Example of PCR in Research: Making Better Grain Crops
Backstory
Corn and sorghum are two of the world’s most important grain crops. They are biologically designed to make seeds that are packed with stored starch. As wild plants, the starch would be carbohydrate reserves for the germinating seed. This is also a key role for stored starch in the domesticated versions of these plants. The global popularity of these grain crops, however, is the value of sorghum and corn seeds as a carbohydrate food source for animals.
The proteins made and stored in corn and sorghum seeds have the primary function of providing a framework for starch storage. Genotypes grown to be fed as a carbohydrate source for livestock or people are selected because they can pack starch tightly and form hard kernels. These seed storage proteins (kafirins for sorghum, zeins for corn) are chains of amino acids that contain 18 of the possible 20 amino acids. The two amino acids they lack, tryptophan and lysine, happen to fall into the ‘essential’ category -- if you are a monogastric animal living entirely on sorghum of corn grain, your growth will be stunted because of a tryptophan or lysine amino acid deficiency.
Researchers have discovered genotypes of sorghum or corn which lack the ability to make the kafirin or the zein storage proteins their seeds. These mutants make more copies of other seed proteins which contain tryptophan or lysine but they also make softer seeds that store less starch. A better genetic improvement is needed to create sorghum and corn genotypes that can both store starch in densely packed seeds and produce seed proteins that contain some of the essential lysine and tryptophan amino acids. Figure 9 shows the title and authors of one group of geneticists who discovered a way to create a ‘better’ mutation with the use of a technique called gene editing.
Figure 9. Kafirin genes 11 through 16 on a chromosome. As an abbreviated example of 20 repeats of the kafirin gene, kafirin genes numbers 11 through 16 are shown one after another on a double-stranded segment. The research article below is by Li et al. titled “Editing of an Alpha-Kafirin Gene Family Increases Digestibility and Protein Quality in Sorghum”. Created by D. Lee, 2023.
Sorghum makes a lot of copies of the kafirin protein in the seed, in part because it has 20 copies of the gene repeated in the chromosome. The geneticist’s goal was to edit some of these copies so that they no longer coded for the seed storage protein. These are called knockout mutations. The seed would make less of these proteins but still make enough for good starch storage. One of the gene edits the team created was a knockout mutation in kafirin #14 (Fig. 10). The edit deleted three nucleotides from the gene sequence.
Figure 10. Kafirin gene #14 edits. The normal version of the kafirin gene is Tx430. E6 is the mutated version of the gene that has a deletion of “TGC” in the 27th, 28th, and 29th bases of the kafirin gene. Created by D. Lee, 2023.
Using PCR and Gel Electrophoresis
By using PCR primers that targeted this gene, they could make copies of this kafirin gene. The copies would be 100 bp in the original Tx430 sorghum genotype and three nucleotides shorter (97) in the gene edit generated mutant they called E6.
Gel electrophoresis could then be used to visualize the DNA copies and determine if a plant has the normal kafirin, the E6 mutant or both. A hypothetical result is shown in Figure 11.
Figure 11. Gel electrophoresis detection of DNA from the kafirin gene. Lanes include a ladder, sample 1, sample 2, sample 3, a positive control, and a negative control. Created by D. Lee, 2023 using work by M. Hanneman.
We can see that lanes 1 and 4 have only a 100 bp DNA copy, lane 3 has only the 97 bp copy and lane 2 has both copies. The positive control is a PCR test done on the original Tx430, the negative control is a PCR test with all the components except of the sorghum DNA sample.
Question #2 to test your understanding
Another PCR + Electrophoresis analysis the geneticists applied was to use the specificity of the PCR primers to differentiate the normal from the mutant kafirin alleles (Fig. 11).
Figure 11. Gel electrophoresis detection of DNA from the kafirin gene. Lanes include a ladder, sample 1, sample 2, sample 3, a positive control, and a negative control. Created by D. Lee, 2023 using work by M. Hanneman.
Figure 12. The forward and reverse primers for detecting kafirin edits. Notice the reverse primer on the right outside of the edited region and the forward primer within the region where the edits take place. The forward primer sequence will target the normal Tx430 allele sequence and generate a PCR copy of about 50 bp using the same reverse primer. The primer will not bind well to the DNA sequence in the E6 mutant allele as the deleted nucleotides change the sequence. Thus, the primer pair will make copies of the normal allele but not make copies of the E6 mutant. Image created by D. Lee, 2023.
Assume you have samples of sorghum plants that are homozygous for the normal Tx430 allele, homozygous for the mutant E6 allele with the three-nucleotide deletion and one sample of a heterozygous plant. Describe what you would observe on the electrophoresis gel if these PCR tested samples were run in lanes 1, 2 and 3 on the Figure 11 electrophoresis gel. Remember to include the expected nucleotide lengths of each band in the gel.
Keep reading when you're ready for the answers.
Answers:
- Homozygous Tx430 plant = a single band at about 50 bp
- Homozygous E6 plant = no bands
- Heterozygous Tx430, E6 plant = a single band at about 50 bp