Question

This is genetics, please give detailed answers,

QUESTIONS:

1. In looking at the class data, do you think the experiment was overall successful or not. Describe in a few sentences for your reasoning.

2. Groups 3 and 4 had lawns of Ade+ yeast suggesting that their culture was not mutant. Interestingly, group 10 also use a colony from this same mutant, which they had isolated earlier in the semester. From group 10’s previous work, this mutant was found to be a C>T mutation that changed a glutamine to a stop codon. Develop a hypothesis based the occurrence of a second mutation that could explain the occurrence of Ade+ colony arising from the original ade2 mutant. (Do not consider a simple back mutation.)

PART III CRISPR-Cas9 mediated repair by Homology Directed Repair (HDR) The experiment done in class was designed to use CRISPR-Cas9 to promote Homology Directed Repair of selected ade2 mutations. The following conditions were 1) 10 μ| g RNA + 25 μ1 fragment + 10 μ1 carrier DNA 2) 10 μ1 gRNA + 10 μ1 carrier DNA 3) 25 μ1 fragment + 10 μ1 carrier DNA 4) 10 μl carrier DNA only 1. If HDR was successful what would the expected result be. 2. In looking at the class data, do you think the experiment was overall successful or not. Describe in a few sentences for your reasoning. Bonus question (up to 6 points added to final score) Groups 3 and 4 had lawns of Ade+ yeast suggesting that their culture was not mutant. Interestingly,group 10 also use a colony from this same mutant, which they had isolated earlier in the semester. From group 10's previous work, this mutant was found to be a C>T mutation that changed a glutamine to a stop codon. Develop a hypothesis based the occurrence of a second mutation that could explain the occurrence of Ade+ colony arising from the original ade 2 mutant. (Do not consider a simple back mutation.)

Answer 1

1)according to the data groups 10,11,13,17,18, had positive results and showed conversion of ade2 wild type cells from mutant cells which are capable of producing adenine. But overall the results were low in percentage as compared to the whole data. Maybe the efficiency of the guide rna or the guide rna, target sequence would be repetitive leading to negative.   

2) group 10 had used a cell which was converted to mutant because of c> t , and now a mutant to ade 2.

The group 10 had 2 colonies growing for just the presence of carrier fragment which is not possible to happen.

It could be because mutation.

The types of mutation that could have lead to such sequence change are,

1) insertion or deletion because of the rapid multiplication of the genetic material. Or because of the chemicals in the media (eg ethidium bromide, etc)

2) DNA replication lag, which occurs when the single strand is replicated and due to some mechanical or enzymatic error the RNA polymerase detaches and starts replicating again from the already replicated part leading to increase in the no of particular nucleotides present in that area.

And finally leading to increase pyrimidine more than purine.

As in most of the functional genes the open reading frame has more A & T than cytosine and guanine. As they consume more energy to hydrolyse and form bond again.

The above can be the reasons of random, autonomous mutation which can lead to production of wild type strain spontaneously from mutant.