My question is: Do all strands of the same length have the same sequence of base
ID: 31937 • Letter: M
Question
My question is:
Do all strands of the same length have the same sequence of base pairs? I wouldn't think so, and this would be a problem in the next step, determining the sequence of one size fragment by using special base pairs that are lacking a hydroxyl group. Isn't it possible I could have two strands that are 50 base pairs long (for example) that have the same beginning and same end and are thus cut by the restriction enzymes to a length of 50, but are most assuredly not the same within their sequence
The text says that once the different size strands are separated by electrophoresis, you can just cut out one of the bands and work with that. That makes sense if you are guaranteed to only have 1 strand at that level of the gel agar or if the multiple strands are all the same exact sequence.
If you read this far, thank you. I think more generally I need to know how many of these segments are produced when you use restriction enzymes and how long the average one is. And is there any guarantee for one chromosone (or all 46 chrmosones) having all the segments be unique sizes (I wouldn't think so)?
And even more generally :), I need additional sources that explain DNA sequencing in simple to understand terms.
Explanation / Answer
You are correct: strands of the same length do not necessarily have the same sequence. The separation of DNA in gel electrophoresis is purely based on the hydrodynamic size.
The number of fragments produced by restriction enzyme digestion depends on the number of sites for that particular enzyme in your input DNA. Restriction enzymes only cut at specific nucleotide sequences. There is no "typical length" of fragment produced for this reason.
The idea of separating DNA on the basis of an RE digest is that a mutation that changes the sequence where the RE binds and cuts will reduce the number of fragments because the RE can no longer bind and cut at that site: 2 smaller fragments will now appear as one large fragment. It will be visible on the gel because of the difference in number and size of fragments.
Typically, when running a gel, you are using DNA that has been selectively amplified to bias the readout to a particular (known) region of the chromosome. This electrophoretic approach works because you're not looking at all 46 chromosomes at once.
As far as sequencing is concerned, I'd recommend starting with Wikipedia. Sequencing techniques are changing rather rapidly, so there's not one good source of which I am aware. To begin with, you can read about Sanger sequencing method. If you understand PCR then you won't find it hard to understand.
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