I am going to manipulate KRAS gene from mammalian cancer cell lines for vaccine production, and the gene codes for the KRAS protein (189 amino acids). I've already chosen my own antigenic region which consists of 15 of the amino acids.
I will be doing reverse transcription-PCR (RT-PCR) later, and my question is should I only amplify the antigenic region (45 bps) itself, or the whole gene (567 bps)? Will there be any difference between both? Because I learned that KRAS mutation might occur in multiple codons (ie codon 12, 13, 61), depending on the type of carcinoma. Is it possible for one cell line to harbour multiple KRAS mutations?
FYI, I will clone the amplicons into a bacterial plasmid vector after performing RT-PCR.
Hi. i did not amplified 45 bp fragment until now but i synthesized 60 bp fragment. you can isolate your cDNA and after sequencing, (find mutation on it) synthesis your interest fragment (45 bp) it is more economical.
If your purpose is to produce antibodies (Ab) against the KRAS proteins (based in the transcript sequences) in the specific cell lines then I suggest you the following:
Perform the RT-PCR not with the whole gene, neither your antigenic fragment, design primers that could amplify about 200 bp that include your antigenic position in between (because short fragments are a little tricky to amplify).
When you got the whole sequence, translate your antigenic fragment and synthesize the peptides which as you say, are about 15 AA.
With the peptides you can produce the antibodies, in the system of your preference, Chicken eggs, rabbit etc etc (for ethical issues I have tried With chicken eggs and they have performed very good.)
Do a western blot to validate your peptides.
I hope it helps you to find the answer,
JUAN
Thanks Juan, I understand that amplifying short sequences is quite tricky, but my concern is that I'm afraid the genetic sequence from cell lines will mutate and change as the passage number increase. FYI I will be culturing various cell lines by myself too before doing RNA extraction, and without doing authentication on those cell lines, amplifying longer DNA sequences might be risky. That's just my thought.
- Badges
- Science topic