Diversity of Fishes
Case Study: Dr. DeSalle Identifies Sturgeon Species

Altering PCR: The Testing Begins

Because PCR can generate billions of copies of selected DNA stretches, Rob had up until now used PCR for two purposes. First, he had used it to amplify sections of sturgeon DNA that he then sequenced to find where genetic differences occur between sturgeon species. Then, later, he had used it to amplify the sections of the mystery caviar eggs so he could then sequence them and compare their sequences to the sequences of the known samples.

Rob reasoned, however, that if he included primers that attached to only one species' DNA during PCR, he wouldn't need to sequence the DNA copied. This is because he would design one primer so it would only attach to a base pair unique to one species. Because the primer must attach to DNA for the polymerase to begin adding on more nucleotides, the reaction would fail for any other species.

For example, the sturgeon that produces sevruga caviar, Acipenser stellatus, has a C at a particular spot in its DNA where every other sturgeon species has a T. If Rob tested a caviar egg with a primer specific to sevruga (it is designed to match up precisely with that C) and no DNA was amplified, then he could confidently say that the egg was not from sevruga. If, on the other hand, the DNA was amplified then he knew that the egg must come from sevruga.

Rob also made a second primer specific to each species that would attach a specified number of base pairs away from the first primer. For A. stellatus, he designed another primer that would attach 234 base pairs down. After he ran an unknown caviar egg through his altered PCR (using a primer specific to sevruga) and it produced a mass of DNA segments, he would check their base pair length with gel elecrophoresis to determine whether it corresponded with the expected 234 base pair lengths for the sevruga species. With any other species, he'd get nothing.

So Rob created species-specific primers for each of the three major caviar-producing sturgeon species that make up 80% to 90% of imported caviar. He would test eggs from each of the caviar tins with these primers. If the tests came up negative for all three he would move on to the lengthier process of sequencing the DNA to find out which of the other 22 species of sturgeon the caviar came from. This allowed him to quickly determine which of the 25 species of sturgeon the egg came from‹or whether it's a sturgeon egg at all.

Also, one trick smugglers use is to mix two kinds of caviar together, blending cheap or illegal caviar with authentic beluga, sevruga, or osetra, the most costly varieties. To detect such fraud, Rob wanted to separately check 10 to 20 eggs from each can.

The beauty of Rob's altered PCR technique is that you don't need to analyze the exact sequence of the DNA you copied—you only need to know its length. If the reaction produces a mass of DNA of the right length, it means both primers attached to the template, and the DNA between the primers was copied. The length of the product will be exactly the number of nucleotides between the anchors of your two primers.

The length of DNA fragments is easily determined by gel electrophoresis.

Multiple tests are run simultaneously to control for contamination.
Results from gel electrophoresis.
Multiple tests are run simultaneously to control for contamination.

One lane is reserved for a prepackaged mixture of pieces of DNA of known length, called a standard. If the standard contains DNA fragments that measure 100 base pairs, 150 base pairs, and 200 base pairs, they will be separated by electrophoresis into three distinct groups. Each group shows up under UV light as a stripe in the gel. These stripes serve as a yardstick against which to measure the other samples.

For example, suppose Rob put the above standard in the left lane, and the product of his altered PCR test in the lane next to it. If the PCR product makes a stripe that lines up with the second stripe of the standard, you would know it is about 150 base pairs long. To look for a product 2,000 base pairs long, your standard might include pieces ranging from 1,000 to 3,000 base pairs.

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