I have a CRISPR guide which targets near to the end of Exon 1 of a gene. When I am analysing the sequences my mutants, I can see that there are insertions and deletions in Exon 1, but since the guide targets near its end, the premature stop codon (PTC) is being introduced further downstream beyond Exon 1.
Theoretically, if I assume that the intron between Exon 1 and 2 is spliced out, the PTC gets introduced in Exon 2. But I am not sure whether that actually happens in reality. Can anyone explain?
Thanks in advance.
Are you changing the reading frame? To verify, you will need to either do western blot to show the absence of the protein, or analyze the mRNA by making a cDNA and sequencing to show the spliced mRNA now has altered the reading frame to essentially delete your protein.
Hi,
Insertions and deletions in one exon can indeed introduce a premature stop codon in the mRNA downstream of the mutation, as you alter the open reading frame (you shift it unless insertions or deletions are 3 bp of the same sign (+3, -3 or multiple of 3)..
Gregory suggested the most commonly used methods to study the effects of these mutations. You may also do a Protein truncation test (PTT), by performing RT and then PCR using a forward primer that contains T7 RNA pol promoter (very short sequence) and then doing in vitro transcription translation: analysis of the protein translated in vitro gives you an idea if a premature stop codon is inserted in your sequence.
Hi Paola, thanks for your suggestions. My query was whether a STOP codon can be inserted in the next exon (after the deletion takes place in the previous exon). Its more of a conceptual question. I eventually found out that it is possible using prediction tools.
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