Sequencing: Revealing the DNA code
DNA sequencing is now routinely undertaken in a huge range of research projects. A step-by-step guide to sequencing DNA in the lab prepared by by science, mathematics, and technology teacher fellow Peta Poletti.
DNA sequencing requires a range of different molecules and solutions.
Take the piece of DNA that is to be sequenced and add the following in any order:
- A buffer solution that keeps the pH constant.
- An enzyme that makes new DNA pieces complementary to the DNA that is being sequenced.
- Free DNA nucleotides that are needed to make new DNA strands.
- Special DNA nucleotides, called terminators.
- And finally a primer, this is a short piece of DNA, which is needed to start the process.
The first thing that happens is that heat is used to separate the two strands of the DNA double helix. Next, new DNA sequences are made.
First the primer binds to the DNA, and the polymerase enzyme then makes a matching or complementary strand. It does this using the DNA nucleotides that were added.
Sometimes, instead of the enzyme adding an ordinary DNA nucleotide, it adds a terminator. Whether or not a terminator is added is random, and once a terminator is there, the DNA chain can’t be extended any further.
Over time, a whole range of DNA sequences are made. The length of each sequence will be different, but they will all be complementary to the DNA piece that is being sequenced.
The next step is to separate the DNA pieces using gel electrophoresis. The solution is placed in a gel, and the DNA pieces are pulled through the gel by an electric current.
In this case, the DNA pieces will be running from the top of the gel towards the bottom.
Notice, too, that there is a laser beam at the bottom of the gel.
The gel contains up to 100 lanes where different DNA samples can be separated.
Let’s take a closer look at what will be happening in one of the lanes in the gel. The blue bar represents the place where a small amount of the DNA solution is added.
The DNA pieces are then pulled through the gel by an electric current. The smaller pieces of DNA are able to move more quickly through the gel than the larger pieces. This is because they’re able to squeeze through the small pores in the gel more easily.
At the bottom of the gel there is a laser. This laser is able to pick up the signal from the terminators at the end of each DNA sequence. Notice that each terminator is associated with a different colour. A computer can measure these colours in the laser beam and interpret them as being A, T, C or G.
The final result looks something like this. Each peak represents a nucleotide in the DNA sequence. The base code can then be read off – T, A, C, A, T, C and so on.
- 27 November 2007
The University of Waikato