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DNA sequencing: definition, methods, examples

Science 2021

Nucleotides are the chemical building blocks of life and occur in the DNA of living organisms. Each nucleotide consists of a sugar, phosphate and a nitrogen-containing bae: adenine (A), thymine (T), cytoin

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Nucleotides are the chemical building blocks of life and are found in the DNA of living organisms. Each nucleotide consists of a sugar, phosphate and a nitrogenous base: Adenine (A), thymine (T), cytosine (C) and guanine (G). The specific order of these nucleotide bases determines which proteins, enzymes and molecules are synthesized by the cell.

Determining the order or sequence of nucleotides is important for studying mutations, evolution, disease progression, genetic testing, forensic examinations, and medicine.

Genomics and DNA sequencing

Genomics is the study of DNA, genes, gene interactions and environmental influences on genes. The secret to deciphering the complex inner workings of genes is to identify their structure and location on chromosomes.

The blue of living organisms is determined by the order (or sequence) of the nucleic acid base pairs in DNA. When DNA replicates, adenine pairs with thymine and cytosine pairs with guanine. Mismatched pairs are considered Mutations.

Since the conception of the double helix deoxyribonucleic acid (DNA) molecule in 1953, dramatic improvements have been made in the fields of genomics and large-scale DNA sequencing. Scientists are working diligently to apply this new knowledge to the individualized treatment of diseases.

At the same time, the ongoing discussions allow researchers to stay one step ahead of the ethical consequences of such rapidly exploding technologies.

Definition of DNA sequencing

DNA sequencing is the process of discovering the sequence of different nucleotide bases in snippets of DNA. Whole gene sequencing enables the comparison of chromosomes and genomes that are in the same and in different species.

Chromosome mapping is useful for scientific research. The analysis of the mechanisms and structures of genes, alleles and chromosomal mutations in DNA molecules suggests new ways of treating genetic disorders and stopping the growth of cancerous tumors, for example.

DNA sequencing: early research

Frederick Sanger's DNA sequencing methods The field of genomics has developed significantly since the 1970s. Sanger felt ready to tackle DNA sequencing after successfully sequencing the RNA while studying insulin. Sanger wasn't the first scientist to look at DNA sequencing. However, his sophisticated DNA sequencing methods, which were developed together with colleagues Berg and Gilbert, were awarded the Nobel Prize in 1980.

Sanger's greatest ambition was sequencing large, entire genomes, but sequencing the base pairs of a tiny bacteriophage pales in comparison to sequencing the 3 billion base pairs of the human genome. Still, learning to sequence the entire genome of a lower bacteriophage was an important step in putting the entire human genome together. Because DNA and chromosomes are made up of millions of base pairs, most sequencing methods separate DNA into small strands and then the DNA segments are put together; It just takes time or fast, sophisticated machines.

DNA sequencing basics

Sanger knew the potential value of his work and often collaborated with other scientists who shared his interests in DNA, molecular biology, and life sciences.

Although slow and expensive compared to today's sequencing technologies, it was at the time that Sanger's DNA sequencing methods were lauded. After trying it, Sanger found the secret biochemical "recipe" for separating strands of DNA, creating more DNA, and identifying the order of nucleotides in a genome.

High quality materials can easily be purchased for use in laboratory studies:

DNA sequencing methods: Sanger methods

Sanger found out how to use the enzyme DNA polymerase to cut DNA into small segments.

He then made more DNA from a template and added radioactive tracers to the new DNA to delineate sections of the separated strands. He also realized that the enzyme needed a primer that could attach to a specific point on the template strand. In 1981, Sanger made history again by discovering the genome of the 16,000 base pairs of mitochondrial DNA.

Another exciting development was the shotgun method, in which up to 700 base pairs were randomly sampled and sequenced at the same time. Sanger is also known for his use of the dideoxy (dideoxynucleotide) method, which inserts a chain-terminating nucleotide during DNA synthesis to mark sections of DNA for analysis.

DNA sequencing steps

The temperature must be carefully adjusted throughout the sequencing process. First, chemicals are placed in a tube and heated to disentangle (denature) the double-stranded DNA molecule. Then the temperature is cooled so that the primer bonds.

Next, the temperature is increased to promote optimal DNA polymerase (enzyme) activity.

The polymerase typically uses the normally available nucleotides, which are added in a higher concentration. When the polymerase gets to a dye-bound "chain terminating" nucleotide, the polymerase stops and the chain ends there, which explains why the colored nucleotides are called "chain terminating" or "Terminators" are called.

The process continues many times. Finally, the dye-bound nucleotide was placed at every single position in the DNA sequence. Gel electrophoresis and computer programs can then identify the dye colors on each of the DNA strands and figure out the entire sequence of the DNA based on the dye, the position of the dye, and the length of the strands.

Advances in DNA Sequencing Technology

High throughput sequencing - commonly referred to as Next generation sequencing - uses new advances and technologies to sequence nucleotide bases faster and more cheaply than ever before. A DNA sequencing machine can easily process large sections of DNA. In fact, using Sanger's sequencing techniques, the entire genome can be created in hours instead of years.

Next generation sequencing methods can perform high volume DNA analysis without the additional step of amplification or cloning to get enough DNA for sequencing. DNA sequencing machines perform multiple sequencing reactions at the same time, which is cheaper and faster.

In essence, the new DNA sequencing technology performs hundreds of Sanger reactions on a small, easy-to-read microchip, which is then run by a computer program that puts the sequence together.

The technique reads shorter fragments of DNA, but is still faster and more efficient than Sanger's sequencing methods, allowing large projects to be completed quickly.

The human genome project

The Human genome project, completed in 2003, is one of the best known sequencing studies to date. According to a 2018 article in Science newsthe human genome consists of approximately 46,831 geneswhich was a daunting sequencing challenge. Top scientists from around the world have worked together and advised for almost 10 years. Led by the National Human Genome Research

Institute, the project successfully mapped the human genome with a composite sample from anonymous blood donors.

The human genome project relied on artificial bacterial chromosome (BAC) sequencing methods to determine base pairs. The technique used bacteria to clone fragments of DNA, resulting in large amounts of DNA for sequencing. The clones were then reduced in size, placed in a sequencing machine, and reassembled into sections representing human DNA.

Other DNA sequencing examples

New discoveries in genomics are fundamentally changing the way we prevent, detect and treat diseases. The government has allocated billions of dollars to DNA research. Law enforcement relies on DNA analysis to resolve cases. DNA test kits can be purchased for home use to examine origins and identify variants of genes that may pose a health risk:

Ethical Implications of DNA Sequencing

New technologies often harbor the potential for social benefit and harm. Examples of this are defective nuclear power plants and nuclear weapons for mass destruction. DNA technologies also involve risks.

Among the emotional concerns about DNA sequencing and gene editing tools like CRISPR is the fear that the technology could make human cloning easier or result in mutant transgenic animals created by a rogue scientist.

More often than not, ethical issues related to DNA sequencing have to do with informed consent. With easy access to DNA tests performed directly on the consumer, consumers may not be able to fully understand how their genetic information is used, stored and shared. Laypeople may be emotionally unwilling to learn about their defective gene variants and health risks.

Third parties such as employers and insurance companies could potentially discriminate against people who carry defective genes that can lead to serious medical problems.

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