Brother, i will be expected to present and explain a machine from a company which i utterly want them to hire me, i decided to present 454 sequencing and this video saved my life. thanks from turkey
Good video but your explanation of the emulsion PCR step is a bit off. You seem to suggest the strand complementary to the original template (created by extension of the bead oligo) breaking off after denaturation and annealing to another one of the bead oligos. This is not what happens as 1.) this strand is attached to the bead and therefore cannot move and 2.) it wouldn't bind to the bead oligos as it is not complementary, it's identical. Instead, after the first denaturation step it is the original template that breaks off and binds to another bead oligo (as before) and again a complementary strand is created. At the same time, a PCR primer will bind to the 5' end of the first complementary strand (the end away from the bead) and will be extended towards the bead to generate another copy of the original template strand. This process is repeated until the bead is covered in clonally amplified copies of the original template.
Thanks. I'm wondering: How exactly do you make sure only one DNA sequence lands on a bead, without others landing there at the same time and the bead therefore being incosistant?
mashaAllah very nice and helpful explanation thank u so much u r genius really though you look very young , may Allah bless you ... I watch you lectures since last year and thy ve been always a great help to me .. thank you dear
Excellent explanation !! your every lecture's video is so nicely explained, Good to helps other students or researchers ! Good luck for future !! Go ahead....... :)
Thank you for informative video but am confused with primer addition at the end. As I know first we use primer with adaptor sequences to make copies of complementary DNA stand by pcr and then run on 454 sequencer. But in your video you explain that we don't need pcr to make copies. Then how we will add primer and adaptor sequence to target dna for sequencing. Please if possible make it more clear for me and please also draw a schematic work flow of this whole process in Sequence from 1 to end. Thank you you did very job for us. GOD BLESS YOU MORE SUCCESS
Hi Shomu, thanks for great video. As far as I understood the method reads nucleotides in order they actually appear on the strand. What I don't understand is how the method prevents the same nucleotides from different parts of the strand from being read all at once. So if we have the following sequence ATTGCAA and we add Thymine we detect just one fluorescence as T bonded to the first A. How does it not bond to the final AA as well?
Hey man, I like your video its helped me out and is very clear. The only bit I'm confused about it the end. So you have all these fragments (I'm assuming there are thousands of fragments) of double stranded DNA composed of the target single strand and the fluorescent single strand from which we derived the target sequence. I don't understand how these fragments are put together into the correct order of the actual full length DNA molecule of whatever we sampled?
hello! i have a question, isn't 454 same as pyrosequencing? becouse here you explain like each nucleotide has a flourescent tag, but pyro uses luciferase....
Awesome bro, you are a genius! One question, is the release of pyorphosphate after each nucleotide is added the way by which the fluorescence is detected?
Hey! Thanks for uploading those videos. I'm a bit confused. Is this really the 454 sequencing? 454 is the same as pyrosequencing which uses luciferase, or am I wrong? This video looks like the cycle-sequencing. In any case, thank you!
how is 454 different from pyrosequencing? i notice that 454 uses two adapters while pyro uses one, but isn't using one adapter a lot more easier? whats the advantage of 454 and pyro method?
wait i wanna know something Dr Shomu there is a video that u uploaded by the tittle "pyrosequencing" and this one of "454 sequencing"...is there a difference between pyrosequencing and 454 sequencing since i feel as if what u have explained is the same for both videos ...please help.thank u
I am confused. How target strand is used as complementary strand in sequencer? I think complementary strand ( produced in beads) is used as complementary strand in sequencer.
I am a little bit confused that which strand was going where and which was target strand. Just messy man... please, make another clear concept video,please.
My Man always there when needed
Thank you so much for appreciating my efforts
Brother, i will be expected to present and explain a machine from a company which i utterly want them to hire me, i decided to present 454 sequencing and this video saved my life. thanks from turkey
You're the best , I can't undersand in my class, and you make things so easy to understand , thank you !
