@@zarlashtkhan3540 it is just because incorporation of ddgtp is a random process. It is not necessary that every complementarity of A is DDG. G can also be complementared. ddntps are provided in low concentration in the mixture than regular nucleotides( A, T,G,C). It means regular nucleotides are also always present. So incorporation of ddgtp is a random probability( sometimes in the beginning complementary to 2nd C, sometimes to the 3rd C, sometimes to the last C). Hope it helps.
There are all 4 dNTPs added in each reaction and also one of each ddNTP in each well is added because we want to produce fragment with different length. Otherwise the reaction would have stop at the first complementary ddNTP added after the primer. and there will be only 4 length fragments.
Yeah, thanks for clearing that. I was wondering how he got 2 GTP & 3 GTP as well. It should have terminated at 2 GTP position only if 3 dNTPs & one ddNTP were used.
This is by far the most helpful explanation of Sanger Sequencing; it's a whole walk-through of how/why the process works (rather than just pieces/bits of information from other videos). Thank you very much!
I am a second yr medical student and i really can't express how helpful this and your other videos on genetics have been to me ! You are a wonderful teacher ! Thanks a lot for teaching the topic so beautifully.
Shomu knows how to explain DNA sequencing very very effectively. I could not understand the ins and outs of the Sanger DNA sequencing until he went into explaining the method step by step, beginning with the explanation of dideoxy chain termination. I plan to watch all of his videos on DNA sequencing.
Excellent explanation.. before watching this lecture, I tried to read a review paper on sequencing published in the Genomics journal, watched a lecture by Worcester Polytechnic Institute but you, my friend, are the real deal.. Thanks!!
Hellow sir I am from Sri Lanka.You are the best biology teacher that I have ever seen.I and my almost all colleagues in our university are following your channer and have achieved highest grades because of your fantastic teaching ability.I highly appriciate it.GOOD LUCK SHOMU'S BIOLOGY!!!!!!!
4th sem biotech student here. You're awesome. College mein shaam ke 6 baje padhaya thha Sanger Sequencing, kuch nahi samjha. Now I'm quite clear about Sanger Sequencing. Thanks a lot.
The best teacher ever and every complicated lecture becomes easy and crystal clear after watching ur videos. I thank u from the bottom of my heart prof.
I am here again because my professor has sent your link but I have already subscribed to your channel since high school times. You make me understand better. Thank you so much for all these years.
Nice presentation. However, as a colleague I would suggest you to add 4 dNTPs plus limiting amount of ddNTP in each reaction tube. Otherwise, you will only get the chain terminated at the first nucleotide such as G, and no next G can be detected....
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in all the tubes all dNTP's are added, otherwise how would the chains be of different length. For eg in 1st tube according to your method, the chain should terminate at the 1st A attachment only. Thus all chains would be of same length
Each tube needs one ddnucleotide sequence for termination. Different sequence undergoes different length if they have fluctuating sequence. This is only applied for short sequences, and worth of a Nobel price😊
abhishek yadav the quantity of di deoxyNTP are lesser as compare to the dNTP. Hence there are chances of getting dd on the right place as well as d. During electrophoresis it can give larger bands if the dNTPs are added everytime but ddNTP can also bind
shomu's biology each tube should contain all four dNTPs not 3 dNTPs and very less amount of one ddNTP ... btw thanks for all videos .. they are simple and easy to understand
After reading from notes which is given by sir and also from slide share ... but not clear with concept... n have confusion .. here i am at 2 am watching ur video... n nw 100% clear with basic concept of method.... n clr with all my doubts.... thank u soooo much... 💕
It's a great explanation, but as other comments have mentioned, you are a little ambiguous when explaining how the sequence can continue once the ddNucleotide is added. It is necessary to emphazise that each of the four mixtures contains ALL FOUR dNucleotide PLUS one of the four ddNucleotide. Otherwise, thanks for the clear explanation.
very useful.most of the lectures well describes the steps untill autoradiography,for an unexpert person from that step hurdles starts.this videos thoroughly clarified such a gassy knowledge.thank you so much
Hey bro ur question is valid. Shomu has taught in a little wrong way in 1st test tube there should be both ddATP and dATP thn by probability either of them would attach
Instead of using only ddNTP we also use the same nucleotide otherwise a reaction for ddGTP will stop at 2nd step only and it will not move further to 3rd or 7th step without a free OH group.
