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I'm wondering how to confirm the specific T(which is the unmethylated cytosine) after PCR? And also thanks for your video which is clear and easy to understand!
You wonder how distinguish between the “new“ T (unmethylated cytosine) and the “normal“ T in the DNA, correct? This works only by comparing the untreated DNA with bisulfite treated DNA... (sequencing) e.g. Untreated DNA A T C A G G Treated DNA A T T A G G the first T was originally also a T the second T was initially an unmethylated C
I know this question is a few years old but it’s possible to sequence whole genome with low concentrations of DNA ie extracted and not amplified DNA with next gen sequencing. So you can compare the sequence prior to bisulfite conversion and after.
A CpG island is a region in the DNA with a high C G dinucleotide frequency and density. They are often located in promoter regions. Indeed, here we find an important part of methylation. You probably talk about CpG *sites*? Mainly methylation occurs at CpG sites, true. However, there is also methylation at CpA, CpT and CpG sites. See -> www.ncbi.nlm.nih.gov/pmc/articles/PMC5485512/
Yes. You compare post PCR sequence to pre. it’s possible to sequence whole genome with low concentrations of DNA ie extracted and not amplified DNA with next gen sequencing. So you can compare the sequence prior to bisulfite conversion and after.
The video is very nice. However, I had not understood at first glance why U is converted into T in PCR. In another video, I saw that a complementary sequence is generated in a first-round, where U is paired with A. Then, in a second-round, the primary sequence is generated, but with T paired with A. It is equivalent to the conversion of U into T. I had confused because, in the cell, the U in DNA appears to be corrected into C. If the video showed the conversion of U into T in PCR it will be perfect!
I agree! I decided to skip the step you mentioned ´cause I put the focus on the bisulfite conversion itself. But you described it correctly! I said "certain rounds of PCR" to simplify everything, but U is paired with A and this newly synthesized strand is paired with T then! True
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Great explanation. Very comprehensive summary with good animation👍👍👍
Amazing explanation, right on the point. Thanks :)
Great video! Thank you for explaining it so fully.
Great video, short and simple, nice graphics.
Thank you!
Beautifully and quickly explained. Thanks for the video, you earned a sub!
I really like these kinds of videos where you understand every part of the topic. It was really helpfull.
You way of teaching is amazing
This is super informative and easy to understand thank you
Very nice video thanks for sharing
Very clear explanation. Thank you 😊
Amazing crisp explanation. Thanks a lot
Explanation was clear. thank you.
I just donated about 2 dollars for the video. But the video deserve much more than that
Well done explanation!
But I have to confess that the "arrows" you showed ( C > C or C > T) looks like "greater than". 3:34
I agree! This is a bit misleading..
Very helpful video. Nice and easy to understand
This was very useful thanks a lot!
Excellent video!
Helped a lot, many thanks!
Great explanation, thank you for your help
Thanks for this easy explanation!
Thanks for watching!
Awesome 🙏🏻 thank you
well explained, thanks.
Valeu!
Not again... stop it, Mario! Thank you, man!
Thank you so much for clear explanation ❤
I'm wondering how to confirm the specific T(which is the unmethylated cytosine) after PCR? And also thanks for your video which is clear and easy to understand!
You wonder how distinguish between the “new“ T (unmethylated cytosine) and the “normal“ T in the DNA, correct?
This works only by comparing the untreated DNA with bisulfite treated DNA... (sequencing)
e.g.
Untreated DNA A T C A G G
Treated DNA A T T A G G
the first T was originally also a T
the second T was initially an unmethylated C
@@henrikslab Do you know a way to compare the bisulfite converted sequence with a non-converted sequence? A software maybe?
I know this question is a few years old but it’s possible to sequence whole genome with low concentrations of DNA ie extracted and not amplified DNA with next gen sequencing. So you can compare the sequence prior to bisulfite conversion and after.
Nice channel and nice videos 👌
Very helpful.
so grateful sir
Thank u so much. This is helpful.
Amaaaaaazing thank u
Excellent
Good exp.
Thank you so very much
Well, how are we gonna use the same primers for the template whose Cs are changed to Ts in PCR?
I thought methylation mostly occurs at CpG islands alone?
A CpG island is a region in the DNA with a high C G dinucleotide frequency and density. They are often located in promoter regions. Indeed, here we find an important part of methylation. You probably talk about CpG *sites*? Mainly methylation occurs at CpG sites, true. However, there is also methylation at CpA, CpT and CpG sites. See -> www.ncbi.nlm.nih.gov/pmc/articles/PMC5485512/
Thank you for clarifying my question
Good.
How do you know a given base was an un-methylated C and not a T? Do you to compare it the original sequence somehow?
Yes. You compare post PCR sequence to pre. it’s possible to sequence whole genome with low concentrations of DNA ie extracted and not amplified DNA with next gen sequencing. So you can compare the sequence prior to bisulfite conversion and after.
The video is very nice. However, I had not understood at first glance why U is converted into T in PCR. In another video, I saw that a complementary sequence is generated in a first-round, where U is paired with A. Then, in a second-round, the primary sequence is generated, but with T paired with A. It is equivalent to the conversion of U into T. I had confused because, in the cell, the U in DNA appears to be corrected into C. If the video showed the conversion of U into T in PCR it will be perfect!
I agree! I decided to skip the step you mentioned ´cause I put the focus on the bisulfite conversion itself. But you described it correctly! I said "certain rounds of PCR" to simplify everything, but U is paired with A and this newly synthesized strand is paired with T then! True
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What an honor!
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Thank you ,it was helpful.