Today’s complicated genomics issues need a level of detail that standard DNA sequencing technology cannot provide. Next-generation sequencing (NGS) has filled that need and has become one traditional method for answering these concerns. So what is DNA sequencing, and what types are currently available?
What is DNA Sequencing?
The term “sequencing” in biology refers to determining the order of units in a linear polymer. The application of methods and technology to establish the identity and order of the four nucleotide bases (adenine, guanine, cytosine, and thymine) in a segment of DNA is hence referred to as DNA sequencing.
According to the National Human Genome Research Institute (NHGRI), the human genome contains around three billion base pairs of DNA. Because the sequence of bases includes the instructions for generating proteins and regulating gene activities, the ability to interpret genetic sequences is instrumental in scientific study. For example, sequencing is a valuable technique in forensics because DNA and RNA sequence changes may classify creatures down to species and individual levels. It may also be necessary to define various illnesses, identify therapeutic targets, and tailor therapies. Although sequencing is seen as a relatively recent innovation, it has a long history.
Since its advent in the 1970s, DNA sequencing continues to evolve. Today, there are vital techniques researchers turn to when working to understand the DNA constructs of various plant and animal species.
Sanger sequencing is an option for researchers needing low-throughput, targeted, or short-read sequencing. Sanger sequencing is the gold standard in sequencing technology today due to its sensitivity and relative simplicity in workflow and methodology. It is utilized in many applications ranging from focused sequencing to validating mutations detected using orthogonal approaches.
Sanger sequencing employs a chain-termination approach. Chemical analogs of the four nucleotide bases are used in this approach. These analogs, known as ddNTPs, lack the hydroxyl group essential for expanding the polynucleotide chains that make up the DNA molecule. When radiolabeled ddNTPs are mixed with template DNA, strands of every possible length are formed when the ddNTPs are randomly integrated, thus ending the chain.
Capillary Electrophoresis And Fragment Analysis
Capillary electrophoresis (CE) equipment may do Sanger sequencing and fragment analysis. Fragment analysis is a technique in which fluorescently labeled DNA fragments are separated by CE and sized by comparison to an internal standard. While CE is used to establish the precise base sequence of a single fragment or gene segment, it may also offer size, relative quantification, and genotyping information for fluorescently labeled DNA fragments generated by PCR using primers intended for a specific DNA target.
Analysis of DNA fragments provides applications ranging from cell line verification to aneuploidy diagnosis. While sequencing techniques enable these uses, researchers prefer fragment analysis because it is quicker, has greater sensitivity and resolution, and is less expensive.
Next Generation Sequencing
Next-generation sequencing provides researchers with a better way to obtain high-throughput results. Although similar to Sanger, the critical difference in NGS is volume.
While the Sanger technique only sequences a single DNA fragment at a time, NGS is massively parallel and may sequence millions of fragments per run. This method allows for the simultaneous sequencing of hundreds to thousands of genes. As a result, NGS provides a better discovery capacity for detecting novel or uncommon variants with deep sequencing.
Researchers and scientists use the type of DNA sequencing that best suits their needs and ultimate goal. As the types develop, this process may become even more beneficial.