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3 years ago
Hi Folks,

I'm really struggling with a PCR & restriction enzyme question that's part of my formative (unmarked) work for the summer months in preparation from an Applied Biology course at University.

The entire set of questions are set out on an instruction sheet so I'll try to replicate them here as I'm struggling to fully understand how PCR works and exactly what it's asking me in terms of the restriction enzyme affected by a point mutation.

Just to give additional information this PCR and restriction enzyme work is specific to identifying cAIS (Complete Androgen Insensitivty Syndrome) in a 17-year old.

The PCR primers used to amplify the gene that I'm looking at are: -

Primer 1  (forward):          5’   gggcc ccgcagcagc    3’

Primer 2  (reverse):            5’   cggatactgcttcctgc   3’

The position of the point mutation within exon 2 is shown in bold below along with the primers in Orange:

5'  .. cacgttgtcc ctgctgggcc ccgcagcagc tgccagcacc tccggacgag gatgactcag ctgccccatc cacgttgtcc ctgctgggcc ccactttccc cggcttaagc agctgctccg ctgaccttaa agacatcctg agcgaggcca

gcaccatgca actccttcag caacagcagc aggaagcagt atccgaaggc agcagcagcg ggagagcgag  …..3'

The size of the PCR product that should be generated is 180 bp (base pairs) and I've identified a restriction enzyme that would be affected too (BspTI).

The section I'm stuck on is the following:

11. Your chosen enzyme (as it is affected by a change in this nucleotide) could be used in a test digest of the PCR product in order to detect individuals that carry the point mutation   

Give the size of the DNA fragments that would be generated after digestion with your chosen enzyme of PCR samples amplified from:-

12a.   The patient

12b.   The patients father

12c.   The patients mother.

I just don't have a clue how to work this out and I'd appreciate any help that could be given in answering this in a way that I can clearly see how PCR works and what I'm doing in this digest in order to get the right answers. NB: I'm not just after the right answers here, I'd like to really understand how to arrive at these answers.
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