Pretend you are a genetech scientist working on a cystic...

Pretend you are a Genetech scientist working on a Cystic Fibrosis (CF) treatment. You want to create a mutant DNAse I in order to cut the DNA found in the mucus of CF patients. The goal is to decrease the viscosity of the mucus in the lungs through the chopping of DNA. Your project partner comes to you with the following proposals for mutating the DNAse protein. Which set of mutations would you move forward with if you could only choose one?

Transcript

00:01 So for this problem, we are to use the given pcfd3 vector to express single guide rna in drosophila to create a knockout of this nipp1 gene given in the problem statement.

00:18 So the first part of this problem, part a, asks us to find the two pam sites within the sequence.

00:26 We know from the blurb before this problem that the canonical pam site is 5 prime, n -g -g, n being any nuclear tide.

00:39 So all we need to do is scan through the sequence to find any n -g -gs.

00:46 The two that are present in this sequence are near the end.

00:50 So at the end of the exxon, we have t -t -t -g, and then in the first intron we have another t -g -g.

01:05 This first part of the problem also asks us which site we would use to produce a null allele and why we would prefer that site.

01:14 So this first pam site would be the ideal one for us to design our kass9 system around because it would cause a bubble within this exxon for it to cut.

01:29 And hopefully we would get a break causing a frame shift somewhere within this coding sequence and leave.

01:35 Us with a null allele.

01:38 The next part of this problem, part b, asks us to determine the percentage of imprecisely repaired genes that we could say with confidence would be null alleles.

01:52 So the first possibility is that it repairs exactly where it broke, so right at zero, and it repairs without any addition.

02:01 However, this problem statement said that we are only to consider the percentage of the imprecisely repaired chains.

02:09 So that's eliminated as a possibility.

02:12 The next option is that it can add up to six nucleotides.

02:17 However, if it adds three or if it adds six, these could possibly still maintain function because this would cause a frame shift of a full three nucleotides, which would be a single amino acid.

02:32 Now the same goes for the removal of three or the removal of six.

02:38 Now that could still maintain some function.

02:42 It's unlikely, but it could be possible.

02:45 So now we know that with the addition of 1, 2, 4, or 5, and the subtraction of 1, 2, 4, or 5 nucleotides, we would have a frame shift that would most certainly disrupt function.

03:03 So that means we had 12 possibilities.

03:06 So the addition of six or the subtraction of six nucleotides and then eight known nulls.

03:15 So now it is just simple math.

03:17 8 over 12 is equal to 66 .6 % of the imprecisely repaired genes would be null alleles.

03:30 Next we are to diagram the cut pcfd3 vector.

03:39 And where to ignore the blue segment that would be removed.

03:43 Now this would be cut using the bbs1 recognition site.

03:48 So we have five prime end and three prime ends.

03:57 So this would be the left site or the orange side.

04:02 So we've got t, t, t, a, c.

04:08 C.

04:09 And then we've got our matches.