The Tools of Biotechnology

Molecular markers - Polymerase chain reaction

Undisplayed Graphic Most methods for keeping track of DNA sequences in a genome use an enzymatic reaction known as the Polymerase Chain Reaction (PCR). In PCR, enzymes called DNA polymerases can be used to make millions of copies a DNA sequence in a test tube containing DNA from any species. To determine which sequence, out of the entire genome, will be amplified, short DNA molecules called primers are added to the reaction. Wherever the primers (top right) occur by in the genome, the DNA sequence between those primer sequences will be replicated millions of times. In the example, the priming sites occur by random chance at four sites on three different chromosomes. At each site, the primers occur at different distances from one another, so different sized DNA sequences are amplified.

To distinguish among the different sequences amplified, the DNA fragments produced by PCR are separated by gel electrophoresis. The DNA mixture is loaded to a slab of agarose gel (a little like thick Jello), and an electric current is applied. The negatively-charged DNA migrates towards the positive pole (anode). However, the longer the DNA molecule, the more its movement is impeded by the gel, and the more slowly it migrates. Consequently, smaller DNA molecules will run rapidly, and large ones will run slowly. In this example, four different size classes of DNA are shown, representing the four different sequences amplified at each of sites a, b, c and d.

We can distinguish one plant from another, or one cultivar from another on the basis of differences in DNA sequence. In the example, DNA from plant 1 allows the amplification of sequences a, c and d, but not b. This indicates that in plant 1, priming sites for the primers used are not present at sequence b. Similarly, a DNA change (mutation) at one of the priming sites for sequence a has prevents it from being amplified when DNA from plant 2 is used.

Most importantly, the DNA differences between two plants, cultivars, or species can be visualized by comparing the patterns of DNA bands on a gel.