DNA Sequencing – 3D

DNA Sequencing – 3D


DNA sequencing is the process of working out the order of the building blocks, or bases, in a strand of DNA. Before we can sequence the DNA, it has to
be cut up into smaller pieces that are inserted into plasmid DNA and then put into bacterial cells. This makes it possible to produce lots and
lots of copies of it as the bacterial cells multiply. The DNA is then isolated from the bacteria
and sent for sequencing. The isolated DNA is transferred to a plate
where the sequencing reaction will take place. A mixture of ingredients is added. These include free DNA bases (A, C, G, T), DNA polymerase enzyme and DNA primers. Modified DNA bases labelled with coloured,
fluorescent tags are also added. These are called terminator bases. To start the sequencing reaction, everything
is heated to 96 degrees Celsius. This separates the DNA into two single strands. The temperature is then lowered to 50 degrees. This enables the DNA primers to bind to the
plasmid DNA. The temperature is then increased to 60 degrees and the enzyme DNA polymerase binds to the primer DNA. DNA polymerase starts making a new strand of DNA by adding unlabelled DNA bases to the target DNA. It continues to add DNA bases until a terminator
base is added. These terminator bases have been chemically altered so that no more bases can be added to the new strand of DNA. Once a terminator base is added, the DNA polymerase enzyme stops making DNA and falls away from the strand. Everything is then heated to 96 degrees Celsius again to separate the new DNA strand from the original strand. This process of heating and cooling is repeated again and again to produce lots of fragments of DNA of different lengths. The length of each fragment depends on when
a terminator base got added. To read the sequence of the DNA the various
fragments are separated by length using a process called electrophoresis. A capillary tube is lowered into each well
of the plate and an electrical charge is applied. This causes the negatively-charged DNA molecules
to move through the capillary tube. Each capillary contains a porous gel. The shorter fragments of DNA move through
the gel more easily than the longer DNA fragments. As a result, the fragments become arranged
by size, from the shortest to the longest. As the DNA fragments come to the end of the capillary, a laser makes the terminator bases light up. The colour is detected by a camera and recorded. Each terminator base is labelled with a different colour. ‘A’ fluoresces green, ‘C’ blue, ‘G’ yellow, and ‘T’ red. The shortest DNA fragments will be read first,
and the longest read last. The sequencing machine records the colour
of the terminator bases as a series of coloured blocks. Each coloured block represents the labelled,
terminator base at the end of each fragment of DNA. By converting the colours into letters we
get the sequence of our piece of DNA.

85 comments

  1. Very nice explanation. Very smart techniques and learnt a lot about DNA sequencing process. Thanks! Looking forward for more of your video.

  2. Hi would it be ok for me to please use a snippet of this video for a project I am working on regarding the associated bacteria of coral reefs and how this might help them respond to climate change??

    Thanks

    Paige

  3. Wait do you mean theres a camera at the end of the electrophoresis anode side which takes snaps the dna structures.

  4. Thanks! I was curious how a machine can read the DNA. One question: if the DNA is cut into small pieces, how do you know what's the correct order when it's put together into one piece again? You said it goes from the shortest to the longest but that's not necessarily the original order is it?

  5. awesome video.
    quick question: if the terminator bases are added randomly by the polymerase enzyme, how does lining up the DNA by length (using electrophoresis) with the terminator bases on the end get the DNA to be arranged in the original sequence?

  6. I have never studied biology, but this vid is so awesome that even i understand it.

    It amazes me the huge waste of resources needed to get this task done.

  7. For those who didn't understand: Even though the terminator bases are added randomly, the process is repeated enough times that there are multiple copies of each length strands and all possible length of strands are produced enough times. As a result, when the electrophoresis is finally done the strands get arranged in short to long order, the last/terminator base of each strand marking the end of each strand. Thus when you put together the end base of each strand, you get the original sequence of that original piece of dna. (Remember the beginning portion of each length strand here is always the same because the primer binds to a specific location only, so the produced strands will look like this hypothetically: ATT< ATTC< ATTCG< ATTCGT etc.)

  8. Why are only the terminal bases read by the detector? What happens to the rest of the fragment? Doesn't somehow make sense to me. Isn't every nucleotide supposed to be detected by fluorescence and then joined to be read as a whole sequence?

  9. To the interested student, the following statement is misleading: "Inserted into plasmid, and then put into bacterial cells". While Bacterial Artificial Chromosomes [1] have been used in many large-scale sequencing projects, including the Human Genome Project, they certainly aren't mandatory. A small chunk of DNA can be directly sequenced via the Sanger method, and a whole genome can be sequenced via a shotgun method ("next gen sequencing"); in either of these cases, no plastid is used.
    [1] https://en.wikipedia.org/wiki/Bacterial_artificial_chromosome

  10. Very helpful, just adding, some experiment lab tend to use PCR first instead of bacteria transport. Just another method, mentioned in my textbook but doesn't matter anyway.

  11. wait, how do you get the whole piece of DNA, if it only reads the terminators color, not the rest of the chunk

  12. I get how you can get a copy from all elements the complete DNA strand, in order, using the terminators, but how does this deal with duplicates?
    I can imagine that purely by chance multiple strands of n elements might be formed, which will appear together in a group when reading them in with the laser. How do you know these are actually identical when you only have a single terminator at one end? If there was some second indicator to get the strand length you could just throw away all but one strands of the same size but I don't see anything like that in this animation. So how do they do that?

