Sanger Method - DNA Sequencing

ddNTP

Sanger Method

1. Start off with a single strand of DNA.  Occurs through denaturation of DNA, be being heated.
2. Radioactively labeled primer attached to four copies of single strand DNA.  (to help identify the starting point)
3. Each strand is put into individual test tubes.  Each test tube is filled with an abundant amount of dATP, dGTP, dCTP, dTTP.  
4. In each separate test tube, a small amount of ddNTP is added to each test tube.  One type of ddNTP for each test tube, e.g. ddATP in one, ddGTP in one, etc.  ddNTP stands for dideoxy nucleoside triphosphate.  It has NO oxygens on the carbon chain, and therefore cannot bond with any other dATPs, which terminates the DNA replication.
5. Polymerase 3 is added.
6. DNA replication occurs.  In each test tube, there will be strands where the replication automatically stops as a result of ddNTP.
7. Each individual test tube is loaded into four individual lanes on a gel.  The gel is run through an electrophoresis machine, and the DNA sequence can be read from bottom to the top, because it is radioactively labelled.