at 0:35 please note that the 3'-end of the primer should point towards the region, which is to be sequenced. because the polymerase elongates the chain in 5'--->3' direction.
Note that this is not quite how modern Sanger sequencing is done. Rather than using radioactively labeled dNTPs, the ddNTPs each have a different fluorphore on them. There is only one reaction, and the sequence is read by looking at the color of fluorphore on the end of each consecutively longer strand.
yes but if kids at school are struggling to understand anyway then might as well just learn the right one ! it would be nice to have seen the nucleotides actually add in a short animation or at least series of diagrams so its not all in your head. nice video though.
I only started watching these videos for my Science class because I didn't take the basic course in the previous year...Now, I'm getting recommendations on MCAT exam tips...TT
When you digest your Dna it is always done with a restriction enzyme that s widely available and we'll known, which includes information on the specific sequence the enzyme cuts at.
I don't understand how knowing one base (the detectable one) helps determine all the bases on the strand. I've seen several videos like this that just end with this comment but don't explain it. Confused.
usually this is done on DNA fragment that have been formed using restriction endonucleases. These enzyme only cleave the DNA at specific sequences so we can use these "restriction sites"as sites to add the primer.
+Deena Haroon I typed all this on my phone and only now realized all the typing errors. sorry. I hope you can read past it and it was able to explain it properly. (as a note, although it may look like the 5' end is the end shortening, it should be the 3' end that is being removed and shortened.)
+Jessica Wong hey im not entirely sure myself but the video mentions that the gel can separate molecules by even one base. And usually, when you use a gel you always have a marker (I'm assuming these markers mark different intervals across the length of DNA). So if you combine this with the fact that each lane has a band corresponding to a termination by a base of that lane, you can read off (using the marker) where these bases are on the fragment of DNA. Not too sure I made any sense there hah
+Jessica Wong actually, you probably don't even need a ladder here. I think the main fact is that you have a primer, so you can make a defined start point. So the template bound to the primer should be the smallest molecule. On a gel, this should be at the bottom. So now, if you have all other molecules in the well separated by a single base, you can pretty much read the order in which they appear, from the primer sequence onwards (or upwards in the gel). Again, hope that makes sense. I'm busy studying for a genetics exam :-p
+Borat Sagdiyev To rid of any confusion: DNA Poly replicates in the 5' to 3' direction complementary to the template strand which will appear 3' to 5'. Edit: Removed PolyIII as Poly I is used in this method.
I thought ddNTP is radiolabled, not 'one of dNTPs'. if one of 'dNTP' was instead radiolabled, then you wouldn't be able to assess the length accurately.
1. He fails to explain that it is the primers that got labeled so that X-ray can be used to see all sequences. 2. His accent is annoying. 3. He fails to mention the sequences differ by one base only in gel electrophoresis. Therefore I gave the video a thumb down.
In reply to your critisms: 1) Radiolabelling of nucleotides mentioned at 1:02. Also, at 2:09; "The gel is dried down and an X-ray taken. Since we radio-labelled a dNTP we can now see it show up on the X-ray film." 2) A matter of opinion. I would point out that Irish accents are consistently ranked as some of the most attractive, but I understand that some people are just hard to please. 3) This is incorrect. At 1:47 I mention how polyacrylamide gels can separate fragments differing in length by 1bp. Also I would point out, contrary to your point, that this is not true for all gels. Agarose gels for example, can typically resolve differences no smaller than ~40bp between fragments but this does depend on the gel percentage and other variables.
at 0:35 please note that the 3'-end of the primer should point towards the region, which is to be sequenced. because the polymerase elongates the chain in 5'--->3' direction.
Conor here is putting out the BEST video on the Sanger method!!!
Was trying to read this out of a textbook which was hard to understand conceptually. The video made things clearer. Thanks!
I have been a tricky time with DNA Sequencing and you have provided brilliant help! Thanks!
Note that this is not quite how modern Sanger sequencing is done. Rather than using radioactively labeled dNTPs, the ddNTPs each have a different fluorphore on them. There is only one reaction, and the sequence is read by looking at the color of fluorphore on the end of each consecutively longer strand.
