It would be quite useful for breeding traits that are technically difficult or costly to be determined, such as disease resistance, mineral content. It's a quite simple, cost-effective, and high-throughput technique. To begin, place four leaf discs into each well of a 96-well PCR plate.
Add 50 milliliters of buffer A solution into each well. Freeze the plate in a minus 80-degree Celsius freezer for 10 minutes. Then, defreeze the plate at room temperature.
Incubate at 95 degrees Celsius for 10 minutes. Add 50 microliters of buffer B to each well, and mix well by vortexing. Centrifuge the plate for one minute at 1, 500 times g.
Collect the supernatant as DNA for PCR. First, use the DNA supernatant from one well as PCR template to optimize the annealing temperature. Add reagents and one microliter of DNA supernatant into each PCR well.
Place the PCR tubes in a gradient-capable thermal block, and adjust the heating program according to the manuscript to determine the optimal annealing temperature for each target fragment. Perform electrophoresis for the PCR product on 1%agarose gels to examine amplicons. Determine the optimal annealing temperature that enables specific amplification of the target fragment.
To perform HRM analysis, combine reagents, including one microliter of 10 times fluorescence dye and one microliter of the DNA supernatant in each well of HRM-compatible plates. In each plate, include one wild-type parented sample and one negative control without DNA. Add a drop of mineral oil to each well to prevent evaporation.
Then, seal the plate with adhesive film, and centrifuge at 1, 000 times g for one minute. Run the PCR using the optimized annealing temperature. Now, remove the adhesive film from the plate, and insert the plate into an HRM machine.
In the software, select New Run from the file menu, or press the Run button at the top of the screen. Specify the starting and ending temperatures for the melt from 55 to 95 degrees Celsius. Select samples for high-resolution melting analysis, and exclude samples similar to the negative control.
Normalize the melting curves to have the same beginning and ending fluorescence. Visually confirm that the lower minimum and lower maximum temperature cursors are in a region of the curves. Keep the delta F, difference of fluorescence, level at the default setting of 05.
From the Standards selection list, select the common versus variant, and choose the normal sensitivity. Select H12 to use the wild type as the control. Samples with a delta F value greater than 05 are considered to contain mutant plant.
Then, using a CTAB method, extract DNA from each of the four plants from the mutant pool with the melting curve significantly different from the wild type. After quantification using a spectrophotometer, adjust the DNA with the NanoDrop 2000 to a final concentration of approximately 25 nanograms per microliter. Add 0.4 microliters of PCR primers into the DNA sample in the PCR tubes, place them in a thermal cycler, and adjust the program to amplify the target fragment.
Then, send the PCR products to a company for sequencing. After Sanger sequencing, compare the molecular lesion by comparing the sequences between the M2 plant and the wild type. In this study, HRM scanning and analysis of M2 seedlings for mutations in OsLCT1 or SPDT genes showed most samples had melting curves not significantly different from the wild type with delta F less than 05.
The mutants showed significantly different HRM curves with delta Fs greater than 05 at temperatures of 90 to 92 degrees Celsius and 81.5 to 83.5 degrees Celsius, respectively. The mutation type and position on the genes from 4, 560 M2 seedlings were confirmed by the sequencing chromatograms. A heterozygous site with a mutation of G to A at 4, 304 base pairs of OsLCT1 was identified.
One single nucleotide A insertion appeared at the 4, 240 base pairs of OsLCT1, and a TTC trinucleotide deletion was observed at the position of 5, 948 to 5, 950 base pairs of SPDT. I optimize the PCR before HRM analysis. The dye in the gel is a bit harmful.
Remember to put on gloves when running the gel.
