Question

What are the 3 primer pair combinations that are considered standard when setting up a genotyping reaction to test for a mutation in an organism that is represented by a putative insertion?

Answer 1

Primer pairs consist of the upper and lower primers. Although two different primers may make up the primer pairs, it is important that the primers both have similar melting temperatures in order to facilitate a successful PCR reaction. Software is available which identifies potential primer pairs, providing their melting and annealing temperatures, along with other details, and also suggesting alternative options of various primer pairs.

Primers are always specified 5' to 3', left to right.
Primers for PCR and sequencing should be between 18 to 25 nucleotides in length.
Primers for PCR and sequencing should have a GC content between 40 and 60%, with the 3′ of a primer ending in C or G to promote binding.
The 3' end of the primer should be an exact match to the template DNA, because extension by DNA polymerase, during PCR, depends on a good match at the 3' end.
In the last 5 bases at the 3′ end of the primer, make sure that there are at least 2 G or C bases (GC clamp). G-C base pairs have a stronger bond than A-T base pairs (3 hydrogen bonds versus 2).
Where a restriction site has been added onto the end of a primer, typically, 5-6 nucleotides are added 5’ of the restriction enzyme site (aka a “leader sequence”) in the primer to allow for efficient cutting.

widely used strategy for detecting DNA sequence variants is allele-specific PCR in which one or both primers are designed to anneal at sites of sequence variation. Ideally, a primer whose sequence matches a specific variant should selectively amplify only that variant; however, in practice, significant mismatched amplification typically occurs. It is common practice to anchor the 3′ end of the allele-specific primer at the mutant base in order to selectively amplify the mutant template. This strategy reduces but does not eliminate amplification of the wild-type allele. The amount of this non-specific amplification has been found to vary widely depending on the particular base mismatch between the allele-specific primer and the wild-type sequence .The variability of non-specific amplification typically requires a process of trial and error when designing highly selective mutation assay.The assay method reported here utilizes a combination of allele-specific PCR primers, a blocker oligonucleotide to suppress amplification of the wild type allele, and a set of reagent design rules that consistently produce highly selective assays for a wide variety of single point substitutions, insertions, or deletions.

Modified assay by the acronym ASB-PCR (Allele-Specific Blocker PCR). Features of the method include the ability to detect mutations in either DNA or RNA with a high level of sensitivity and selectivity.