Simeon Andrews yeah but this is what it's based on, and if you're able to understand this, the fuorescent method is easier do decipher imo.
yes but if kids at school are struggling to understand anyway then might as well just learn the right one ! it would be nice to have seen the nucleotides actually add in a short animation or at least series of diagrams so its not all in your head. nice video though.
Students taking a genetics course need to know the old way sequencing was done as well as the newer ways.
nothing like an intensive purpose
That's Automated DNA sequencing, not Sanger method
Your voice make me feels calm.
Sorry for being weird.
the accent made this vid even better
No you can't barely understand him at some points which makes 50% of these videos a pain in the ass to watch
Exactly
Not really. He kinda swallows half of the words.
Simple and straight forward. Thank-you!
You've helped me so much, thank you. Even though I'm german I understood it very well. This video is definitely the best one you can find on RUclips.
Insha'Allah Allah has allowed me to find this video mashallah i will do well in my exam
All clear Now and the accent made it a lot better Thank you !
i love your accent oh my gosh i watched for that mostly i kinda zoned out lol
I only started watching these videos for my Science class because I didn't take the basic course in the previous year...Now, I'm getting recommendations on MCAT exam tips...TT
Nice short video. Just what I needed, thanks so much
When you digest your Dna it is always done with a restriction enzyme that s widely available and we'll known, which includes information on the specific sequence the enzyme cuts at.
Roy from the IT crowd!!! (Really great video by the way!)
You add the primer from 3' to 5'. Shouldn't it be the other way around or is that just the case for DNA polymerization ?
I don't understand how knowing one base (the detectable one) helps determine all the bases on the strand. I've seen several videos like this that just end with this comment but don't explain it. Confused.
primer annealed to wrong side of strand in video as annoted
I think ddNTPs are labbled not dNTPs.
Am I right??
usually this is done on DNA fragment that have been formed using restriction endonucleases. These enzyme only cleave the DNA at specific sequences so we can use these "restriction sites"as sites to add the primer.
I'm still confused why reading across the bands will tell you the dna sequence. am I missing background info that would help?
Did you find the answer yet ?
I have the same question 😑
+Deena Haroon I typed all this on my phone and only now realized all the typing errors. sorry. I hope you can read past it and it was able to explain it properly. (as a note, although it may look like the 5' end is the end shortening, it should be the 3' end that is being removed and shortened.)
+Jessica Wong hey im not entirely sure myself but the video mentions that the gel can separate molecules by even one base. And usually, when you use a gel you always have a marker (I'm assuming these markers mark different intervals across the length of DNA). So if you combine this with the fact that each lane has a band corresponding to a termination by a base of that lane, you can read off (using the marker) where these bases are on the fragment of DNA.
Not too sure I made any sense there hah
+Jessica Wong actually, you probably don't even need a ladder here. I think the main fact is that you have a primer, so you can make a defined start point. So the template bound to the primer should be the smallest molecule. On a gel, this should be at the bottom. So now, if you have all other molecules in the well separated by a single base, you can pretty much read the order in which they appear, from the primer sequence onwards (or upwards in the gel).
Again, hope that makes sense. I'm busy studying for a genetics exam :-p
Thank you!! Awesomely simple! So many crap explanations of this out there...
Super helpful for my biochemistry midterm! Thanks :D
@JPFamisan so you add the primer to the complimentary strand, in order to replicate the sense strand with the mixture of free dNTPs and ddNTPs?
Thanks . it's good illustration video for DNA sequencing technique .
Great animation! Thanks for uploading
Easily explained so much useful
Hi, sorry when I'm wrong, but DNA ploymerase goes from 5' to 3', so is the primer in the video on the wrong side, isn't it ?
+Borbála Horváth DNA pol goes from 3' to 5'
+Borat Sagdiyev To rid of any confusion: DNA Poly replicates in the 5' to 3' direction complementary to the template strand which will appear 3' to 5'.
Edit: Removed PolyIII as Poly I is used in this method.
+Peter Allen Thats awkward phrasing. DNA polymerase travels in a 3' to 5' direction, producing a NEW DNA strand in the 5' to 3' direction
You're correct, sorry about that! Thanks for rephrasing!