Good video but your explanation of the emulsion PCR step is a bit off. You seem to suggest the strand complementary to the original template (created by extension of the bead oligo) breaking off after denaturation and annealing to another one of the bead oligos. This is not what happens as 1.) this strand is attached to the bead and therefore cannot move and 2.) it wouldn't bind to the bead oligos as it is not complementary, it's identical. Instead, after the first denaturation step it is the original template that breaks off and binds to another bead oligo (as before) and again a complementary strand is created. At the same time, a PCR primer will bind to the 5' end of the first complementary strand (the end away from the bead) and will be extended towards the bead to generate another copy of the original template strand. This process is repeated until the bead is covered in clonally amplified copies of the original template.
Thank you, William Kerry.
Was searching for this explanation and couldn't find - indeed makes sense, thank you.
I was thinking this exact thing as he was explaining it
@Q Suraiya you are correct
Thanks. I'm wondering: How exactly do you make sure only one DNA sequence lands on a bead, without others landing there at the same time and the bead therefore being incosistant?
Thank you with my all heart. i watched almost all of your videos. please know there is a person who pray for you :)
Thank you Shomu sir, your way of explaining concepts is excellent.
Thank you so much for appreciating my efforts
Helped me to actually make sense of the textbook, thank you!
Very well explained especially for a beginner like myself. Thanks for sharing!
Glad to hear that you are getting benefit from
Your video is perfect ! I understood everything, thank you very much :)
You're welcome
Very nice explanation, I learn a lot. THX
You're welcome
mashaAllah very nice and helpful explanation thank u so much u r genius really though you look very young , may Allah bless you ... I watch you lectures since last year and thy ve been always a great help to me .. thank you dear
Thank you so much Sir. It helped me so much. The way you explain is so understandable for everyone. Thanks a bunch. God bless you
+Fatima Batool that's great. I am glad that it helped you
Sir ji you're great ... u provide exactly those material which we want.. hats off.. keep doing it...
Excellent explanation !! your every lecture's video is so nicely explained, Good to helps other students or researchers ! Good luck for future !! Go ahead....... :)
+Praveen Prajapat thank you. Glad you liked my lectures
well done prof! thank you for this great explanation :)
no hablo ingles pero entiendo mas o menos y usted lo hizo tan sencillo, que comprendí muy bien, muchas gracias :D
Well presented...
Thank you
Thank you for informative video but am confused with primer addition at the end. As I know first we use primer with adaptor sequences to make copies of complementary DNA stand by pcr and then run on 454 sequencer. But in your video you explain that we don't need pcr to make copies. Then how we will add primer and adaptor sequence to target dna for sequencing. Please if possible make it more clear for me and please also draw a schematic work flow of this whole process in Sequence from 1 to end. Thank you you did very job for us. GOD BLESS YOU MORE SUCCESS
Very informative
Thank you
Thank you for the indepth explanation, appreciated it.
+Alex Star thank you. Glad you liked my lectures
Thank you! From México.
Thank you. This video is well understandable. But when the primer is actually added?
Primer is added after loading the beads into wells.
Good vedios sir very informative.but the only problem is with sound please update sound system .thank you
Excellent
Thank you
You r the best 👍🏻 keep it up
Plss make a video about emulsion PCR...
It will be very helpful
Okay
Thank you so much sir
You're welcome
Great explanation, thanks.
+Jürgen Penn thank you.
Hi Shomu, thanks for great video. As far as I understood the method reads nucleotides in order they actually appear on the strand. What I don't understand is how the method prevents the same nucleotides from different parts of the strand from being read all at once. So if we have the following sequence ATTGCAA and we add Thymine we detect just one fluorescence as T bonded to the first A. How does it not bond to the final AA as well?
I also have the same problem. If you got your answer kindly reply.
Hey man, I like your video its helped me out and is very clear. The only bit I'm confused about it the end. So you have all these fragments (I'm assuming there are thousands of fragments) of double stranded DNA composed of the target single strand and the fluorescent single strand from which we derived the target sequence. I don't understand how these fragments are put together into the correct order of the actual full length DNA molecule of whatever we sampled?
Thank you, these are great videos!!!
hello! i have a question, isn't 454 same as pyrosequencing? becouse here you explain like each nucleotide has a flourescent tag, but pyro uses luciferase....
Meche Landeira even i have the same question and confused in between pyro and 454
yahh exactly... please rectify the explanation.
