nadaezraturner
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Hi guys! a quick question (Edexcel A2)
I was copying down notes from different resources and I'm kinda confused by PCR. When does it take place?
1- Do we first cut the DNA sample obtained from, say, blood by restriction endonucleases and then use the cut fragment in PCR to make many copies of it (before gel electrophoresis)
OR
2- Do we use PCR after gel electrophoresis?

P.S. could someone please just say the process titles in order?
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flowerscat
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Hi

You would use PCR before gel electrophoresis (usually).

If someone voluntary donates their DNA (cheek swab), then you have enough cells to analyse the DNA without PCR.

If you are analysing DNA from fossils or from a crime scene, then there might not be enough DNA or some of the DNA might be of poor quality (fragmented). In this case, we would do PCR first, check it has worked by electrophoresis, then use the DNA for other techniques like fingerprinting, sequencing etc,

Electrophoresis is a method of determining
- how many fragments the DNA breaks into, if you cut it with a restriction enzyme
- checking if the PCR has worked -i.e. did you get a fragment of the right/expected size?

Electrophoresis need not always follow PCR, and vice versa.

In some rare instances, we would cut the DNA sample with restriction enzymes, run on a gel to separate the fragments, then cut out the desired band, and use it in PCR to get many copies of the DNA that were present in the band. This is usually done when we know the exact size of the gene/allele we are trying to amplify, and we know it appears as a band of certain length when we run it on the gel.

Hope this helps.
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Asklepios
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(Original post by nadaezraturner)
Hi guys! a quick question (Edexcel A2)
I was copying down notes from different resources and I'm kinda confused by PCR. When does it take place?
1- Do we first cut the DNA sample obtained from, say, blood by restriction endonucleases and then use the cut fragment in PCR to make many copies of it (before gel electrophoresis)
OR
2- Do we use PCR after gel electrophoresis?

P.S. could someone please just say the process titles in order?
Your confusion probably comes from not fully understanding what these techniques are.

PCR amplifies DNA, but more importantly only amplifies a gene/sequence of interest (between the two primers). Gel electrophoresis is used after PCR to see if the gene is there or not (if it wasn't there would be no PCR product) and how many base-pairs long it is.

There's different ways to visualise DNA on a gel. You can use a stain that just stains DNA in general (e.g. SYBR green). In this case you would have to do PCR before if you're using genomic DNA otherwise you'd just get a big smear!

Another solution is to only stain the DNA of interest so you don't necessarily need to do PCR before. This uses a short piece of ssDNA complementary to a small sequence of interest, called a probe. And the technique is called a Southern blot in case you want to look it up. This is usually after a restriction enzyme digest. Restriction enzymes cut at specific sequences, and we can look for alleles that are highly variable between individual as they'll have a different restriction digest pattern.


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