All dNTPs along with 1 particular ddDNTP is added in each tube. As a result of chain termination DNA fragments of different lengths are formed. Otherwise, if we are adding only ddNTP along with 3 dNTP, termination will occur only in the first one.
How different length fragments are formed by adding same ddNTP in particular reaction mixture the first fragment is terminated due to particular ddNTP attachment,why don't the same ddNTP attached at the same position again, why it's not happening? Will u plss explain to me
Great lecture sir but i have a confusion When the unknown chain have two CC so once the chain terminate at the first C theb how it go farward toward the 2nd C
Q.you mentioned that termination of complementary sequencing nucleotides occurs after binding of dideoxynucleotide. How could the sequencing continue after binding of this residue to further complementary nucleotides to generate larger fragments?!
Salah El-Den Exactly! The only way for this to work is to have all 4 nucleotides present in d form, in addition to a repeat of the target nucleotide in the dd form. Then, you get random numbers of the extended DNA.... but I have to confirm this procedure.
Shomu's Biology How can the sequence extend and we get so many fragments once the ddNTP is added if termination occurs occurs as soon as it is added. please explain ..
in every test tube there will be dNTP (in excess) and small amount of ddNTP(ddGTP, ddCTP, ddATP and ddTTP respective to the tube) so that the chain will be extended untill and unless there is incorporation of ddNTP
no it is not like that. since u have dNTPs in excess then the chain extension will be the major event while incorporation of ddNTP is by chance, so you will get fragments of different length depending on the incorporation of the ddNTP. For example, in the test tube where u have put ddGTP, the termination will be at G so you will get fragments of different length but all are terminating G. On running of all the 4 tubes on the gel u can get the exact sequence...
Thank you sir.... I can't understand this experiment in my class.... I'm struggling to understanding this but not able...but after watching this video I understand this clearly
A lot of Thank sir i have no idea for dna sequencing but now i feel confident bcoz of you sir...... When i have any problem i see ur videos sir then my problem is solved by u indirectly .... Tysm again sir U r so intelligent sir
You are a wonderful teacher of Biology.. Whenever i feel any topic difficult to teach I come to ur channel...it always helps me out.. God bless you boy..
Thank you so much sir.. You're simply amazing.. Your way of teaching is so relaxing... And makes every complications so easy within minutes.. Much respect for you sir ❤🙏
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Excellent !
Good explanation
But voice is little unclear
Sir please explanation chemicals to indentiy activesite arg, his, lys, his
@@shomusbiologyofficial in case of ddgtp how can be a 3 nucleotide sequence formed if both the consective gtps ar ddgtp please answer
@@zarlashtkhan3540 it is just because incorporation of ddgtp is a random process. It is not necessary that every complementarity of A is DDG. G can also be complementared. ddntps are provided in low concentration in the mixture than regular nucleotides( A, T,G,C). It means regular nucleotides are also always present. So incorporation of ddgtp is a random probability( sometimes in the beginning complementary to 2nd C, sometimes to the 3rd C, sometimes to the last C). Hope it helps.
There are all 4 dNTPs added in each reaction and also one of each ddNTP in each well is added because we want to produce fragment with different length. Otherwise the reaction would have stop at the first complementary ddNTP added after the primer. and there will be only 4 length fragments.
Exactly and thanks for mention this, otherwise it does not make sense. So I suggest to edit this video.
Yeah, thanks for clearing that. I was wondering how he got 2 GTP & 3 GTP as well. It should have terminated at 2 GTP position only if 3 dNTPs & one ddNTP were used.
Thanks .now i am understand
Exactly... Thanx for supplementary this point.
Absolutely right 😊.. I want to write the same thing
This is by far the most helpful explanation of Sanger Sequencing; it's a whole walk-through of how/why the process works (rather than just pieces/bits of information from other videos). Thank you very much!
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I am a second yr medical student and i really can't express how helpful this and your other videos on genetics have been to me ! You are a wonderful teacher ! Thanks a lot for teaching the topic so beautifully.
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Shomu knows how to explain DNA sequencing very very effectively. I could not understand the ins and outs of the Sanger DNA sequencing until he went into explaining the method step by step, beginning with the explanation of dideoxy chain termination. I plan to watch all of his videos on DNA sequencing.