So many thanks. It is really helpful. :)
+Rumeysa Doğan thank you. Glad you liked my lectures
Awesome bro, you are a genius! One question, is the release of pyorphosphate after each nucleotide is added the way by which the fluorescence is detected?
Thank you! you are saving me !
Thanks for the great video!
Thank you -- very well done!!
+Nitin Saxena thank you very much
Thank you!
You're welcome
Where is the bead present? Is it in the tube naturally?
Thankyou sir
You're welcome
Sor kindly suggest a book for ngs study
at minute 5:57 why is it necessary to use adaptor B if it is not useful to fix the DNA to the particle?
How do we know which dna fragment (on the bead) the light belongs to?
well done brother
Thank you.
Thank u very much 😍
great explanation, Thanks XD
would be cool to know how fluorescent was generated.
oh nevermind it in a pyrosequencing vdo XD
Thank you for your explanation! you're saving me ahah
Sir is it same as Solid D sequnc?
Hey! Thanks for uploading those videos. I'm a bit confused. Is this really the 454 sequencing? 454 is the same as pyrosequencing which uses luciferase, or am I wrong? This video looks like the cycle-sequencing. In any case, thank you!
No its differ
Thanks
You're welcome
Thanks for the video. So, how do the adapters bind to the dsDNA? is it just a random event?
Random event but they bind finding the complementarity
So it’s basically ion sequencing but instead of protonlevels you just measure the fluorescence?
Sir please make a vedio on signature sequences
Okay
how is 454 different from pyrosequencing? i notice that 454 uses two adapters while pyro uses one, but isn't using one adapter a lot more easier? whats the advantage of 454 and pyro method?
Well done
Kindly give lecture on 3-C, 4-C 5-C, ChIA-PET AND Hi-C TECHNOLOGIES.
ty for saving my xms :)
HI sir i have a question is pyro sequencing and 454 sequencing are same if not ?AND why we use the second beads on the other end of template dna
why it has got the name as "454" sequencing? Is there any significance to this number?
Brilliant!
Thank you! You're the boss
Thank you!!
You're welcome
very well explanation sir
+either way thank you.
wait i wanna know something Dr Shomu there is a video that u uploaded by the tittle "pyrosequencing" and this one of "454 sequencing"...is there a difference between pyrosequencing and 454 sequencing since i feel as if what u have explained is the same for both videos ...please help.thank u
Thanks Prof
what is the special purpose of using bead. Please someone answer and clarify to me that single point.
Why it is called as 454 sequencing?
@subhalaxmi sahu
454 life science is the name of company that has given the technique...thats why it is said so
I am confused. How target strand is used as complementary strand in sequencer? I think complementary strand ( produced in beads) is used as complementary strand in sequencer.
Thank you
You're welcome
hi sir hope you will be fine ,why 454 sequence is called ,454 ????? please explain it.
Plz make video for SOLiD sequencing
Okay
sir what is the difference between pyrosequencing and 454 sequencing
please explain sir
are they same ???
perfect, thank you! :)
Muchas gracias!!
I think above explained method is ABi SOLID sequencing method..Not the 454 sequencing method..
THANKS!!
+Maite De María thank you
Muito very good
Why 454 sequence is called 454 sequence??
as per my knowledge 454 and pyro are same sequencing method.
I am a little bit confused that which strand was going where and which was target strand. Just messy man...
please, make another clear concept video,please.
I'm curious to know how the machine inputs one type of dNTP at a time and then washes it and adds the next one so damn quickly
why it is called 454 sequence?
454 is the name of the company now called Roche/454
Only ONE fragmentof DNA is caught per bead - otherwise this wouldn't make sense
so what are the software programs involved in this?
Thank you very much for helping me in every single exam (✦థ ｪ థ)
really i love you
Thank you.
Video not accurate.The statement there is no PCR is not correct; emulsion PCR. Other inaccuracies as noted below
🙏🙏🙏🙏🙏🙏🙏🙏🙏🙏🙏
You're welcome
send
must h....
confusing
You have been more celar thad mi
+Leonardo Mohamed thank you.
dude go inside