Why DNA sequencing does not detect deletions - help! - Med student lost on her hw. Breast cancer gene (BRCA1) mutations (Nov/05/2008 )
Hi everyone 
I'm a med student who's in the depths of learning molecular methods and I'm lost on my homework. Go me! Hopefully one of you biology experts can help me out!
The question reads: Among families with a strong history of breast/ovarian cancer, DNA sequencing succeeds in identifying a mutation in approximately 70% of cases. The sequencing approach fails in cases in which there are large genomic deletions that include an entire exon(s). Why would the standard approach to DNA sequencing fail to detect an entire deletion of an exon?
I don't understand how sequencing can't detect deletions, especially in light of the fact that the sequence is going to be compared to a known "normal" sequence. It would seem to me that a deletion would be more than apparent. I'm guessing it has something to do with the sequencing method, and that's what they're asking in the question.
If it helps (or maybe not), the maximal length of sequence that can be read by cycle sequencing is 800-1000 bases (though the average read length is 300-600 bases); the BRCA1 gene is 5,500 bases, so 36 different cycle sequencing reactions must be executed and read at minimum.
Someone want to give me (or at least help me figure out) the missing puzzle piece that I'm just not getting? hehe.
Thanks!
Cristina
I'm a med student who's in the depths of learning molecular methods and I'm lost on my homework. Go me! Hopefully one of you biology experts can help me out! The question reads: Among families with a strong history of breast/ovarian cancer, DNA sequencing succeeds in identifying a mutation in approximately 70% of cases. The sequencing approach fails in cases in which there are large genomic deletions that include an entire exon(s). Why would the standard approach to DNA sequencing fail to detect an entire deletion of an exon?
I don't understand how sequencing can't detect deletions, especially in light of the fact that the sequence is going to be compared to a known "normal" sequence. It would seem to me that a deletion would be more than apparent. I'm guessing it has something to do with the sequencing method, and that's what they're asking in the question.
If it helps (or maybe not), the maximal length of sequence that can be read by cycle sequencing is 800-1000 bases (though the average read length is 300-600 bases); the BRCA1 gene is 5,500 bases, so 36 different cycle sequencing reactions must be executed and read at minimum.
Someone want to give me (or at least help me figure out) the missing puzzle piece that I'm just not getting? hehe.
Thanks!
Cristina
I suppose it would depend on how they are obtaining the DNA to sequence - but if this were a case in which patients are heterozygous for the deletion then you might not see it by sequencing because it is there on the other chromosome. Presumably if you were to PCR around this exon (and use the PCR product for sequencing) you would see a smaller product because of the deletion, but if the PCR doesn't work in the case of the deletion or the product is too small to see, then you wouldn't sequence the smaller product because you didn't know it was there. You would sequence the normal PCR product and be fooled into thinking that both chromosomes are the same. The same would be true if you did a PCR reaction with a primer inside the deleted exon - you wouldn't get a product from that chromosome and you would end up sequencing only the normal chromosome.
This is just my thought - I'm not at all sure if this is how they do genotyping for things like this.
Are you sure they didn't say the "deletion of an entire intron"? That would make sense, since the sequencing is done on cDNA, made from reverse-transcribed RNA, and the introns are spliced out of the RNA. Thus, a genomic deletion of an intron would not be detected by cDNA sequencing.
Yeah, they said exons.
I'm just going to put that if a huge amount of sequence is deleted, there might be nothing for primer to bind to. At least it's something.
Thank you to the people who responded, including the people who PMed me!! You all are awesome!
-Cristina