Excellent explanation.. before watching this lecture, I tried to read a review paper on sequencing published in the Genomics journal, watched a lecture by Worcester Polytechnic Institute but you, my friend, are the real deal.. Thanks!!
Thank you so much for appreciating my efforts. Glad to hear that you're getting benefit from my lectures.
Hellow sir I am from Sri Lanka.You are the best biology teacher that I have ever seen.I and my almost all colleagues in our university are following your channer and have achieved highest grades because of your fantastic teaching ability.I highly appriciate it.GOOD LUCK SHOMU'S BIOLOGY!!!!!!!
Doing a masters in cancer and I can't tell you how much you have helped simplify topics.
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4th sem biotech student here. You're awesome. College mein shaam ke 6 baje padhaya thha Sanger Sequencing, kuch nahi samjha. Now I'm quite clear about Sanger Sequencing. Thanks a lot.
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The best teacher ever and every complicated lecture becomes easy and crystal clear after watching ur videos. I thank u from the bottom of my heart prof.
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I am here again because my professor has sent your link but I have already subscribed to your channel since high school times. You make me understand better. Thank you so much for all these years.
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shomu is the best thing that has ever happened to my education. Thankyou Shomu
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Nice presentation. However, as a colleague I would suggest you to add 4 dNTPs plus limiting amount of ddNTP in each reaction tube. Otherwise, you will only get the chain terminated at the first nucleotide such as G, and no next G can be detected....
EXACTLY .I WAS CONFUSED THERE!!
Hi sir...Without you and your channel i couldn't get pass in the subject..For each and every semester I'm learning from u..ur trying hard to make us understand so easily..Thanks a lot for your effort...I became your fan since 2017....If u reply i ll be happy...Thank u sir...
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I couldn't understand it when my professor was explaining it. But you've just explain AMAZING!!! You are the best!!!!
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Wow, I wish my professor taught this way. Now I understand what I read in the textbook and slides. Thank you
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I agree that you are the best teacher I've ever seen in online. I can keep enjoy of studying biology thanks to you. You help me a lot! Thank you ,sir. From Korea
Today is my exam(3rd. yr. B.Tech) & I have completed my whole syllabus a night before in just 3.5 hrs. by watching your videos on various topics !!😁 Thank you sir .. U are the best 👍 simplicity 💯🌟
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@@shomusbiologyofficial in first test tube why don't we add all the dntps n only three please clear my doubt , appearing for my final MD exams
Thank you so much ...this is the first time I got the Sanger sequencing ..you put the whole process together in one lecture now its easy to remember the whole story...before I used to learn parts of Sanger sequencing and could not connect different parts..👍
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yes he is😊
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YOU ARE AMAZING!! thank you so much mister Suman. Plz keep up what you do. You help so many students from all over the world by explaining these bio topics so easily. I have binge-watched a lot of your video's and passed my exam. Thank you thank you thank you.
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It's really help me to understand the actual process of Sanger sequencing...so clearly & easily...
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@7:00 here not only 3dntps in each reaction centre but all 4dntps and one of each ddntp in each well is added
point to be noted.
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Thank you very much, i study biomedical lab.teknologi, and i startet in 2017, and your videos have been very helpful for me, i appreciate it ,and thank you very mig wee love you
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You are really saving my ass in my clinical imaging course with these awesome lectures man. Thanks for the good and easy to understand explanations! Liked and subscribed.
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in all the tubes all dNTP's are added, otherwise how would the chains be of different length. For eg in 1st tube according to your method, the chain should terminate at the 1st A attachment only. Thus all chains would be of same length
same remark !
Exactly
Each tube needs one ddnucleotide sequence for termination. Different sequence undergoes different length if they have fluctuating sequence. This is only applied for short sequences, and worth of a Nobel price😊
Each tube have all narmal dntps but one specific ddntps.
abhishek yadav the quantity of di deoxyNTP are lesser as compare to the dNTP.
Hence there are chances of getting dd on the right place as well as d.
During electrophoresis it can give larger bands if the dNTPs are added everytime but ddNTP can also bind
Awesome. Tough concept explained very simple. Got confused a bit with why we got 2 and 3 GTP. But after reading posts below I understood the concept.
