signal to be released.
Specialists must examine the nucleotide sequence of covered portions to gather the sequence of a large number of nucleotides in a large piece of DNA, such as a genome. This allows the longer sequence to be assembled from small parts in a similar way to a sequential puzzle piece. Each foundation should be reviewed once in this DNA sequencing technique, but at least a couple of instances in the wrapping parts to ensure correctness.
DNA sequencing may be used by researchers to seek for genetic variants and abnormalities that may have a role in the course of occurrences or the progression of an illness. The illness alteration might be as minor as a single base pair substitution, deletion, or insertion, or as large as a loss of hundreds of bases.
What are DNA Sequencing Methods?
Frederick Sanger, an English scientist, discovered Sanger sequencing in the 1970s. The Sanger method is a traditional DNA sequencing method that prevents the addition of another nucleotide sequencing by using fluorescent ddNTPs (dideoxynucleotides, N = A, T, G, or C).
Because of benefits such as strong bandwidth, cost savings, and speed, next-generation sequencing (NGS, also known as massively parallel sequencing) has primarily replaced Sanger sequencing. NGS can concurrently identify the sequence of billions of pieces. NGS is a kind of brief sequencing that entails building a tiny fraction collection, deep sequencing, unprocessed data preparation, DNA sequencing method, assembling, tagging, and subsequent DNA sequence analysis.
Third-gene sequencing, also known as long-read sequencing and incorporating PacBio SMRT sequencing and Oxford nanopore sequencing, can look at billions of DNA and RNA templates at once and find varied methylations without biases. Lengthy approaches can discover additional changes, including those that aren't visible with brief sequencing alone.
What is Automated DNA Sequencing?
Rather than using a radioactive isotope to mark the nucleotides, automated DNA sequencing uses a fluorescent dye. The luminous dye is non-hazardous to the ecosystem and requires no particular treatment or removal. Rather than utilizing X-ray films to detect the pattern, the fluorescent dye is stimulated with lasers. The fluorescence emission is measured with a charge-linked sensor that can identify the frequency.
Compared to manual DNA sequencing, automated DNA sequencing produces more dependable study results, preserving the research's integrity.
DNA Sequencing Applications
A DNA fragment, a full genome, or a complex microbiome can be sequenced to expose the genetic information contained within it. Scientists may deduce what genes and regulation signals are included in a DNA strand using sequence data. Gene-specific characteristics like coding sequences (ORFs) and CpG islands can be examined in the DNA sequence. For evolution studies across subspecies or groups, identical DNA sequences from various organisms may be compared. DNA sequencing, for example, can show alterations in gene sequencing that could cause a disease.
DNA sequencing has been utilized in healthcare for a variety of purposes, including illness diagnosis and therapy, as well as epidemiological investigations. Sequencing has the potential to transform food safety and environmental sustainability, as well as animal, plant, and public health, by enhancing agriculture through effective plant and animal breeding and lowering disease breakout risks. DNA sequencing may also be used to help conserve and sustain the ecosystem.
(Image Will be Updated Soon)
Fun facts
“DNA sequencing applications are done in a variety of fields from medical, to biology, to social science. It can be used to analyze the factors that are involved in the conservation of species. Latest DNA sequencing applications were used in making COVID-19 vaccines, as DNA sampling is used in making plant/animal-based vaccines that stimulate immunity.”
FAQs on DNA Sequencing: Complete Guide for Students
1. What is DNA sequencing and what is its primary purpose?
DNA sequencing is a laboratory technique used to determine the precise order of the four nitrogenous bases—Adenine (A), Guanine (G), Cytosine (C), and Thymine (T)—in a specific DNA molecule. Its primary purpose is to 'read' the genetic blueprint of an organism, which provides fundamental insights into its biological functions, genetic predispositions to diseases, and evolutionary history.
2. What are the main steps involved in the Sanger method of DNA sequencing?
The Sanger method, also known as the chain-termination method, is a foundational DNA sequencing technique. The main steps are:
- Denaturation: The double-stranded DNA is heated to separate it into two single strands.
- Primer Annealing: A short, single-stranded DNA primer binds to the template strand at the starting point for sequencing.
- Chain Elongation & Termination: The DNA polymerase enzyme adds normal nucleotides (dNTPs) to the growing chain. Specially modified nucleotides called dideoxynucleotides (ddNTPs) are also present. When a ddNTP is incorporated, the chain elongation stops.
- Gel Electrophoresis: The resulting DNA fragments of varying lengths are separated by size using gel electrophoresis. The sequence is read by detecting the fluorescently labelled ddNTP at the end of each fragment, from shortest to longest.
3. What are the major applications of DNA sequencing in modern biology and medicine?
DNA sequencing has revolutionised various fields. Its key applications include:
- Medical Diagnostics: Identifying genetic mutations responsible for diseases like cystic fibrosis, sickle cell anaemia, and various cancers.
- Pharmacogenomics: Customising drug treatments for individuals based on their genetic makeup to maximise effectiveness and minimise side effects.
- Forensic Science: Identifying individuals from biological samples (like blood or hair) in criminal investigations.
- Evolutionary Biology: Comparing DNA sequences between different species to understand evolutionary relationships and construct phylogenetic trees.
- Agriculture: Developing genetically modified crops with desirable traits like drought resistance or higher yield.
4. How does Next-Generation Sequencing (NGS) differ from the traditional Sanger sequencing method?
The primary difference lies in the scale and speed. While Sanger sequencing reads one DNA fragment at a time, Next-Generation Sequencing (NGS) is a high-throughput method that allows for the parallel sequencing of millions of DNA fragments simultaneously. This makes NGS significantly faster, cheaper, and more efficient for sequencing entire genomes. Sanger sequencing is still used for smaller-scale projects or to verify NGS results due to its high accuracy for single reads, whereas NGS excels at providing a massive amount of sequence data quickly.
5. Why are dideoxynucleotides (ddNTPs) essential for the chain termination in Sanger sequencing?
Dideoxynucleotides (ddNTPs) are crucial because they lack the 3'-hydroxyl (-OH) group that is present on normal deoxynucleotides (dNTPs). In DNA synthesis, this 3'-OH group is required for the DNA polymerase to form a phosphodiester bond with the next incoming nucleotide. When a ddNTP is incorporated into the growing DNA chain, the absence of this hydroxyl group prevents the addition of any further nucleotides, effectively terminating the chain. This controlled termination creates DNA fragments of every possible length, which is fundamental to reading the sequence.
6. What is a DNA sequencer and how does it automate the sequencing process?
A DNA sequencer is a scientific instrument that automates the DNA sequencing process, particularly the final step of reading the sequence. After the chain-termination reactions (as in the Sanger method), the resulting fluorescently-tagged DNA fragments are loaded into the machine. It uses a laser to excite the fluorescent dyes attached to the ddNTPs at the end of each fragment. A detector then captures the specific colour of light emitted, which corresponds to one of the four bases (A, T, C, or G). By processing the fragments from smallest to largest, the sequencer rapidly and accurately reconstructs the complete DNA sequence and outputs it as a digital text file.
7. Can DNA sequencing explain why two individuals have different traits, like eye colour?
Yes, DNA sequencing can explain such differences. Traits like eye colour are determined by genes, which are specific segments of DNA. By sequencing the relevant genes in two individuals, scientists can identify variations, known as alleles or polymorphisms, in their DNA sequence. For example, a single nucleotide difference (a Single Nucleotide Polymorphism or SNP) in a gene associated with melanin production can result in one person having blue eyes and another having brown eyes. Therefore, DNA sequencing provides the fundamental data needed to link genetic variations to observable physical traits.
