[rleung]

Hi,

I am a little confused about the process of ASO (allele-specific hybridization) as a means to detect a SNP.  According to my understanding, I always thought that you are taking a segment of target DNA under investigation, exposing it to an oligonucleotide probe, and seeing whether or not the probe binds to the target DNA.  If binds perfectly, there is no SNP, but if it doesn't, there is a SNP. 

But as I am reading this book, it mentions that you use 2 probes, one with a polymorphic base in one position and the other without.  I do not understand why you would do this.  If you are trying to find out whether a segment of target DNA has a specific SNP, wouldn't you use only 1 probe that is complementary to the wild-type target DNA and see whether that probe hybridizes with the target DNA under investigation.  If it does hybridize, then you have the wild-type target DNA with no SNP, but if it doesn't, then you must have a mutant segment of target DNA with a SNP, right?

[Yggdrasil]

An additional control.  A wild type allele should hybridize with the wild type probe but not the polymorphic probe.  On the other hand, the polymorphic allele should hybridize with the polymorphic probe, but not the wild type probe.  This system is arguably more resistant to noise and variation in the amount of DNA you load.  For example, if you use only the wild type probe, a low signal from hybridization to the probe could be the result of one of two factors: 1) poor hybridization (i.e. the DNA is polymorphic) or 2) there is not enough DNA.  If you run an experiment with both probes, you can eliminate DNA concentrations as a variable because no matter how much DNA there is the difference in signal between the two probes should reveal whether the DNA is polymorphic or not.

[rleung]

Ahh, I see, thank you.  As you can tell, I have never actually done these assays myself