Several

organisms

Possible relatives 1 2 3

Possible relatives 1 2 3

2 is a correct match 1 and 3 not related

FIGURE 23.12 Randomly Amplified Polymorphic DNA

The first step of RAPD analysis is to design primers that will bind to genomic DNA at random sites that are neither too rare or too common. In this example, the primers were sufficiently long to bind the genomic DNA at a dozen places. For PCR to be successful, two primers must anneal at sites facing each other but on opposite strands. In addition, these paired primer sites must be close enough to allow synthesis of a PCR fragment in a reasonable time. In our example, there are three pairs but only two of these pairs were close enough to actually make the PCR product. Consequently, this primer design will result in two PCR products as seen in the first lane of the gel (marked "First organism"). The same primers are then used to amplify genomic DNA from other organisms that are suspected of being related. In this example, suspect #2 shows the same banding pattern as the first organism and is presumably related. The other two suspects do not match the first organism and are therefore not related.

pattern of bands that will vary in different organisms, depending on how closely they are related. Although we do not know in which particular genes the PCR bands originate, this does not matter in measuring relatedness. Diagnosis therefore relies on having a primer (or set of primers) that reliably give a band of a particular size with the target organism and give different bands with other organisms, even those closely related. RAPD results using such a primer are shown in Figure 23.13. Grey mold, due to Botrytis cinerea, is one of the most destructive infections of strawberries and also attacks other plants. Classical diagnosis involves culturing the fungus on nutrient agar. It is slow and difficult due to the presence on the plants of other harmless fungi, which often grow faster in culture. As can be seen, RAPD analysis clearly identifies the pathogens from other related fungi, including other species from the genus Botrytis.

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