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DNA is the unique genetic code found in most cells in humans as well as in organisms such as bacteria, many viruses, parasites, and plants. It is structured like a twisted ladder, with two sides and rungs made up of two pieces that fit together. These rungs are created from two of four possible bases: adenine (A), cytosine (C), guanine (G), and thymine (T). Because of their shape, adenine always pairs with thymine and guanine always pairs with cytosine to form a complete rung. These bases are bonded at the sides of the ladder to a sugar and phosphate, which form the vertical backbone of the DNA double helix (the "sides" of the ladder). The base, sugar, and phosphate form a unit called a nucleotide, which is referred to by the base (A, C, T, or G) it contains.

Specific segments of DNA called genes serve as templates to make (transcribe) RNA. The information contained in the RNA is then often translated by tiny molecular machines into proteins. There are approximately 20,000 genes in the human genome. The information contained within these genes allows our cells to produce an enormous variety of proteins that serve as the building blocks for our bodies and that govern how the body works.

DNA sequencing is a laboratory method used to determine the order of the bases within the DNA. Differences in the sequence of these 3 billion base pairs in the human genome lead to each person's unique genetic makeup. In medicine, DNA sequencing is used for a range of purposes, including diagnosis and treatment of diseases. In general, sequencing allows healthcare practitioners to determine if a gene or the region that regulates a gene contains changes, called variants or mutations, that are linked to a disorder.

When considering or undergoing genetic testing, it is important to seek help in interpreting these results from a genetics expert such as a medical geneticist or genetic counselor to better understand the test results, implications of the results, and any potential risk of having or passing a genetic condition on to your children.

How is DNA sequencing performed?

While methods for DNA sequencing have evolved over the years, the technique generally consists of breaking long strands of DNA into many small pieces, using one of several types of tests to determine the order of the nucleotide bases that make up those pieces, and then reassembling the data back in the order of the original DNA strand.

Sanger Sequencing
Developed in the 1970's, this is the method that was used in the Human Genome Project from 1990-2003 to completely sequence the DNA of a human for the first time. Sanger sequencing relies on chemicals called dideoxynucleotides, which are also known as 'chain terminating' nucleotides. When one is incorporated into a growing copy of DNA sequence, no other nucleotide can be added onto the chain after it. Each dideoxynucleotide has a unique fluorescent "tag" that allows A, T, C, and G to be clearly identified.

Incorporation of a dideoxynucleotide occurs at random, resulting in multiple copies of the DNA template, all different lengths. These fluorescently-labeled DNA fragments are then separated by size in a process called electrophoresis. 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 has been the gold standard for clinical DNA sequencing to look at single genes or a few genes at a time. Sanger sequencing is reliable, but it can only read one short section of DNA from one person at a time. Sanger sequencing also has a limited ability to detect changes if they are greatly outnumbered by normal copies of a gene, which can happen when some cells have a variant or mutation (disease-causing variant) and some don't. For example:

If a cell has undergone a genetic change that allows it to grow in an uncontrolled fashion (a tumor), the genetic code of this cell and any that come from division of this cell will have a variant that is not present in any of the other cells in a person's body.
A person may have two different genetic codes in normally dividing, non-tumor cells that are present in mixed proportions throughout the body; this is a situation referred to as somatic mosaicism.
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).

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