1.1 Genotyping with PCR
1.2 Sanger Sequencing
2 Material and Methods
1.1 Genotyping with PCR
Manipulating an organism's genome usually includes the insertion of an DNA fragment (Strecker et al., 2019). This insertion can be of many different kinds, with several effects on the gene expression of the organism. Depending on the position of the insertion, it can result in a knock-out, knock-down, no effect or even an overexpression of the gene it was inserted into. To detect the resulting changes of the genome genotyping is used. With genotyping it is possible to detect differences in the genome of an organism compared to another individual. Therefore, no DNA-sequence is defined, but only the difference between two genomes. Also, it is not necessary to define an individual's genes. If one compares the genome of a genetically engineered organism with the wild type, it is possible to determine if and where the insertion took place, since a difference in their genomes should be detected at the locus of insertion (Kennedy et al., 2003).
There are many different methods with which genotyping can be performed. For this experiment, we used the polymerase chain reaction (PCR). This method includes the exponential amplification of very small DNA amounts in a series of temperature cycles. These cycles of low and high temperatures are performed by a machine called thermocycler. The reactants that are loaded together into the thermocycler include a double-stranded DNA template that should be amplified, nucleotides that are the building blocks of the DNA, DNA polymerase that assembles the nucleotides and primers that bind the template DNA and define the starting points for the polymerase. In the first step, the thermocycler heats the reactants to denature the DNA and therefore separate the double strands into single strands. In the second step, the temperature is lowered to allow primers to bind the single stranded DNA. These single strands now serve as templates for the DNA polymerase that binds the primers and assembles new complementary strands using the free nucleotides in the solution. Then, the thermocycler heats up the reactants and the cycle starts all over again, but with a doubled amount of template DNA, which therefore exponentially amplifies (Saiki et al., 1985).
The resulting amplification products are then compared on an agarose gel to detect differences in the genome of the organisms.
1.2 Sanger Sequencing
Some mutagenesis methods like CRISPR/Cas9 sometimes only result in single base insertions or deletions (Veres et al., 2014; Cradick et al., 2013), or point mutations (Wang et al., 2013). These are difficult to detect with genotyping. To obtain a more detailed impression of where the insertion of DNA took place after genetically manipulating an organism, sanger sequencing used. It was developed in 1977 by Frederick Sanger (Sanger et al., 1977). The method requires single-stranded DNA that should be amplified, deoxynucleotides that are the building blocks of the DNA, modified di-deoxynucleotides that terminate the DNA strand elongation, DNA polymerase that assembles the nucleotides and primers that bind the template DNA and define the starting points for the polymerase. The chain terminating di-deoxynucleotides lack a 3'-OH group that causes the polymerase to cease extension of the DNA (Sanger et al., 1977). Nowadays, they are usually radioactively or fluorescently labelled to be easily detected by automated systems (Smith et al., 1986).
In general, the methodical procedure is similar to the PCR, with a thermocycler separating the double stranded DNA with heat and then cooling down the reactants to allow the assembly of complementary DNA strands by the polymerase. The difference lays in the modified di-deoxynucleotides that cause the elongation to stop. For sanger sequencing four different DNA amplifications are performed, each containing all four normal nucleotides and one of four modified nucleotides. When amplifying the template DNA, the resulting copies will only be elongated up to the point a modified nucleotide is built into the strand. At this point the polymerase stops and the end of the product is marked with the modified nucleotide, which can later be detected via its radioactivity or fluorescence. Because this reaction takes place millions of times during amplification, one ends up with lots of DNA strands that have different lengths, depending on where a modified nucleotide was inserted. Repeating this process with the modified versions of all four nucleotides separately results in a collection of DNA strands with different lengths that are marked at their end and therefore contain the information which base is located at the end point of the product. Aligning these strands at their start and analyzing the marked nucleotides results in a fully sequenced DNA, where the location of every nucleotide can be determined exactly (Sanger et al., 1977).
2 Material and Methods
For the experiments, samples from Arabidopsis thaliana plants with two different genotypes were used. The wild type Col-0 as a control and the rs31 knock-out mutant that was transformed with the CRISPR/Cas9 system. Then genomic DNA was extracted from both Col-0 and rs31. These DNA samples were amplified via PCR using different primers to achieve specific PCR products. Cas9 was amplified using the primers JS99 and JS103, the target gene RS31 with the primers TW108 and JS133 and the selection marker hygromycin with the JS155 and JS156 primers. The resulting DNA fragments were compared using a 2% agarose gel.
Subsequently, the PCR products of the RS31 gene were cleaned up, but only for plants where Cas9 could not be detected. Then they were sequenced at eurofinsgenomics.com and the sequencing results of Col-0 and rs31 were compared to identify mutations.
Afterwards, RNA from rs31 containing Cas9 and Col-0 was isolated using a Roboklon kit. The RNA was reverse transcribed to complementary DNA which is suitable to perform the following PCR. Here, Cas9 and the hygromycin resistance gene were amplified and then compared with the genomic DNA PCR products on an agarose gel. More detailed information about the methods is found in the script on pages 38-46.
3.3 Sequencing Analysis
Figure 3 shows the results of the sequencing analysis of one Col-0 Arabidopsis thaliana wildtype and five rs31/rs31a mutants in comparison. In these plants no Cas9 was detected in the genome. Figure 3 atRS31 shows the DNA sequences that were amplified in the PCR with a AT3G61860 primer, that binds at the beginning of the RS31 gene. Here, there was a difference in the sequences between Col-0 and rs31/rs31a. All five rs31/rs31a plants had an additional cytosine base at the same locus, compared to Col-0. Figure 3 atRS31a shows the DNA sequences that were amplified in the PCR with a At2G46610 primer, that binds at the beginning of the RS31a gene. Here we also detected a difference between the Col-0 and rs31/rs31a plants. In all five rs31/rs31a samples an adenosine base was missing at the same locus, compared to Col-0. These mutations were located close to the protospacer adjacent motif, which is the binding site for Cas9.
- Quote paper
- Falk Deegener (Author), 2020, Plant mutants, arabidopsis genotyping and RNA. Generation and analysis, Munich, GRIN Verlag, https://www.grin.com/document/1131628