DNA (deoxyribonucleic acid) is the polymer that encodes the instructions for making and maintaining all living things in long strands of nucleotides. Two nucleotide strands wind together to form a double helix structure, which is then wound up upon itself in an orderly fashion to form chromatin. When a cell divides, the chromatin condenses further to form chromosomes. Most of the DNA in a eukaryotic cell is found in the nucleus, but cells also have DNA (called mtDNA) in organelles called mitochondria, and plant cells have DNA in their chloroplasts (cpDNA). The DNA in mitochondria and chloroplasts is often joined into a circle, whereas nuclear DNA is linear.

The nucleotides that join up to make DNA are composed of three parts: the five-carbon sugar deoxyribose, three phosphate groups, and a nitrogenous base. There are four bases: adenine, thymine, cytosine and guanine. These are abbreviated as A, T, C and G when listing their sequence in a DNA molecule. The bases pair up specifically when the double helix forms (A with T and C with G); this means that each strand of the double helix can serve as a template for the synthesis of a new strand. The strands have different structures at their ends and thus have direction: typically, the sequence is written in the 5' to 3' direction. Their orientation relative to one another is antiparallel. For example,

5' - G-C-T-G-T-T-A-G-T-G-A - 3'

3' - C-G-A-C-A-A-T-C-A-C-T - 5'

DNA polymerase, the enzyme that links nucleotides together to make new DNA molecules, uses this strategy. DNA polymerase catalyzes the reaction in which the 3' -OH group of one nucleotide attacks the alpha phosphate of another nucleotide to make the next link in the chain. This results in structures that look like (C) above.

Dideoxy sequencing, the most popular method of DNA sequencing, uses the specific reaction catalyzed by DNA polymerase as a strategy for discovering the sequence of bases in a DNA strand. This is basically how it works (also see the illustration below):

• Four slightly different reactions are set up in separate tubes. All four tubes contain the DNA that you want to sequence, DNA polymerase, primers (very short strands of DNA that bind to the DNA strand and help DNA polymerase to "get started" in the right location), and nucleotides A, T, C and G. The only difference between the reactions is that each one contains a small amount of one of the nucleotides which is missing the 3' -OH group on its sugar group (a dideoxy nucleotide). Without this -OH, DNA polymerase can't catalyze the next reaction and the strand stops growing. Because many copies are made of the strand, they will turn out to be different lengths, but all of the strands in a tube will end with the same nucleotide.
• The reaction mixtures are then loaded into the top of a polyacrylamide gel inside an automated sequencing machine. The gel allows smaller molecules to move through it at faster rates than larger ones, so strands of different lengths separate and form "bands" in the gel. The primer attached to the end of each strand is fluorescent. As the molecules flow down the gel, they eventually pass a laser which "sees" them fluoresce, and in this way the machine creates a computer image of the bands in the gel.
• The sequence of the DNA strand can be read right from the image of the gel! The computer will "auto-sequence" the gel for you; that is, it will interpret the image as well as it can and give you a tentative sequence. This sequence then has to be proof-read, but it gives you a good head-start.
• Many different kinds of software exist for helping you to understand the sequence you've obtained. You can decode protein sequences, compare DNA sequences between different organisms, and even use the sequences to decipher the evolutionary relationships between groups of organisms.

 

LI-COR sequencers. These types of sequencers are typically used in small to medium-sized labs that do moderate amounts of sequencing.

Click here to see what I did with DNA sequencing during my internship at the Academy of Natural Sciences during the summer of 1999: