Separating DNA Fragments by Gel Electrophoresis. Are all the strands for one size the same?

Question

My apologies if my question is too basic, and please point me to a more appropriate forum. I am reading the textbook "Essential Cell Biology" by Alberts et al, and am consulting other sources as well. I am reading Chapter 10 about DNA technology and have read how DNA is separated into strands of different lengths by restriction enzymes. The different size strands can then by separated by Gel Electrophoresis. I believe I understand these basics. 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.

Thanks,

Dave

Answer 1

The answer to the part of your question concerned with average lengths of restriction fragments is: if the DNA molecule being digested is of random sequence, and is 50% GC/ 50% AT, then the probability of finding any given short sequence at any position is 1/4^N where N is the length of the short sequence. So, for example for a restriction enzyme with a 4-base recognition sequence such as AluI (AGCT) the probability is 1/256 and so the average length of an AluI fragment would be 256 bp. For an enzyme with a 6-base recognition sequence such as HindIII (AAGCTT) the corresponding number is 4096 bp. Please note that this is the average length, and is not an 'expected' length.

The answer to the part of your question about fragments of the same size co-migrating, how would you purify them on a gel for further analysis is trickier. I think the first thing to say is that you wouldn't do this for sequencing, and this is reflected in the various answers you have already received - sequencing no longer requires this sort of approach.

If you were forced into this position for some reason then I can think of two things that could help: the first option would be to run the analysis on an acrylamide gel when you can get much better resolution of different fragments (all 50 bp fragments are not the same molecular weight), but this is really only useful for small fragments (100 bp or less). However the most likely way out of the co-migration problem would be to purify the two comigrating fragments as a mixture and then ligate them into a vector whereupon each recombinant would carry one or other of the two fragments. This is really going back to the original reason why molecular cloning revolutionised things - it allows the 'biological' purification of DNA fragments followed by production of these fragments in large amounts.

You mention "special base pairs that are lacking a hydroxyl group" so clearly you are thinking in terms of Sanger (dideoxy-) sequencing, in which case you could clone directly into an M13 phage vector so that you could make ssDNA for direct sequencing. By sequencing a number of clones you would find two sequences, one for each fragment. You would still have to find a way to decide which one was the fragment that you were actually interested in, however.

Like I say, things just aren't done in this way anymore.

Answer 2

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.

Answer 3

As I understand, you are confused how one would be sure that a band in a gel is the sequence one is looking for, seeing as sequences can be different and still have the same length (e.g. ACTTAT is the same length as GGCCGT but entirely different). So your actual question might rather be something like "How does one ascertain that a gel band contains only the sequence one is looking for?"

It is true that in practice, restricting a long sequence can lead to similarly long segments which are not the same sequence. It is indeed possible to cut out a small square from the gel which contains the band, and then dissolve the gel and extract the genetic material. Sequencing the result would give you one of two outcomes: 1) If the band contained only your desired sequence, your sequencing technique should render exactly that sequence as a result. 2) If the band contained more than one different sequences of the same length, your sequencing method will probably leave you with ambiguous results.

So sequencing would probably only confirm whether or not the band contained a single distinct sequence. It would not be useful as a method of separating the sequence you want for future experiments from any other sequence of the same length. One method to do this could be PCR (polymerase chain reaction), a procedure that produces vast numbers (billions) of copies of a specific target sequence. If a PCR was carried out to amplify the target sequence contained in the gel band, any other sequences of the same length would not have any copies produced. They would still be present, but being vastly outnumbered by the sequence relevant for your experiments should prevent the other sequences from having any significant effect.

However, as dd3 noted, you would not normally run a the result of a restriction on a gel and then try to separate your target sequence from the rest. Rather, you would simply use the PCR method described above directly after using the restriction enzymes, and then run the result on a gel. You would then get a very strong band for the sequence you were looking for and much weaker bands for everything else.