Homozygous Heterozygous by PCR/Southern blot - (Apr/02/2012 )
Could somebody please explain me how to distinguish between homozygous and heterozygous by PCR and/or Southern blot?
I know that when an individual is homozygous for a gene, it will have only one PCR band. I found some textbook saying that a homozygous could also have 2, 4 PCR bands!
How is this possible?
In the figure attached is an example.
It is said:
With one band: homozygous
2 bands: also homozygous
3 bands: heterozygous
How 2 bands make homozygous, if we speak about the same gene?
If there is only a substitution of one base (to make a restriction site), does this change the size of gene?
Hint: it's about the pattern, not the number of bands.
What do you mean?
The number of bands doesn't determine a given pattern?
A pattern is not a "given shape" based on some visible description?
In your example you have a variant recognised by a specific restriction nuclease. If this variant is present, the enzyme cuts the product in half. If it's not, it won't. On the gel you either see uncut product, two cut products or both cut and uncut. It's not about the number, it's about identifying the bands.
In more complicated cases your restriction endonuclease may cut on other sites present in sequence as well, so there may be more bands, but band pattern for homozygous variant 1 and 2 would differ, heterozygous would have bands from both patterns.