Thank you
Please explain
shomu's biology each tube should contain all four dNTPs not 3 dNTPs and very less amount of one ddNTP ... btw thanks for all videos .. they are simple and easy to understand
After reading from notes which is given by sir and also from slide share ... but not clear with concept... n have confusion .. here i am at 2 am watching ur video... n nw 100% clear with basic concept of method.... n clr with all my doubts.... thank u soooo much... 💕
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It's a great explanation, but as other comments have mentioned, you are a little ambiguous when explaining how the sequence can continue once the ddNucleotide is added. It is necessary to emphazise that each of the four mixtures contains ALL FOUR dNucleotide PLUS one of the four ddNucleotide.
Otherwise, thanks for the clear explanation.
You Clarified further, Kudos!
thaks .. u cleared my confusion .. about d/ff sizes of sequence.....
thanks！
very useful.most of the lectures well describes the steps untill autoradiography,for an unexpert person from that step hurdles starts.this videos thoroughly clarified such a gassy knowledge.thank you so much
You're welcome
When the chain get terminated at 2nd position ( C is present)then why will it again proceed to terminate at 3rd position ( again C is present)?
Hey bro ur question is valid.
Shomu has taught in a little wrong way in 1st test tube there should be both ddATP and dATP thn by probability either of them would attach
@@amishbutle4887 yes bro 👍
Same question I wanted to type but after seeing your answer I understood 👍😊
@@amishbutle4887 thanks bro..I also have same question..
👍
Very well taught up till now I was unable to understand sequencing but after watching this video I cleared my concepts thanku shomu
Instead of using only ddNTP we also use the same nucleotide otherwise a reaction for ddGTP will stop at 2nd step only and it will not move further to 3rd or 7th step without a free OH group.
This is the best lecture. I understand every step very well when you explain . Thank you very much for making it easy and understandable!
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All dNTPs along with 1 particular ddDNTP is added in each tube. As a result of chain termination DNA fragments of different lengths are formed.
Otherwise, if we are adding only ddNTP along with 3 dNTP, termination will occur only in the first one.
How different length fragments are formed by adding same ddNTP in particular reaction mixture the first fragment is terminated due to particular ddNTP attachment,why don't the same ddNTP attached at the same position again, why it's not happening? Will u plss explain to me
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I wonder how cud DNA is still synthesized, even after adding ddNTPs in case of C
amazing! I have biology paper tomorrow. Your lecture is so helpful
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Great lecture sir but i have a confusion
When the unknown chain have two CC so once the chain terminate at the first C theb how it go farward toward the 2nd C
Sir, You really really helped me in my exams..Words are not enough to express my gratitude...
Q.you mentioned that termination of complementary sequencing nucleotides occurs after binding of dideoxynucleotide.
How could the sequencing continue after binding of this residue to further complementary nucleotides to generate larger fragments?!
Salah El-Den Exactly! The only way for this to work is to have all 4 nucleotides present in d form, in addition to a repeat of the target nucleotide in the dd form. Then, you get random numbers of the extended DNA.... but I have to confirm this procedure.
That makes sense.. he needs to explain this point more.
+Salah El-Den watch the video once again
Shomu's Biology How can the sequence extend and we get so many fragments once the ddNTP is added if termination occurs occurs as soon as it is added.
please explain ..
I also do not get this part. How can one get 3GTP for example? Wouldn't it have stopped at 2GTP?
OMG! Just easily explained! You just gained another subscriber!
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sir in 2nd case where two CC is present when termination is there then termination always occur from first C how it proceed next C
My doubt exactly
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I have a question. In the tube that has ddGTP, there are no dGTPs, so won't the reaction always stop at the 2nd position ?
same question
exactly my question as well... :/
in every test tube there will be dNTP (in excess) and small amount of ddNTP(ddGTP, ddCTP, ddATP and ddTTP respective to the tube) so that the chain will be extended untill and unless there is incorporation of ddNTP
Ravikant Shekhar
If this event happens then we will get the correct termination for very few times
no it is not like that. since u have dNTPs in excess then the chain extension will be the major event while incorporation of ddNTP is by chance, so you will get fragments of different length depending on the incorporation of the ddNTP. For example, in the test tube where u have put ddGTP, the termination will be at G so you will get fragments of different length but all are terminating G. On running of all the 4 tubes on the gel u can get the exact sequence...
Thank you Sir..You simplify and explain the concepts very nicely!!
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I can explain the Sanger method now. Thank you!
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Precise and straight to the point, well understood
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Explained in very easy and grasping terms. Thank youu!
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