PCR Use in Genetic Testing

[mideasyrole]

I understand the process of PCR and how it is performed, but I am struggling to make the link to genetic testing.

Currently, I understand that you take a sample of DNA from a person and amplify it using PCR (make multiple copies of a specific segment of DNA) What is the reason for amplifying DNA if it is the same identical segment as the original? Can't we just test the taken sample?

I have also learnt that you can screen for mutations in that amplified DNA using specific restriction enzymes which will fragment the DNA to then be detected using gel electrophoresis. But how do these genetic mutations arise during PCR, if the section of DNA is amplified to give identical copies?

Thanks!

 

[Fili]

I understand the process of PCR and how it is performed, but I am struggling to make the link to genetic testing.

Currently, I understand that you take a sample of DNA from a person and amplify it using PCR (make multiple copies of a specific segment of DNA) What is the reason for amplifying DNA if it is the same identical segment as the original? Can't we just test the taken sample?

I have also learnt that you can screen for mutations in that amplified DNA using specific restriction enzymes which will fragment the DNA to then be detected using gel electrophoresis. But how do these genetic mutations arise during PCR, if the section of DNA is amplified to give identical copies?

Thanks!

It's to produce enough copies that it can be analysed. If you only have one piece of DNA and run that through a gel electrophoresis, nothing will show up. You need A LOT of DNA so that they can actually be seen on the gel.

Normally, 2 restriction enzymes should produce 2 pieces of DNA that you can see on the gel. A mutation in one restriction site would mean that we will only see one band as only one restriction enzyme will work. Similarly, if a mutation resulted in the addition of DNA, we will see an increase in size of those 2 bands and can compare it to the control subject.

I don't think "mutations" arise from PCR unless someone's culture was accidentally contaminated with DNA and it so happens that the restriction enzymes and primers used bind the contaminant to the desired DNA that is amplified.

 

[Breg]

I have also learnt that you can screen for mutations in that amplified DNA using specific restriction enzymes which will fragment the DNA to then be detected using gel electrophoresis. But how do these genetic mutations arise during PCR, if the section of DNA is amplified to give identical copies?

If you're looking for this information be aware that "screening mutants" means something very different in molecular biology, and indeed is about generating mutations. I don't think that's what you mean, as you want to detect mutations rather than generate them. In the case of looking at DNA and trying to detect mutations the technique would depend on the type and size of mutation, you may be able to detect it with a simple gel electrophoresis; in that case you could amplify the sample DNA fragment where the mutation is believed to be and compare the band to a known WT. There's also denaturing gradient electrophoresis and of course, sequencing.