DNA sequencing is the process of determining the sequence of nucleotide bases in a piece of DNA. While this may sound straightforward, DNA sequencing is a complex process with many steps and chemical processes.
DNA sequencing is used in various research fields and industries. It also has a wide range of uses and applications. It is frequently used in biochemistry, genomics, biological anthropology, evolutionary biology, medicine, virology, forensic science, and the burgeoning ancestry testing industry.
A Brief History of DNA Sequencing
Although DNA was first isolated in 1869, its importance wasn’t discovered until decades later. The first DNA sequences were done over a hundred years later in the 1970s, using a laborious process called two-dimensional chromatography.
This developed into the process known as Sanger sequencing.
Early DNA sequencing was first performed on viruses. The first complete DNA sequence was published in 1977 and belonged to bacteriophage φX174, a virus that attacks E. coli bacteria. Viruses have single-stranded DNA instead of the double-stranded DNA of both prokaryotes and eukaryotes, which made them much easier to sequence in the early days of DNA sequencing.
From there, DNA sequencing technology developed rapidly. In the 1990s, the NIH ran several sequencing projects for various species of bacteria with the goal of sequencing the human genome. The Human Genome Project was formally launched in 1990 where it took 32 years to complete a gapless DNA sequence for the human genome.
The Human Genome Project officially published the complete human DNA sequence in January 2022.
What Is Involved in DNA Sequencing?
Sequencing DNA means determining the order of the four nucleotide bases that make up the DNA molecule. The order of these bases lets analysts figure out the kind of genetic information that is carried by a particular segment of DNA.
To identify the order of the base pairs in a segment of DNA, it must first be manipulated, amplified, and processed. There are several ways to complete this.
The oldest and simplest of these is known as Sanger sequencing. This involves making multiple copies of the DNA region to be sequenced.
In Sanger sequencing, the DNA sample is combined in a tube with both normal DNA nucleotides and dye-labeled, chain-terminating dideoxy nucleotides. The DNA is heated to denature it, then cooled to let the primer bind to the single-stranded template.
After this, DNA polymerase synthesizes new DNA. It continues adding free nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a normal one.
At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide.
The next step is to run the DNA fragments through a gel matrix in a process called capillary gel electrophoresis. The smallest fragment, ending just one nucleotide after the primer, finishes this process first.
It’s followed by the next-smallest fragment with two nucleotides, all the way to the final fragment. The dyes are then illuminated by a laser and recorded by a detector to put the original DNA sequence together one nucleotide at a time.
This process works, but it is extremely tedious and expensive compared to modern methods.
Modern DNA sequencing methods are much more sophisticated than the Sanger sampling developed over 40 years ago. These advanced methods are known as next-generation sequencing.
There are several next-generation sequencing methods that use different technologies, but they all have a few things in common.
Next-generation sequencing reactions are parallel and use short nucleotide sequences that are run at the same time. This means the results are ready much faster.
They are also done on a chip instead of in a tube or using gel electrophoresis. This allows large quantities of DNA to be sequenced much more quickly and cheaply than with Sanger sequencing.
One of the most crucial parts of next-generation sequencing is the various algorithms that are used to assemble the DNA sequences. Since a large volume of data is generated from a single sequencing chip, computer programs like Phred and Phrap evaluate raw sequence data.
Trimming algorithms are then used to clean up the sequences and transform the raw data into usable material.
Other next-generation sequencing technologies include nanopore technology, which threads single DNA strands through tiny pores in a membrane. The DNA bases are read one at a time and identified by measuring differences in their electrical effects on ions as they flow through the pore.
Nanopore DNA sequencing is extremely fast because the same DNA molecule can be reused. The scientists who use this method do not have to replicate large batches of DNA before studying it.
Who Uses DNA Sequencing?
DNA sequencing is used for many different applications and areas of research. The following examples are only a handful of the potential applications of DNA sequencing.
DNA sequencing is one of the best ways to identify the causes of rare genetic disorders. Only 4,000 of the more than 7,800 identified genetic disorders are linked to a specific gene.
Thus, looking at the entire genetic sequence of a person with a genetic disorder can provide information about which alleles cause a particular illness.
DNA sequencing is also vital for treating cancer. Understanding the genetic basis of a tumor or cancer makes diagnostic decisions easier.
It also provides a better understanding of the potential treatment options a cancer patient may respond to.
Most biology laboratories now include DNA sequencing as an integral part of laboratory operations. Gene sequencing is used to study variations in the genetic compositions of all kinds of model organisms and to verify the characteristics of cell lines and cloned tissue cultures.
DNA sequencing is the primary method of studying regulatory elements within cells and how variations in these regulatory sequences affect cells, tissues, and organisms. Being able to identify which genetic regions are responsible for gene expression is a valuable purpose of DNA sequencing in any molecular biology laboratory.
DNA sequencing is the basis of modern forensic science. The ability to use low concentrations of DNA to obtain reliable genetic data through next-generation sequencing is extremely useful for crime scenes.
DNA sequencing can even be used to look at human remains to determine the cause of death. Poisoning deaths cause damage to the DNA in affected organs, which can’t be seen in a standard autopsy.
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