I think you are correct - new nucleotides add on to the 3' end - so that would make it the wrong way round
Sanger method is used to determine dna sequence and requires a primer. Then how, a primer sequence is known?
Now why couldn't my lecturer explain it like that? Always adding useless side notes! Thank you for the clear explanation!
Is the sequence of the DNA shown on the gel CGGTAGCTACATGACGTTCTGA?
Are you Canadian by any chance? The way you said "aloow" was very French Canadian, but I like your accent a lot.
Why are you making this from the hills of Ireland?
Thanks, this made it so clear!!!
This video was very helpful. Thank you :)
Thank you. Very helpful.
I'm going to fail A2 biology 😭 thanks though
At start of the process, we don't know the sequence of DNA ...then how the primer is designed?
Kindly answer this question
AMAZING EXPLANATION!!!
thanks, its help me to understand my lecture and make it easierr !! Aplause 4 you :)
Thank you!! Very clear
The primer needs to be on the 3' end not the 5'end. Remember, DNA is synthesized 5' to 3'.
Thanks for the video
Thank you so much sir
Should the primer be annealed to the three prime end? Great video by the way :)
The primer anneals to the 3' end of the template strand becoming the 5' end of the new strand.
The ending totally leaves you hanging.....
very helpful video, thanks you!
Very nicely made video, one question not related to the subject.. vere is the accent from ?
Sounds Irish to me.
I LOVE YOU YOU ARE WONDERFUL AND I LOVE YOU. Woo! Now back to studying.
it is very informative
Thank you so much! It helped alot!
Perfectly explained! Thank-you :)
@gefulostfoever tf bro
@gefulostfoever 😂
Thank you so much.
I thought ddNTP is radiolabled, not 'one of dNTPs'. if one of 'dNTP' was instead radiolabled, then you wouldn't be able to assess the length accurately.
1:34 what did he say? "The DNA must now be separated onsite"?
+Vivi33 The DNA must now be separated on size.
why u don't have more videos like this?
is this a computer generated voice?
+Irakli Kalichava Irish country voice
you sounds like you are in a giant saucepan
Thanks so much
thank for you 💖
Thank you
really good science pls
You forgot to mention that you need to heat the ds DNA first. And than the DNA polymerase can add dNTPs and ddNTPs..
No they didn't..
watch again
The primer is on the WRONG SIDE.
I think primer is attached on wrong side!please explain this your video confuse me further!
how to dowanlod this
we want french traduction pleaze
The primer is on the wrong end!
I noticed that too *Kickass nickname btw*, and got a little confused. Nevertheless, great video. Thanks
great :)
eire stand up !
V.nice..
He put the primer on wrong end!
thumbs up if u rchs
thanks for the good video but dear god you mumble at times
ooohh haychee gruup
interesting
old-fashion way in at this omics era.
or 35S? LOL
1:02 - reaction 'mixters', meaning mixtures? thanks for the video, but my ears bled listening to this accent, no offence really.
german narrator
Javier M thanks for the video ^^
Javier M Irish actually
I feel like this is a fake accent...
YOUR VOICE IS DISTURBING.
1. He fails to explain that it is the primers that got labeled so that X-ray can be used to see all sequences.
2. His accent is annoying.
3. He fails to mention the sequences differ by one base only in gel electrophoresis.
Therefore I gave the video a thumb down.
In reply to your critisms:
1) Radiolabelling of nucleotides mentioned at 1:02. Also, at 2:09; "The gel is dried down and an X-ray taken. Since we radio-labelled a dNTP we can now see it show up on the X-ray film."
2) A matter of opinion. I would point out that Irish accents are consistently ranked as some of the most attractive, but I understand that some people are just hard to please.
3) This is incorrect. At 1:47 I mention how polyacrylamide gels can separate fragments differing in length by 1bp. Also I would point out, contrary to your point, that this is not true for all gels. Agarose gels for example, can typically resolve differences no smaller than ~40bp between fragments but this does depend on the gel percentage and other variables.
Thumb up for the effort.
wilson Liu u got rekt
your comment is unnecessary
if you understand it so well then why don't you make your own video?
thank you
thank you...