In 1977, two laboratories, one at Harvard, the other in England, invented two new procedures for determining the sequence of DNA. Previously, sequence determination was very time consuming and laborious. For their work, Walter Gilbert and Fred Sanger received the 1980 Nobel Prize.
The two procedures that they developed are different, although they share some properties. Sanger's technique is particularly elegant. It also is the easier of the two to understand. Moreover, it has become the one that is used by the vast majority of molecular biologists today. We'll therefore cover the Sanger procedure -- also called dideoxy sequencing -- in some detail, with an eye toward understanding its theoretical underpinnings as well as how it works in practice. We'll begin our discussion with the components that are required to carry out the procedure.
1. The Template -- In dideoxy sequencing one begins with a stretch of DNA that is to be sequenced. It should be in the form of single stranded DNA. How do you get single stranded DNA? In two ways. You can begin with double-stranded DNA and denature it (using heat or other agents to disprupt the hydrogen bonds that hold the two strands together). Alternatively, you can can clone the DNA that you want to sequence in a vector that produces single-stranded DNA. We didn't cover such vectors, but there are several available.
2. DNA Polymerase -- Sanger sequencing is a technique that requires DNA synthesis. The DNA mentioned above is used as a template to make a copy of a second strand. Since the synthesized strand is a complementary copy of the template, its sequence can be used to deduce the sequence of the template. DNA synthesis requires an enzyme called DNA polymerase, as well as several other components noted below. DNA polymerase, as its name implies, knits together -- polymerizes -- a DNA chain from monomers.
3. Nucleotide triphosphates -- These small molecules come in four flavors, one for each of the letters in the DNA alphabet. They consist of a base (either A, C, G or T), a sugar (deoxyribose) and not one, but three phosphates on the fifth carbon of the sugar. The enzyme lines up the proper triphosphate with the template, and joins it with the previous base. The joining releases two of the phosphates, and leaves the base, sugar and one phosphate behind on the newly expanding DNA strand. As we discussed previously, synthesis goes from 5' to 3', that is, the nucleotides are added onto the 3' end of the growing chain.
4. A primer -- All DNA synthesis requires a piece of single-stranded DNA that is complementary to part of the template. This acts as a sort of seed, on to which nucleotides are added. In other words, DNA synthesis can not start by itself from base 1. It requires a piece of DNA to add nucleotides to.
All DNA synthesis follows certain rules. We've encountered almost all of them previously, either just above or in prior sessions. Nevertheless, I'll review them here.
1. The newly synthesized strand is a complementary copy of the template.
2. Synthesis requires nucleotide triphosphates.
3. Synthesis adds on nucleotides to a primer.
4. DNA synthesis is unidirectional. It goes in the 5' to 3' direction. In other words, nucleotides are added on, one by one, to the 3' end of the polymer.
5. Synthesis always proceeds in the opposite direction of the
template. Therefore, if the template is written from left to right,
5' to 3', synthesis (which goes according to rule 4, also in the 5'
to 3' direction) goes from right to left.
