Question

4. qPCR using TaqMan probes have the advantage of being very specific for the target species, even when closely related species could also be amplified (e.g., African vs. Indian elephants). TaqMan probes are often 30bp in length. If you had a genome of 3 billion base pairs long: a) provide how many theoretical times that probe should bind to the genome b) explain how the TaqMan probe makes qPCR so much more specific than just having 2 primers to amplify a locus.

5. You have a control moose sample were your extraction yielded 57ng/ul eluted in 100ul. Create a dilution series of tubes to test how well the primers work for end point PCR for 1ng/ul, 100 pg/ul, 10 pg/ul, and 1pg/ul of DNA. Show your work (in the form of a flow chart) describing your dilutions to create each of the dilution tubes from the stock tube. NOTE: 2ul is the recommended minimum pipetting volume for each step in the dilution series.

Answer 1

as the length of primer increases the selectivity of primer increases. for example

if we create primers of 12 bp and 18 bp long and anneal them to human genome.

the specificity of 12 nt long primer = (4)¹² = 16777216 bp. means it will bind once if the sequence is this much long (16777216 bp).

means The times to find the same sequence into the genome is = 3*10⁹ / 16777216 = 196.68

and for 18 nucleotide long primer = (4)¹⁸ = 68719476736 bp. means it will bind once if the sequence is this much long

= 3*10⁹ / 68719476736 = 0.0477.

now if the primer length is 30 bp then it will surely bind once in a genome. that is why it is so specific.

2)