Question

You are developing primers for a wildlife forensic case and want to identify both species and distinguish between individuals.
a) For which type of analysis would you want to develop/use degenerate primers? Why?

b) If the primer you developed for species ID is 14bp, the mitochondrial genome is 18500 bp, and the nuclear genome is 3.2x109bp, how many times would you expect it to bind to each of the respective genomes assuming the same primer could bind to both the mtDNA and nDNA genomes? (2marks)

c) Comment on the specificity of the primer if this primer was intended to amplify only a mtDNA region and not a nDNA region.

Answer 1

a.) For genotypic analysis, degenerate primers should be used. Degenerate primers are the mixture of similar, but unidentical primers that can amplify a same gene from different species. If we don't know the exact genetic makeup of wildlife, then we must use degenerate primers, as it'll be quite difficult for us to design specific primers for each wildtype species, as their genome will vary between different species.

b.) Attachment sites for the given primer is expected to occur, on average, once every 4^14 = 268435456 bp.

(Since there are only four possible nucleotides, hence the base is 4. Similarly, the primer length is 14 bp, hence the exponent is taken as 14)

For mtDNA genome:

The expected frequency for this primer is much greater than the entire length of mitochondrial genome. Hence this primer will bind only once.

For nDNA genome :

Expected Frequency = Genome Length / Attachment Frequency

= (3.2 x 10^9 ) / 268435456 = 11.92

It gives approximately 12 possible sites in the 3.2 x 10^9 bp of nucleotide sequence that makes up the nDNA genome.

c) As mentioned above, the expected frequency for this 14 bp primer is much greater than the entire length of mitochondrial genome. Hence this primer will bind only once, and the specificity will be maximum. Therefore the final amplification product will be single and highly specific.