Hi everybody! I am carrying out an MLPA experiment and I have to confirm my data.
I have chosen Long-PCR approach to confirm the presence of deletions and to define their limits, but I have some doubts: can I achieve my aim if the PCR primers are designed upstream and downstream of the first probe and of the last probe related to the deleted region, respectively?
For example, the deletion encompass the exon 2, 3 and 4: the primer 2f is designed upstream of the probe number 2 and the primer 4r downstream of the probe number 4. So, using this conditions am I able to amplify and sequence the correct region to analyse the deleted region or I have to use the primer 1f and 5r?
I hope it's clear...I am looking forward hearing from you!
I would use both but I think it is more likely to have results for the second alternative (1f-5r). It depends how large your amplicon would be. Overlapped amplicons on either site of the mutation would be another alternative, particularly for very long introns.
Thank you Daniela! I haven't cDNA but only genomic DNA and I have to find a solution using this sample.
I mean: each MLPA probe defines a deletion which is located downstream of the end of each probe, because the presence of the ligation site in DNA sequence is fundamental for probes' annealing...isn't it true?
The DNA complementary sequence to each probe must not be deleted, otherwise the probe will not find the ligation site.
Indeed, obviously, in my MLPA experiment every probes are ligated!
Yes, it is true. However, the probes are located somewhere in the middle of the exon (or close to it) so theoretically the deletion could be in the same exon but in 5' (or 3' respectively) from the recognition site of the probes. Most probably the breaking point is located in the intron and this is why it is more likely to have a better result for the primers containing the first non-deleted exon on each side. Several pairs of primers to cover the entire region might be necessary.
Additional splicing modifications can introduce some intronic sequences in the mRNA and this you will not be able to see at the genomic level. If you detect the breaking point you can use the sequence to predict the potential splicing effects but the proof in this case stays in the cDNA sequencing.
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