Considering that we hypothetically designed an experiment, we have an x gene, and we have the mutant version of this gene with a single nucleotide mutation in one of the exons.
What kind of an experiment should we construct if we want to isolate the wild-type and mutant versions of this gene, as well as the isolations of the wild-type and mutant proteins produced from these genes?
I mean, should we do the PCR of these genes with the same primers or analyze proteins with the same antibody? Should the antibody we use be polyclonal?
Thank you!
If you want to isolate gene segment, PCR with specific primer is fine. Sometimes universal primer does not work, then you have to design your primer. But if you are interested in expression or coexpression analysis, RNA seq or microarray might help you.
I agree with Shamsunnahar Mukta . You should design specific primers which are able to amplify the wt and mutant segment. After that, cloning is necessary to put the gene in the vector which then is amplified through mini and maxi prep.
Regarding protein isolation, I think it's difficult to find an antibody specific for the mutated protein probably mass spectrometry might help you.
If you know the exact position of the mutation within the gene you can easily use allele-specific PCR such as ARMS, and RFLP. These techniques are easy to perform and low cost. But if you have a gene that the exact point mutation position is unknown it's better to sequence the gene with the Sanger sequence technique. However, if the gene itself is unknown better to use high throughput sequencing technique (NGS).
About the protein, it is hard to tell since you must determine the position of the mutated nucleotide within the codon which some alterations make a huge change in the amino acid but some may not change the amino acid.
Dear Shamsunnahar Mukta , thank you for your answer.
In fact, I guess it would be more correct to think of it as an mRNA rather than a gene segment. PCR may not be suitable as the length of the entire gene will be too long, but if we can switch from mRNA to cDNA, it might make more sense maybe. What do you think about this? Since it is a point mutation, it feels like a single primer should work but I am not sure.
Dear Rosa Gallo , thank you for your answer.
As I mentioned in the previous answer, this is a point mutation so I think the same primer should be able to work. For the protein, perhaps a polyclonal antibody can be chosen, or the same antibody can be made to work by doing something like antigen retrieval or something. Do you think this is possible? If I do cloning, how will I get pure protein? Would precipitation with beads be appropriate?
Dear Behrouz Mollashahi , thank you for your answer.
Does it mean that even if we know the exact location of the mutation, we cannot use the same primer?
I agree with you about the protein, but suppose there is a "mutant protein" in this example, assuming the mutation made an amino acid change.
for differentiating mutant gene from wild type, you can use the same pair of primer as long as The position of mutation is flanked by the forward and reverse primer. This work with the RFLP method. for ARMS technique is somehow different, you should design primers for exact mutation and wild type, meaning you need 2 pairs of primers ( one pair of outer primer and one pair of inner primer)
Gülşah Torkay Perhaps if you overexpress the mutant protein, you might be able to isolate it by a bead-based system where you have attached a polyclonal antibody. I am not expert in protein isolation, but try to search some protocols on Nature. Good luck!
It is important to keep in mind the aims of the study to achieve short-term and long-term goals when designing such studies. Over many years, we have been studying functional aspects of proteins and their variants, that associated with a variety of human diseases. Using RT-PCR, we cloned individual protein variants into mammalian expression vectors for a range of in vitro studies. Because antibodies are not likely to distinguish between a wild-type protein and its mutant differing only at one amino acid position, we tagged individual protein variants with small epitopes. In our studies, tags like V5, His, HA and FLAG at the C-terminal end of a protein have worked very well in a range of assays, such as Western blotting and ELISA. Antibodies against these tags are commercially available. His tag provided a good purification option for recombinant proteins that were produced using the baculovirus system.
Dear Nasim Yousaf thank you very much for your detailed answer.
Epitopes could really work for me, I think.
Have a nice week.
Regarding the the genes, if you know the point mutation, one can use PCR - SSP (Sequence specific Primer) using two primers pairs, one pair specific for wild gene and other for mutant gene together with an internal control. You can also use Restriction fragment length polymorphism (RFLP) if there is restriction site specific for a restriction enzyme .If facility availiable,you can sequence it by sangar sequencing. The later is also useful if not know the exact position of the mutation.
Regarding the proteins, clone the wild and mutant genes separately into vectors and the extract them using mini or medi prep. Then do transient transfection for both the mutant and wild types separately and then obtain the proteins. You should determine the method used for colecting these proteins. If the point mutation is not deletion or insertion , you can use same monoclonal antibody for both wild and mutant proteins as long as the antibody is escaping the mutant area . Then do western blot and see how the mutation can affect the protein . For example the mutation may change the size or relative expression of the protein
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