Hi,
I am currently doing a research on Inborn errors of metabolism in Sri Lanka and for that, I need to develop a method to identify few SNPs and a single point deletion using allele specific PCR method. And I am having troubles with primer designing for the deletion. If any of you can give me a explanation, that will be a great help.
Thank you.
for pcr to work the binding of both primers must be perfect at the 3' end of each primer. So when you have a small sequence difference if you design primers that exactly match the base change either at the 3' end or with the polymorphism at the N-1 or n_2 position of the primer then the 3. end of the primer cannot bind to the dna and pcr fails except when the sequences match exactly. It is then a matter of finding an annealing temperature that allows one primer to work and the other primer to fail. Another way of detecting single base changes is the dcaps method where you deliberately add a second mismatch to the primer so that when it amplifies the combination of natural polymorphism and introduced base change creates a restriction site so the identification of an allele is done by cutting the pcr product and whether it cuts or not defines the base changes present. Primer design and an explanation of how it works is found at http://helix.wustl.edu/dcaps/dcaps.html for design or
https://www.ncbi.nlm.nih.gov/probe/docs/techdcaps/ for both design and explanation
for pcr to work the binding of both primers must be perfect at the 3' end of each primer. So when you have a small sequence difference if you design primers that exactly match the base change either at the 3' end or with the polymorphism at the N-1 or n_2 position of the primer then the 3. end of the primer cannot bind to the dna and pcr fails except when the sequences match exactly. It is then a matter of finding an annealing temperature that allows one primer to work and the other primer to fail. Another way of detecting single base changes is the dcaps method where you deliberately add a second mismatch to the primer so that when it amplifies the combination of natural polymorphism and introduced base change creates a restriction site so the identification of an allele is done by cutting the pcr product and whether it cuts or not defines the base changes present. Primer design and an explanation of how it works is found at http://helix.wustl.edu/dcaps/dcaps.html for design or
https://www.ncbi.nlm.nih.gov/probe/docs/techdcaps/ for both design and explanation
Hi Nadeesha,
in addition to Paul's advices, when you will have designed your primers, you should test in silico your primers just to be sure you selected specifically the right position and maybe see if SNP and deletion are known in databases. try to use the UCSC website (http://genome.ucsc.edu), for its tools as "in silico PCR" and its genome browser with optional tracks (for SNP and indels viewing).
fred
In addition, you can try melt-curve based genotyping, if you have access to a qPCR machine. If your PCR products are small enough, you can detect a single base-pair deletion and any SNP variants. This does take a LOT of design, controls, & trouble-shooting.
It might be easiest & cheapest to simply amplify and Sanger sequence.
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