Discussion Questions

Teachers: The following questions summarize the use of the tool, and a discussion in class is recommended.

23. Based on what you have learned in this task about Primer3Plus: what is the principle by which Primer3Plus works? Specify for what purposes the tool can be used for and how.

Teachers: Primer3Plus is a tool for designing and picking primers from DNA sequence, for example for the amplification of a DNA segment (or a complementary DNA sequence) using PCR. Based on an input DNA sequence and default settings, the tool designs primer pairs. The user can also define limitations as for where on the sequence the primers should be located, or for the target segment for amplification within the sequence- namely the region that will be flanked from both sides by primers.

24. In this task, we saw that not any random sequence of 20-25 long nucleotides can be used as a primer. Specify some essential characteristics of sequences that make good primers and explain how they affect the conditions of the PCR.
Teachers: A typical primer has several essential characteristics. First, the sequence of each primer should be complementary to a sequence of one of the DNA strands and in the correct orientation: the left primer binds the anti-sense strand and the right primer binds the sense strand. Second, the melting temperature of each primer, determined by the length and composition of nucleotides of the primer, should be similar for both primers. This temperature sets the temperature of the annealing stage in the PCR and should usually be 54-60°C. The location of the primers over the template defines the ends, and subsequently, the length, of the amplified segment (PCR product). For a longer amplified segement, a longer elongation time is required at the PCR. Also, the primers should not include complementary sequences within within the same primer and between the pair, so they will not bind each other instead of the template strands. Overall, the conditions for a PCR method are generic, and the differences between one PCR reaction and another are usually the annealing temperature and elongation time, set according to the qualities and location of the primers.

25. One of the features of the tool is that apart from its ability to design primers it shows the location of each primer on the template, even if the user determines by itself the sequence of a primer. Can you recall working with other bioinformatics that have similar feature? What do they have in common?
Teachers: In fact, in order to find the location of a primer within the template, Primer3Plus uses a method called “sequence alignment.” This method is used by for example BLAST tools, the searching engines for squery sequences (in this case, it searches and aligns the the nucleotide sequence of a primer against the template sequence). It also used by ClustalW, a tool that aligns multiple sequences, to indicate areas and positions of identical, similar or different sequences (in this case, the nucleotide sequences of the primers and the template). Of note, with both the BLAST tools and ClustalW, the user can analyze either nucleotide (BLASTn) or amino acid (BLASTp) sequences, Primer3Plus analyzes only nucleotide sequences.

In this task, we used the bioinformatics tool Primer3Plus to design primers for a PCR to diagnose a CFTR gene mutation. As mentioned, it is crucial to diagnose the specific mutation in the CFTR alleles in order to adjust the proper medication to a patient. At first, we designed primers from both sides of the mutation site, to amplify the DNA sequence that is different between the normal and mutant alleles. We can sequence the amplification products and compare them to known sequences in the database, to determine whether it is a normal or a mutant allele. Later on, we took the “mutation-dependent amplification” approach, in which the sequence of one primer matches the unique sequence of the mutation site from the mutant allele, and thus an amplification product will only be obtained for the mutant allele (in contrast to a DNA template from a non-carrier subject, referring to the specific mutation).
Primer3Plus allows to design primers according to definitions and settings of primer length, melting temperature, composition, etc. In addition, the user can define limitations, such as the region in the sequence aimed for amplification (Targets) or a region where the primers should be placed in (Included Region) or out of (Excluded Region). The tool also allows the user to dictate the sequence of the primer; Primer3Plus confirms that it meets to the settings of primer and design a pairing primer to match.
Due to the multiplicity of mutations in the CFTR gene, for each mutation, not necessarily the point mutations, there is a specific pair of primers designed for its detection. For genetic diagnosis, the template from each individual should be tested using all the different primer pairs aimed for the detection of the different mutations. Sometimes, for the detection of certain mutations, an RT-PCR method is used, in which the template DNA in use was originated from RNA. This approach works better when the effect of the mutation is more prominent at the RNA level (for example, mutations that affect splicing or RNA stability). Primer3Plus can also be used to design primers for this approach. As we saw in this module, a successful use of PCR depends on choosing the appropriate experiment strategy and on an intellignet and careful design of primers.