DNA sequencing is a laboratory technique, that aims to indicate the order of the nitrogenous bases (adenine, guanine, cytosine, thymine) that makes up the unique DNA makeup. Clinicians use the sequenced DNA to inspect if any region in the genome that regulates gene has any changes or mutations that could lead to diseases.
How DNA sequencing is performed?
Sanger Sequencing (single gene)
This technique was developed in the 1970’s and it was used in Human Genome Project which was the first time DNA of a human was completely sequenced. The principle behind this technique is to secure the DNA chain with a chemical called dideoxynucleotides, so no other nucleotide can be inserted after, and to tag the bases with unique fluorescent with the same chemical, that makes the bases identified and differentiated. This step results in multiple copies of DNA templates that vary in sizes. Then these fluorescently-labeled fragments are separated by their sizes in an electrophoresis process. As each fragment stops in a slightly different spot based on how many nucleotides are in the chain, the color at the end of each fragment shows exactly which base is in each position along the DNA sequence.
For many years, Sanger sequencing was a high standard clinical process for determining single genes or a few genes. It is still reliable but also has limiting sides to it. With Sanger sequencing you can only read one short section of DNA at a time. Also Sanger sequencing detects the variants in the gene if it’s outnumbered by the normal copies of a gene. In order for Sanger sequencing to be able to tell that there is more than one variation of the genetic code present, at least 15-20% of the DNA tested needs to contain the same variant or mutation (disease-causing variant).
Next-generation sequencing (NGS) (whole exome sequencing and whole genome sequencing)
Different from Sanger sequencing, it is now possible to sequence the human genome in matter of days. Sequencing time and cost have declined dramatically thanks to a compilation of sequencing technologies called next-generation sequencing (NGS). NGS methods are faster because they sequence millions of small DNA fragments from different parts of the genome simultaneously, rather than reading one fragment from one region at a time.
The protein-coding sections of genes are called exons, and the intervening areas that separate the exons within a single gene are called introns. The collection of exons in all known genes is called exome. When NGS is used to evaluate the entire exome or genome, it is called whole exome sequencing or whole genome sequencing, respectively.
Another advantage of NGS is that it is highly sensitive to detecting the alterations. In NGS only 2%-5% of the tested DNA needs to show the same mutation in order to be detected.
Some changes are known to cause problems with the structure or function of a gene product, and these are referred to as “pathogenic” variants. Other changes are known to have no effect at all on the final gene product and are considered “benign” (harmless) variants.
Some changes don’t have clear evidence either way (of being pathogenic or benign) and are called “variants of uncertain significance”.
(1)DNA Sequencing. (n.d.). Retrieved from https://labtestsonline.org/dna-sequencing