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.)
HeartThisRush *how do you know that all possible lengths are copied where is the double blind study proofing this* *and i read the margin of error of using this technique to read dna is 23%*
No wonder you will not excel at your job. I'm not even into biology and yet I typed "dna sequencing explained" in the search bar and it showed up near the top of the results. Sure as hell didn't take me 3 hours. More like 10 seconds.
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.
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.
أنا مهتم جدًا بعلم الوراثة ولكني أجد صعوبة في دراسة الحمض النووي. لقد جعلت مقاطع الفيديو الخاصة بك الأمر سهلاً للغاية بالنسبة لي. شكرًا. يرجى الاستمرار في إنشاء المحتوى الخاص بك 💫
THIS IS WHAT SMART STUDY ACTUALLY LOOKS LIKE. THANKS BUDDY😊 WHEREVER YOU ARE I HOPE YOU WILL BE ALRIGHT. AGAIN, THANKS FOR THIS WONDERFUL MASTERPIECE. THIS IS WHAT RU-vid WAS ACTUALLY MEANT FOR.
So, If I understand correctly. 1. Cut DNA by pieces and add each piece to a sample of DNA (plasmid) 2. Pass each piece that was added with plasmid DNA to a bacteria that would multiply. (How do we not get repeated copies that we don't need where?) 3. Add free DNA bases (A,C,T,G), DNA Polimerase, DNA primer and modified DNA bases that will act as terminator basis 4. Then we raise the temperature to 90 for the spiral to spilt in two (like PCR) and then low again we split so that DNA primer can connect and the free DNA basis connect, until it finds a stop sequence in which it adds the modified terminator basis. (How are we keeping the sequence here? Aren't we combining two things that we created, the DNA primer and the plasmid DNA?) 5. Then we raise the temperature again so that we can split the sequence that we just found. And then repeat process 4, 5 for many times.(Again... Aren't we getting repeated sequences?) 6. Apply electroforesis in order to split these sequences by size. 7. Then we get all the sequences and use a laser to light up the terminating base. And save all the basis that light up, this way we get the sequence. ( Why do we only save the terminating base and not the sequence? And how are we sequencing a person DNA if we are keeping one terminating for each gene sequence found?) Added some questions for each phase in brackets. Hope that might be also other people questions. Best Regards,
1. Repeated copies don't matter so much. Same sized fragments travel through the electrophoresis at roughly the same time, so duplicates will be ignored by the scanner. Even so, the process will be repeated multiple times to ensure no errors. 2. I'm not sure what you mean by your second question. The DNA fragment that we're looking to sequence is first added to a plasmid in order to get multiple copies, and then the DNA fragment is removed from the plasmids, as the plasmid is unnecessary. We only wanted multiple of the original fragment. That DNA is then mixed with bases, primers, and flourophore (colour) terminals to get multiple complementary copies of the original DNA fragment. 3. See 1. 4. Because the DNA fragments are all different sizes (varying from one base + terminal, to three bases + terminal, to n number of bases + terminal) we can determine the sequence of the DNA by measuring the fragments as they pass through the scanner. The coloured terminal that attaches to the end of the bases will be complementary to the base on the DNA strand. So, e.g the DNA strand has a C base. The complementary coloured terminal will be a G base - this will be read by the scanner. This tells us that at that point of the sequence, the base was a C. Imagine you have the sequence ATCGATCG (random sequence) The process of adding free bases, primer, and coloured terminals will result in fragments such as: *T*. T*A*. or TA*G*. or TAG*C*. or TAGC*T*. or TAGCT*A*. or TAGCTA*G*. or TAGCTAG*C*. where the starred base is the coloured terminal. The scanner will read only the terminals, in order of smallest fragment to largest, thus the scanner would read: TAGCTAGC we could then figure out the original sequence was: ATCGATCG whether there were a hundred T*A*. fragments, or only one, they're the same mass, two bases big, so will be read by the scanner at the same time and will only be counted once. Hope this helps!
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.
It helped me a lot 😍this type of animation take a very short time,and preventing us from learning....so many books...in less time I understand more things😊
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] en.wikipedia.org/wiki/Bacterial_artificial_chromosome
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?
arielle aiken the terminator bases are complimentary to the original dna bases meaning u can work out the order of original bases by using the length of the molecule. For example if the shortest molecule has an adenine terminator base then you can deduce that the first base on the original is thymine.
The primer is always the same (10 bases) but the count of free basis is always different. In this video, one string has 11 bases added to it, another has 12... etc. and it goes up and up. By that technique you can read part of the original sequence because of the terminator bases.
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?
The first wave of fragments to show up at the laser would be just one base (the terminator base) after the primer. The second wave to show up timewise would be the primer, one regular base and one terminator base. The third wave of fragments would be the primer, two regular bases, and one terminator base. And so forth. So as the terminator bases light up their colors, they can read the first position, then the second, the third, and so on. It is only reading the end position of each fragment, but because the fragments have a common starting point and are sorted by length they can read each base position one by one. I agree this video leaves out too much needed background information. It needs to emphasize the common starting point, for one thing. Also the simplified graphics are actually confusing... the bases hovering around are only the exact bases needed to complete each fragment, and no extras? The terminator bases are waiting there pre-selected, rather than stumbling in randomly? There is only a single fragment of each length when they reach the laser? All this must be untrue, and to me that makes it confusing.
