Deoxyribonucleic Acid, or DNA as it is popularly known, is present in almost every cell in the human body and other organisms. This microscopic hereditary material located in the cell carries all the genetic information for the organism it belongs to. The information the DNA carries determines how the organism grows, functions, and reproduces in its lifetime.
This unique genetic information is stored as a code made up of four basic nucleotides called Thymine(T), Guanine(G), Cytosine(C), and Adenine(A). These four DNA bases pair bond with one another, as in, Thymine pairs with Adenine and Cytosine bonds with Guanine (C & G). These two units are known as base pairs, and together, they create the bond that forms the DNA’s famous “double-stranded helix structure”.
To understand DNA genome sequencing, we must first understand the role of the four basic nucleotides in DNA. Amazingly, human DNA consists of nearly 3 billion bases that carry the necessary instruction to create and maintain a human.
The sequence of the base pairs (A&T, C&G) stores a substantial amount of genetic information, and they form the fundamental platform for how DNA molecules are copied, translated, transcribed and paired. DNA genome sequencing is a highly technical process that determines the sequence of nucleotide bases. Sequencing is a complex task that is now much faster, accurate and less expensive due to the aggressive advancements made in DNA sequencing technologies and methodologies.
DNA sequencing methods
One of the most common and well-established DNA sequencing methods is Sanger Sequencing, invented in the late 1970s by Frederick Sanger, an English biochemist. This sequencing method involves making many copies of a target DNA region by utilising fluorescent ddNTPs or dideoxynucleotides.
Next-Generation Sequencing or NGS:
The best answer to fast and inexpensive sequencing is NGS, also known by the term Massively Parallel Sequencing. Various Next-Generation sequencing techniques have effectively replaced Sanger sequencing. Their benefits include high throughput, accuracy, and speed. With NGS, it is possible to run millions of sequencing reactions simultaneously.
Some of the latest sequencing methods are long-read methods that can read billions of RNA and DNA templates to detect variable methylations. Long-read methods such as PacBio SMRT Sequencing and Oxford Nanopore Sequencing can identify more variations than slow-read sequencing methods.
DNA Sequencing: The methodology then and now
Sequencing has come a long way from what it was in the 1980s. Most sequencing processes involved electrophoresis, a lab technique using electric current through a gel to separate DNA and RNA. During the process, the DNA that is earmarked for sequencing is placed at one end of the gel and electrodes are placed at the other end. An electrical current is passed through the gel, which causes the movement of the DNA molecules. The process segregates the DNA molecules into different bands according to their size.
Sequencing with electrophoresis did not come without drawbacks. It was an exhaustive, time-consuming process that required manual reading of the electrophoresis gels. Due to the technique’s limited capacity, the DNA needed to be cut up into small pieces, and each piece had to be attached to a radioactive label. An X-ray picture was needed to make the DNA bands visible. This tedious process was fraught with error.
DNA Sequencing has grown by leaps and bounds, thanks to the automatic sequencing devices that have been commercially available since the late 1980s. Today’s advanced computer technology and tools have made large-scale sequencing possible with incredible speed and accuracy.
The difference between automatic sequencing machines and capillary sequencers:
Most sequencing labs and facilities have embraced the latest sequencing technologies by investing in sequencing machines that are faster and reliable. To enable large-scale sequencing, many labs utilise automated sequencing machines and capillary sequencers. Although both devices are designed to enable multiple sequencing simultaneously, they differ in their processes.
In automated machines, the gel is manually poured into glass plates, and after it sets, the DNA is loaded into each of the ninety-six lanes. As the DNA moves through the gel, the DNA machine reads the order of the DNA bases and stores the information in the computer attached to the machine.
In capillary sequencers, the DNA is run through ninety-six gel-filled capillaries that measure no more than a hair’s breadth. However, the end process is similar to that of the automated machine, where the collected sequencing information is stored in the computer.
The capillary sequencers are fully automated and require only minimum human intervention and supervision to refill the gel, water, and other solutions.
Yaazh Xenomics is one of Chennai’s largest genomic labs, fully equipped with the latest sequencing technology, infrastructure, and a team of well-trained scientists and support staff. We offer the best kind of support for all sequencing projects and research. To discuss your projects, please get in touch with our team.