BY GRETCHEN NEWBERRY AND EMY MONROE, MIDWEST FISHERIES CENTER
the process. During the lab’s busiest times of the year, the wall racks are filled with
samples, and thousands more are in cold storage. Credit: USFWS
What happens when our Fish and Wildlife Conservation Offices send their water samples to the Whitney Genetics Lab? As the eDNA collection season winds down, we thought we would take a moment to peer into the mysteries behind the lab’s biosecurity doors and show the eDNA lab process step by step.
The water samples containing potential Asian carp genetic material arrive at the Midwest Fisheries Center in Onalaska, Wisconsin. Whitney lab staff use commercial kits to extract DNA from a pellet found at the bottom of each sample. The kits breaks down cell membranes to release cellular contents, including DNA. To purify the DNA, detergents from the kits are used to wash away fats, proteins, and other organic matter from the DNA. The final extract may contain tiny amounts of silver or bighead carp DNA, or DNA from other aquatic organisms.
To make detection of target DNA easier to find, the samples’ contents are duplicated in a process called amplification. Whitney staff use Polymerase Chain Reaction (PCR), a process that uses species-specific strands of DNA called primers that match only silver or bighead carp DNA. These primers, along with the extracted DNA from the water sample are put into a cocktail containing DNA Taq polymerase, an enzyme that copies DNA. These reactions undergo several cycles of heating and cooling to create thousands of copies of any silver or bighead carp DNA that may be in the water sample.
robot to load PCR mixes into plates with 384 wells. Credit: USFWS
To begin identifying the samples’ DNA, Whitney uses quantitative PCR, which involves a third specific strand of DNA called a probe. The probe is labeled with a fluorescent tag on one end and quenchers that prevent the tag from glowing on the other end. When Taq polymerase copies target DNA, it cuts the fluorescent dye free, and as it moves away from the quenchers, it glows. The qPCR machine measures the increase in the amount of fluorescence as it doubles with each round of amplification. Each sample is screened with two markers that detect both silver and bighead carp DNA, and if both markers are detected, the sample is presumed to be a positive match for carp DNA.
Samples are confirmed positive at the Whitney lab in a second set of qPCRs. In one qPCR, two regions of silver carp DNA are copied and in a second qPCR, two regions of bighead carp DNA are copied. In each qPCR, if just one of the regions is detected, that sample is confirmed positive for that species. Water samples testing positive have thus been detected with 3 or 4 regions of DNA specific only to Asian carp.