I am just wondering how possibly a mutation can cause any impact on splicing of a mRNA. I have this gene with 5 exons and I will be amplifying those exons for downstream sequencing application.
So do I have to sequence the introns in order to detect any splicing relevant mutations and if yes, which regions from introns and/or exons needs to be amplified and sequenced and ultimately how do you bioinformatically predict or determine if those mutations have any impacts on splicing?
Extract mRNA, synthesis cDNA, PCR the entire cDNA species (including as much of exon 1 to the final coding exon as possible). Run on gel, if there is aberrant splicing you will see a differently sized cDNA (or even multiple different sized cDNA) compared to a control cDNA, unless the cDNA is not stably expressed (ie undergoes nonsense mediated decay etc) in which case you wont amplify any product and will need to inhibit NMD (which is simple if you have cultured cells to extract the mRNA from) prior to extracting the mRNA. Once you have the gel run, excise the different band species from the gel and gel purify before sequencing. From this you will be able to determine where in the cDNA the aberrant splicing is happening and this can guide you in targetting those particular introns and exons to sequence more thoroughly from gDNA.
Thanks David. Well what my primary intentions are to amplify the exons and see if there are any mutations. Addition to that, if its possible I would also like to see if the splicing process is impaired. Because you may not find any mutations in the coding region, but if the splicing isn't working, you won't probably get the protein expression. So where exactly do you recommend to see the mutations specifically associated with splicing. Please note I'm not really trying to see the mRNA expression, but to predict that certain mutations in the intron region may cause impaired splicing. Thanks in advance and please note I am not a molecular biology person.
Extract mRNA, synthesis cDNA, PCR the entire cDNA species (including as much of exon 1 to the final coding exon as possible). Run on gel, if there is aberrant splicing you will see a differently sized cDNA (or even multiple different sized cDNA) compared to a control cDNA, unless the cDNA is not stably expressed (ie undergoes nonsense mediated decay etc) in which case you wont amplify any product and will need to inhibit NMD (which is simple if you have cultured cells to extract the mRNA from) prior to extracting the mRNA. Once you have the gel run, excise the different band species from the gel and gel purify before sequencing. From this you will be able to determine where in the cDNA the aberrant splicing is happening and this can guide you in targetting those particular introns and exons to sequence more thoroughly from gDNA.
First you need to also sequence your introns. To predict impact of mutation on splicing process you can use many tools. One of the tool ESEFinder predict the exonixcsplice enhancer motifs, which are necessary for splicing. You can find also the tools which predict the the motifs in intronic seqeunce such as branch point polypirymidyne tracts, splice sites, mutation in that motifs can affect splicing, for that purpose I've used ASD project from EBI, but I think isn't working anymore, but you can find some tools on expasy server. Of course this is only bioinformatic prediction, if you want to be sure if mutation affect splicing, you need to isolate DNA and RNA from control and mutated tissue, perform RT for cDNA. After that just do the PCR for all 4 samples with primers designed that way to be in exons, which are next to the intron with mutation. If you observe that product form mutated cDNA is the same length as product from gDNA, you can say that mutation affect splicing. PCR performed on the control DNA and cDNA show you if the gene is not alternatively spliced.
Hello Magdalena, Thanks. Quite frankly I am not really familiar with all the motifs you were talking about. However I will possibly be going with the RT & PCR for cDNA & gDNA to see the impact on splicing. As I had earlier mentioned my chosen primers will target the exons and few base pairs from introns in both sides. So would you kindly be able to tell me where exactly do I need to amplify and sequence in introns. Because sequenceing the entire introns is out of my current research scope. So if you can point me out what could be the possible sites in intron where a potential mutation may interfere the splicing process. So then I can do RT & PCR to compare the mutant one with the control. Really sorry about the trouble.
So the 5' and 3' splice site are the first two and the last two nucleotides in intron sequence (mostly AG-GT). Branch points and polyprimidyne tracts are localized in intron sequence but you need to use some tools to localized them, but for that you need the sequence of the intron. Exonic enhacer are in the exons 6-8 nucleotides motifs, which are mostly at the beginning or the end of exon sequence. If you don't want sequence the intron and only use RT PCR, PCR approach, the best is to design the primers in the two exons, where the interested intron will be in the middle. Of course if your whole gene isn't very long you can amplify whole cDNA and whole gene, or you can try do it this experiment for each fragment of the gene separately.
Thanks, my gene of interest is indeed a long one with really long intronic regions. Would you suggest me a free tool which can be used to identify those motifs and their locations of the gene I am interested in. A simple to use online or computer software which analyses my sequence/ FASTA or accession number of the gene and graphically shows me where exactly those motifs in that gene and how the splicing process works in that particular gene. Thanks in advance
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology