DNA Sequencing Hunter Jones, Mitchell Gage

What’s the point? In a process similar to PCR, DNA sequencing uses a mixture of temperature changes, enzymes (DNA Polymerase), primers, and nitrogenous bases to produce new strands of DNA and determine original DNA sequences by using the produced strands like puzzle pieces. Instead of making many identical copies, DNA sequencing aims to make a specific sequence, which is achieved by randomly cutting off DNA Polymerase’s copying of the template strand using ddNTPs (explained later).

Sanger sequencing Cycle sequencing Uses heat to denature DNA strands apart and then the primers anneal. We then add dNTPs and ddNTPS to the newly created random sequences using DNA Polymerase. Very similar, but the DNA Polymerase used is thermostable, so it doesn’t denature after the first heated cycle, so the process can be repeated using less template DNA and the heating and cooling can be automated using a laser.

dNTPs- deoxynucleoside triphosphates dNTPs are used by DNA Polymerase to attach to the newly sequenced DNA strand. They lengthen the new DNA sequence. dNTPs come in 4 nitrogenous base forms- A, C, T, and G.

ddNTPs- Dideoxynucleotide Triphosphates These molecules are also useable by DNA Polymerase, but they lack a 3’ OH group, so when they are added to the sequence, they terminate it, because no new dNTPs can bond with them. They are in the original mixture at about 1% of the concentration of dNTPs. They are incorporated randomly into a sequence, creating many different-sized DNA molecules by stopping the sequencing wherever it is put in. They also come in nitrogenous base forms (A, C, T, G).

What's with all these test tubes? each of the 4 tests tubes contain a different form of dNTPs and ddNTPs The 4 different test tubes separate the process into four different reactions based on which form of nitrogenous base is used (A, C, T, G) The test tube samples are loaded into a gel after sequencing in order to separate them using gel electrophoresis.

Gel electrophoresis This process allows scientists to separate the DNA samples by size. Because ddNTPs (that terminate sequencing of that particular strand) are added randomly, each strand could be a different length. Gel electrophoresis allows scientists to determine the sequence created, because the different length samples will separate based on where in the sequence the process was terminated.