In general I don´t understand your strategy. Normally you look for point mutations in your candidate gene by sequencing all exons and if you don´t find any in the next step you will look for insertions/deletions.

What about a Southern Blot to determine the length of the deletion? Alternatively you can try a long range PCR with the primers of exon 23 (5´primer) and exon 25 (3´primer) and if you obtain two fragments you can try to sequence the smaller one after cloning.

Thanks for the suggestion: a long range PCR could be the answer and we will start with it. Southern Blot is not an option for now, in our lab. 

Dear Sanja

Why don't you design a MLPA probe for one/some of the adjacent exons yourself?

This is fairly easily done and works fine. Design the probes so they fit into the kit you are using already (most often the is a gap with no probes around 108-120 bp), for example 115 bp.

See Jizu Zhi, BMC Res Notes, 2010; 3:137 on how to do it. Don't bother with stuffer sequence.

Good luck,

Charlotte

You can use the MAPD software from this www

http://www.osa.sunysb.edu/bioinformatics/resources.html

Dear Sanja

I would start with a quantitative PCR of exon 24, using intronic primers, to check the MLPA result. Because it happened to us that we saw deletion in the MLPA but the PCR amplified correctly, so when we sequenced the exon we detected a point mutation in the place where the probe binds to the DNA.

If you detect the deletion with the PCR you should go on by analysing the exons not included in the MLPA kit. Maybe if you have an STR that maps in this region you could use it to limit the deletion.

But if exon 24 amplifies you should sequence that exon only.

Hope this help you

Good luck

Leonela

Thanks for the answers, to Charlotte and Leonela!

To: Charlotte

I have looked for MAPD software, but the link:

http://bioinform.arcan.stonybrook.edu/mlpa2/cgi-bin/mlpa.cgi

doesn't seem to work. Do you know how can I obtain it? Do you often use synthetic probes in your lab? What is your experience?

To: Leonela

When this happened, first of all I could think of was qRT PCR... But, we don't perform RT in our lab and some colleagues suggested sequencing as a first step... 

To; Charlotte and Leonela

By the way, its NSD1 gene and P064 kit and there is another problem: I couldn't find information about existence of exon 24?? RefSeq of this gene says that there is only 23 exons... Do you have any idea about that?

Thank you both in advance.

Kind regards,

Sanja

Dear Sanja

I had the same problem with that link when I tested it, just before writting to you. Never had those problems before. Wrote the guy that designed it and asked if he cancelled the www. Waiting for a reply.

Yes, I have been designing synthetic probes for 5 years in my clinical work to verify arrayCGH data. It work very nice.

Where did you see that the gene (NSD1?) had 24 exons? Was that in a MLPA kit or the databases? UCSC shows two isoforms. One has 23 exons, the other has 24 exons. The DGV track in UCSC also shows that duplications from exon 4 (aprox) to exon 23/24 are not rare in the general population.

I'll be happy to help you, but this discussion is probably too specific for an open forum now. Why don't you email me and I'll see if I can help you if you wish to do custom designed MLPA probes for you patients.

Best wishes,

Charlotte