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?
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)
It describes the Human Genome project that ran from 1990 to 2003. BACs can copy much larger chunks of DNA than a PCR can, and back in the day we couldn't do PCR in high-throughput I guess.
The process which was described (the heating and reheating) was the process of PCR which is used to make billions of copies of the DNA in order to amplify it. Then DNA sequencing process occurs
"Before we start sequencing the DNA it has to be cut in smalled pieces" yet give no explanation as to how do we obtain this DNA, where is it? How is it isolated or is it isolated? And how is it "cut" in smaller pieces, with tiny scissors?
There is no explanation because there are many ways to do it, btw: 1. DNA Source: literally any living thing 2. DNA Isolation: it depends of the starting organism, but essentially you have to disrupt the cell and then isolate DNA 3. DNA Cut: once again there are many ways, but one of these is the one you said: tiny scissor can be used (restriction enzymes). Sorry for my english, i'm italian
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.
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.)
Yes. There is no way to determine the order of a fragment (DNA cut into fragments at 0:15) using this process. Let's say ten G terminators are recorded in a row: how do we know wheter it is just one G repeated ten times or ten G recorded only once each or something inbetween? We have no idea. Hence this method cannot determine anything. There are many more issues. For example, how do we guarantee the sorting order to be 100% accurate? Also note this is a 2D representation that makes it look easy and there is no 3D movement at all as suggested by the title. I conclude the entire thing is a fraud. This doesn't have anything to do with science. Could be the entire DNA genome teaching is flawed or even fraudulent.
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?
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?
it is not put together in one piece again! Yes sorting it from the shortest to the longest gives you the original order. They are all copies of the same piece of DNA, like copies of one book, and every copie has one more letter then the shorter one befor it. You always can only read that one more letter but the letter befor you know already from the shorter copy befor.
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.
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.
So, if someone could help me understand, I'm confused by this: doesn't this just sequence the terminator nucleotides? And if that's the case, then wouldn't that not tell us anything about the DNA strand at all except a random sequence of terminator nucleotides?
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?
Another question: when the DNA is first separated using head, how do you get rid of (or compensate for the effects of) the second strand? The video doesn't seem to mention this.
The video does not emphasize enough the common starting point for all the fragments... they all start at the end of the plasmid DNA, right where the human DNA begins. So yes, you will get multiple fragments of the same length, but they will all be exactly identical and travel through the gel at the same speed and show the same color at the laser. You might get two adjacent base positions that are identical, and therefore two consecutive fragments that show the same color at the laser even though they differ slightly in length... apparently the gel can separate different length fragments with enough resolution to tell them apart and we know it is a repeat rather than just one base taking too long to get by.
Pertinent questions. You might say the video does not do justice to the technology. But I venture the technology does not exist at all and this video is pure disinfo to make people believe into something that is only science fiction.
Apologies for the naive question, but this way won't you just get the base of the terminators, rather than the full sequence of each fragment? what about the unlabelled bases?
But in the sequencing part, they also take the plasmid sequence according to the video. Wouldn't that affect the genome data that you want to sequence?
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
By this way didn't we sequence the placmid and the fragment of DNA together because I see the DNA polymerase putting the nucleotides away from the fragment of DNA?
From where we get DNA polymerase? What does it consist of? Whatever fragment, A-C and G-C alone must pair, if so, which specific sequence held responsible for specific trait in a species (size, colour, position of organ, feeling, emotions, IQ,....)?
Wouldn't it be more simple to just use nucleotids that are color marked and that aren't terminators so we get a strand of DNA that is all color marked and we can then visualize it ?
1. how do you know the primer sequence if you're very objective using the primer is to find out the nucleotide sequences? 2. how can you read the nucleotides after modified nucleotides if the RNA polymerase stops as soon as it puts down the modified nucleotide e.g if the modified ATCG are all placed with base pairs left to be read? 3. how can the nonmodified nucleotides that are placed down before the modified ones be read, there is only a max of 4 modified nucleotides total in all tubes so if a sequence isn't just one of each base pair then a nonmodified nucleotide is placed and cant be read
1. the human genome project started in 1990 and actually in may 2021 the finished sequencing the entire human genome! So we look at references when making primers! 2. there is a low ratio of "terminate bases" to normal bases (about 1:100) so it will randomly insert the terminate bases all the way along the DNA. 3. there are millions of modified nucleotides and billions of strands of the template DNA we are looking at, so they are randomly added and this whole process is done over hours by a machine (thermocycler)! I hope this helps!
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.
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?
That's a good question, but that's why the polymerase used involves a high temp resistance, so the bacteria DNA is disintegrated and the new wanted dna strand from the plasmids are left behind because they are temp resistant
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
