I am investigating the expression of one particular gene in a mouse and am interested in determining the exact mRNA sequence. Is there anyway I can sequence the mRNA transcript of this gene?
For example is it possible to isolate the RNA, reverse transcribe, then use sanger sequencing?
I can't see any examples of this being done and RNA-seq seems like overkill if I am just interested in a single gene.
Any suggestions welcome!
Yes. You can amplify the target gene using PCR. Direct Sanger sequencing is possible, but it is recommended to clone the gene in the cloning vector before sequencing.
Hi Kat
It is possible although you do not need to isolate your target RNA molecule. Simply extract total RNA, then convert them to cDNA then do a two-way sanger sequencing with a pair of specific primers to your desired RNA.
You may do it with a modification in case your target RNA is expressed at a low level. Please add 2pm of your reverse primer when you are making cDNA. It will facilitate to synthesis your specific cDNA.
Good luck
Hi Kat
Sanger sequencing is possible for only one gene, yes.
it depends on the length (500-600bp is the limit for sanger) but you can also design primers to do overlapping amplicons. if it's for human or other "higher" species, go to the UCSC (http://genome.ucsc.edu), the genome browser allow you also to see additional tracks as already designed and published primers.
fred
ps: further help needed, send me a message.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA