What is DNA Sequencing and how does it work?

Most of the money raised will go towards sequencing, an approach that tells us about the molecular mechanisms helping corals acclimate to warmer temperatures. I'm sure you've read some of my posts and thought "What does sequencing mean?", so I wanted to make sure I gave an overview of exactly what it is! 

DNA stands for DeoxyriboNucleic Acid and it contains the genetic information for each organism, to help them grow, function, and reproduce. The genetic code is made up of four nucleotides which organize themselves in a linear fashion. These four nucleotides are Thymine (T), Cytosine (C), Guanine (G), and Adenine (A). Adenine and Thymine pair together, and Guanine and Cytosine pair together creating a bond that forms the double stranded helix structure of DNA (image below). 

 

The collection of all the DNA in an organism is called a genome. The human genome is three billion bases long! In coral genomes, there are only about 300 million bases. The order of the nucleotides in DNA is extremely important. Specific sections or sequences of DNA are called genes, and genes act as instructions for certain proteins that have specific functions throughout an organism. 

DNA sequencing is used to determine the order of bases within DNA. One common method of sequencing is called Sanger Sequencing, named after the scientist who created the method. In Sanger Sequencing, an enzyme called DNA polymerase duplicates short pieces of DNA. In doing so, special nucleotides with fluorescent tags are added. When these special nucleotides are added DNA polymerase stops duplicating that piece of DNA. The polymerase does this millions of times so that there are millions of differently sized pieces of DNA with a fluorescent nucleotide at the end.  

The sequencing machine sorts these DNA fragments by size and then takes a picture of the fluorescent tag on the end. From this data, the machine can determine which nucleotide is in each position of the sequence. The output, like the image below, indicates which nucleotide the machine thinks it saw (color and letter) and the confidence that the nucleotide selection is correct (height of peak).

 

So how will I use this method? As I mentioned, I will be looking at how the coral host responds to acclimation and the symbiont type. I will be use Sanger sequencing to analyze which types of symbionts the coral is associating with and if that helps them acclimate. I will use a universal but specific DNA sequence called ITS2 that has been used to identify symbiont types within a coral. Knowing what types of symbionts are in my corals, I can determine if associating with specific types of symbionts helps the coral acclimate.  

I hope this description helped you understand what I'll be doing! :) I'll keep you updated once sequencing preps have begun!