I would like to know how we can assign a nucleotide of a primer with ambiguity codes.
Take this primer which targets the nosZ gene in bacteria for example;
5'-CGCRACGGCAASAAGGTSMSSGT-3' (Henry et al., 2006).
R can be either G or A; S can be G or C; M can be A or C.
How can I define which base to be chosen for the ambiguous nucleotides?
Hi Muhammad,
Degenerate bases are used to create equimolar mixtures of primers with variation at specific points, so in the example you use above 50% will start CGCG...and 50% will start CGCA...The more degeneracies in a primer, the more complex the variations and the lower quantity there will be for each variant.
You do not need to assign a base to degenerate positions, the company you use to manufacture the primer will produce all the necessary variants based on the sequence you provide which lists the degenerate bases
Hope that helps
Barry
Hi Muhammad,
Degenerate bases are used to create equimolar mixtures of primers with variation at specific points, so in the example you use above 50% will start CGCG...and 50% will start CGCA...The more degeneracies in a primer, the more complex the variations and the lower quantity there will be for each variant.
You do not need to assign a base to degenerate positions, the company you use to manufacture the primer will produce all the necessary variants based on the sequence you provide which lists the degenerate bases
Hope that helps
Barry
You cannot assign a base to any of the positions in advance of your pcr. Degenerate primers are used when there are mixed populations of similar organisms and you want to detect all of them , In your example different organisms have 6 SNPs under the primer position so you get a degenerate primer made which is actually a mixture of 64 different primers which should anneal to an amplify all of the likely sequence variants present in this template mixture , If you were looking for one particular amplimer from a sub species which had a unique sequence under your primer then you could assign specific bases and most likely similar organisms would not amplify
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