The overall goal of this procedure is to rapidly screen for different genotypes, mutations or transgenes in zebrafish. This is accomplished by first extracting DNA from thin clips and then amplifying the DNA by PCR. The next step is to denature the PCR Amplicons while recording the melt curves, which are analyzed by software.
Ultimately, the results show distinguishable melt curves for different genotypes. The main advantage of this technique over existing method like gel electrophoresis volume PCR, is that is sensitive to single nucleotide changes, small insertion, all deletions, and this technique is fast and reliable. Though this technique may be used to rapidly, effectively genotype zebra fish, it can also be applied to other systems and model organisms, including cell lines, mice, and other animals.
On the day of the DNA preparation, add fresh proteinase K to the lysis buffer at a concentration of one milligram per milliliter. Tissue can be collected from an adult fish using a thin clip or from an embryonic fish. First anesthetize the fish in Trica solution.
Wait until the gill movements slow. Then put the fish on a stack of tissues and with a sterile razor blade, cut off a small piece of the tail fin about two to three millimeters long. Quickly place the fish in a labeled tank with fresh water for recovery.
Pick up the fin clip with a sterile pipette tip and transfer it into a tube filled with 100 microliters of DNA lysis buffer. Be sure to label both the animal's tank and the tube, incubate all the collected tissues for at least four hours and up to overnight at 55 degrees Celsius after the incubation. Inactivate the protein ACE K by heating the tubes at 95 degrees Celsius for 15 minutes.
These samples should be used for PCR immediately, but can also be stored at minus 20 degrees Celsius for up to three months. Perform the PCR in a 96 or 384 well plate for each reaction. Combine no more than one microliter of the adult DNA with four microliters of light scanner.
Master mix containing the fluorescent probe and primers at a final concentration of five pico molar each. If the DNA was from an embryo, use up to three microliters. Cover the reactions with 30 microliters of mineral oil and then cap them shut.
Next, cover the loaded PCR plate with an optically transparent adhesive seal. Now optimize the PCR cycling conditions. A typical condition set begins with five minutes of melt time.
This is followed by 30 PCR cycles with ten second melts, 25 seconds of a kneeling time and 30 seconds of elongation. The reaction should end with an added 32nd melt and then cool to 15 degrees Celsius. Analyze the PCR plate by placing it into a melt analysis system in the software.
Set up the temperature and create a new data storage file. Set up a subset. Select the wells with samples on the lower left side of the screen.
Select the normalized tab to eliminate fluorescent variants. Manually position the parallel double line in prem, melt and post melt regions and normalize premel and post melt fluorescent signals of all samples to one and zero respectively. Next, distinguish the genotypes based on their melting temperature by first selecting grouping.
Then select auto group from the standard selection list. Under the grouping section, select normal or high for melting profiles with single transitions or multiple transitions respectively. They're in the sensitivity selection list under the grouping section.
Now select compute groups under the grouping section and then go to the file menu and click save to save the results. The protocol can be performed during a single day or separated in steps over several days. When performing PCR, the temperatures for the melt of the amplicon depends on the size and GC content, but generally start and end temperatures of 60 degrees Celsius and 95 degrees Celsius are appropriate once the melt is performed.
Analysis of the fluorescent melt curves typically requires normalization of the variation of the different sample curves using pre and post melt regions as standards. This improves comparison of results from different samples in which the variation of fluorescence is related to minor experimental variations. Each pair of temperature lines for normalization should be placed approximately one degree Celsius apart.
The data can be represented in two ways by a graph that shows the melt curve profiles files, or by a graph that shows a subtractive difference plot in comparison to a reference sample following HRMA analysis mutations or transgenes can be detected. Two different mutations in the EIF two B five gene are noted by the red and blue curves. These mutant samples are identified by their significant difference in their melt curve, shapes, or change in fluorescence when compared to the standard wild type curves.
This example of four different genotypes in a single collection of embryos illustrates the power and sensitivity of the method. Once master, this technique can be done in less than eight hours. After watching this video, you should have a good understanding of how to perform HRMA for efficient large scale screening of zebrafish genotypes.
