I’ll try to demonstrate the extent of my understanding & hopefully any of you fellow experts can fill in the gaps for me
So we know that we run Gel Electrophoresis, the DNA gets separated into many fragments and are sorted according to their sizes. But……
1) WHAT caused it to separate in the first place? Is it the voltage that breaks the DNA into fragments? Or is it the restriction enzymes that cut at the restriction recognition sites? How does it get fragmented?
2) Why are some fragments larger/smaller than others? I don’t understand how there are many fragments that have the same size?
3) Why does each person’s DNA show different bands?
Besides the above, I am also trying to link between performing a PCR then Gel Electrophoresis for visualisation and Sanger sequencing. I understand that PCR amplifies the region that we’re interested in, and then we use Gel Electrophoresis to confirm that they were amplified and there are no mutations. What would we have seen on the Gel if there had been mutations? What does sequencing do?
Would appreciate some feedback so we can discuss in the comments x
In: Technology
1) The DNA is broken into fragments by enzymes before the voltage gets applied. The voltage is there to pull the fragments through the gel.
2) The cutting enzymes target particular sequences. The fragment lengths will depend how far it is between those sequences for that particular DNA strand. You have lots of fragments of the same length because you have tons of copies of the DNA (thank you, PCR) and it’s all the same and it’s all getting cut at the same locations.
3) Each person’s DNA is different, so the locations of the target cutting sequences are different, so the fragment lengths are different, so the bands show up at different distances through the gel.
A mutation is a change in the DNA; it would have to occur in a way that alters the fragment lengths for it to show up in the gel bands. That may or may not happen.
Sequencing is actually reading off the DNA code, base pair by base pair. You can just compare it to the un-mutated sequence to see if anything mutated.
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