  13. Hi! I found this video very nice..Can I use this in my presentation? I want your permission. Please..

  14. For those who still do not understand. Shortest one will be Primer code- Terminator (first base). Second shortest will be Primer code- Base X- Terminator (Second base). Third one Primer code- BASE X-BASE Y- Terminator (Third base.)

  15. Very cool, although seems quite inefficient, but then again I guess we're not fully in the nanotech/biotech age just yet

  16. This video isn't entirely clear to me, when polymerase creates 5 base pairs and puts a terminator at the end of it, how can this sequence of base pairs be converted to a single nucleotide letter when being read? There's a whole lot of information missing from this video and it doesn't make a lick of sense. When the pieces are filtered by size and read out how is the sequence of dna maintained?

  17. For anyone who wants to know the chemical modification of the terminator bases they're modified by removing the 3'OH group so it cant bond with the 5' phosphate group. These are also known as Di-deoxynucleotides (DDNTs)

  18. But how do ther differentiate between two strands that are the same lenght and those who are not?How do they know that the next terminator base is really the next DNA base or just another copy of the previous base?

  19. I still don't get it fully. This helped a lot, but the end lost me. If each strand is made up of different lengths of base pairs. They why is the whole strand assigned a letter based on the terminator bases? It looks like they took a chain of bonds, and titled it with a single letter. How does this determine anything other than the chain of terminator bases and not the entire chain of each strand?

  20. Well, then it would still read as a single base. The gel differentiates the fragments by size. And since we have already fixed the starting point, each fragment would have the same starting base. But the the terminating base has a very small chance of occurring at the same spot(probability or something). The result will be something like this: the shortest fragment will be the one having a terminating base pair adjacent to the primer. The second shortest will have a terminator base second next to the primer and so on.

  21. How is the DNA isolated from the bacteria, without including bacterial DNA or other chemical substances within the bacteria? Doesn't that add a major source of contamination?

  22. Also, how are the phosphate-ATCG pairs targeted? Is it broken randomly? The video makes it appear as though specific sections are targeted. What chemical breaks these DNA strands into sections, that can be removed completely from the chemical solution and won't adulterate the sample?

  23. I mean, sure, you'll say "an enzyme," and the spacing of the enzyme activation sites could arguably connect to and react with base pairs, but then you run into the problem of targeting again. AT – CG. That's it, 2 pairs, of two relative orientations, which doesn't affect whether or not the enzyme will connect. So, why isn't every AT or CG being split, as an enzyme can react with multiple substances. You're not, by this logic, removing "chains" or what one would consider a "subsection" (as displayed), you're basically dissolving it. Pairs should be split in much smaller subsections, every one of them, arguably.

  24. Awesome presentation👍.
    These terminator bases are dideoxynucleotides, which lack both hydroxyl groups at 3' and 2' carbons of sugar, and hence can't make phophodiester bond i.e terminate dna synthesis.

  25. Am I correct that in real life the DNA fragments of a given length will be very numerous in the capillary tube, something like a large school of tiny fish all swimming abreast, and with substantial separation from those schools of fish that are longer or shorter? It seems like this would be necessary (or at least very helpful) in detecting the fluorescent light color, to have a lot of molecules together vs. trying to detect the feeble light of one lone molecule. It seems like the production of a lot of fragments of each length is something that is bound to happen, since the bacteria produced "lots and lots" of copies of the original DNA. Those identical copies would all be getting sequenced at the same time, and even a single one of them would eventually turn out multiple DNA fragments of each possible length in this random process, provided the heating/cooling cycles were repeated enough times. I suppose the large quantity of copies of the original DNA section produced by the bacteria help reduce the number of heating/cooling cycles required down to a practical figure, as that cycling must be a bit time consuming. This video could stand to be a lot longer and more detailed… for example the average person is not going to know what a plasmid is, and the common starting point for each fragment produced needs to be emphasized more. Also, since this is just one section of the total DNA strand to begin with, how is this information mated up in the proper place with other sections sequenced separately? I presume the sections have enough overlap at each end to provide an essentially unique matching combination of DNA bases? Well, I'm a machinist… don't ask me.

  26. It was so amazing and interesting. Every time I hesitate about choosing med/bio for my future studies I watch these kinds of videos and get reminded what an amazingly beautiful world this field is.

  27. i guess for smaller length of the fragment there will be countless copy, but isn't it possible skip some fragment's length if it is long enought ???? cuz the probability to reach there will be realy low so you could heppen to have the fragment with length 1,000,000,000,000 and the next 1,000,000,000,002,

    and how can you differentiate 2 or more frangment that is next to each other with diferent length but the same terminator base??? like the sequency for the length 1, 2, 3, 4 be AAAA, but it will be thousands of copy for each one.

  28. v will only about the labellled bases not normal how will v know the sequence of the normal dna i mean orgnl dna?

  29. Not really well explained, from what's been said here it's unclear how they match those colors to the actual letters in correct order. It's also unclear what kind of errors there might be and what they do to fix this.

  30. Down below is basically what you get in the end, the process is done so many times that you get millions of strands all lined up from shortest to longest, increasing in size one base at a time. So then you can know the sequence of the full strand.
    (The O's are bases you don't need to know, its only the end bases that they detect)
    OOOOA
    OOOOOT
    OOOOOOC
    OOOOOOOG
    OOOOOOOOA

    So the strand would be ATCGA…keep going so on so on

  31. shouldnt temperature as high as 60*C denaturate the dna polimarase? cause its a protein so it seems like a little too much

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