Pyrosequencing
Pyrosequencing enables real-time DNA sequencing based on the detection of pyrophosphate released during DNA elongation. A specific pair of PCR primers, one of them biotin labeled, is used to generate a locus-specific amplicon of approximately 200 base pairs (bp) while a sequencing primer is used to sequence the region of interest and quantify heteroplasmy levels. The procedure requires denaturation of the double-stranded PCR products and isolation of biotin-linked single strands with streptavidin-coated beads to be used as template for pyrosequencing. After annealing of the sequencing primer, nucleotides are added one at a time into the reaction mix. If the complementary nucleotide is incorporated into the nascent elongating strand, pyrophosphate is released and converted to ATP by ATP sulfurylase. This ATP along with oxygen is used by the luciferase to convert luciferin into oxyluciferin in a reaction that generates light and releases pyrophosphate Light production is proportional to the amount of incorporated nucleotide, and it is represented as a pyrogram peak that provides information that can be used to quantify heteroplasmy levels.
please watch video for more detail
Пікірлер: 77
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You made bio techniques so easy to learn and understand . Thank you ❤️
@BaaYo
Жыл бұрын
Thank you
Tomorrow is my exam and your lecture is life saver thank you ❤
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Great to see such a great teaching style 👐
Just love your teaching ❤
I can easily understand you teach through your lectures. These help me alot during my exams preparation. Thank you 😊
THANKYOU SO MUCH MAAM...
You are the best teacher mam 😍😍
Ap kounsa screen recorder and editor use krte ho.. n ur videos are super awesome 👍
Great explanation ❤
Thank you ma'am 😊
Awesome mam❤🎉
Ma'am concept clear 💃☺️☺️☺️
Thank you Soo much Really helpful for exam held on Monday❤️
Mam you are excellent superb fabulous fantabulous awesome Teacher a lot of thank you dear mam
Cuteness overloaded mam😀
आप खड़े होकर समझाते हैं तो एकाग्रता बनी रहती है मैडम आरती जी , इस तरीक़े से आप समझा अलग रहे हैं और पॉइंटर कहीं रहता है , पॉइंटर आप की कलाम या पेन सही रहता है जी , मेरा अपना सुझाव है कि आप पहले की जैसे खड़े होकर समझाएँ ।❤
Thanks a lot for such informative lecture.... Lots of love from Pakistan.... Stay blessed
Thank you ma'am
thank you guru ma❣
Thanks ma'am
Please explain NGS also
Thank you mam 💖
you are definitely better than my university professor❤
Aap amazing ho mam 🎉🎉
Very easy to understand
@BaaYo
8 ай бұрын
thanks :)
Thankyou ma'am 🙏
@BaaYo
Жыл бұрын
Thank you
thanks mam
I have my genetic engineering exam in 5 hrs and I had no idea of pyrosequencing. Thank you.
@viveksairam1747
Жыл бұрын
How was the paper 📃
@minimishra6703
Жыл бұрын
@@viveksairam1747 soo freaking shitty 🥲🥲🥲
@BaaYo
Жыл бұрын
ya how was your paper
Maxam and Gilbert sequencing method upload kriye mam plzz 🙏
Maxam Gillbert kab upload kry gi video???
You are awesome Teacher 👍👍lots of lov madam
@BaaYo
Жыл бұрын
Thank you :)
Ma'am when Next generation sequencing video will Upload?pls ma'am upload it earlier
MADAM..IF APYRASE ENZYME DEGRADE THE NUCLEOTIDE BEFORE ADD WITH SSDNA THEN ??? PLEASE MADAM...
why dNTPs are not degraded before addition?
Do we get the same intensity when we add CTP and GTP? If yes then how can we get to know that only this particular nucleotide is added on that particular site? ...because intensity is same...
@ChitraK16
Жыл бұрын
It's already mentioned on the graph (x axis) which particular base is going to broken down into pyrophosphates (PPi)
@kishanjilka8820
Жыл бұрын
@@ChitraK16 suppose we got two same intensity points for two break down of two nucleotides...then by the intensity how we can get to know that this intensity is for this nucleotide and this intensity is for this nucleotide.
@ChitraK16
Жыл бұрын
@@kishanjilka8820 don't get confused. Let say GTP and CTP is broken but two dNTPs of each simultaneously. So as ma'am already explained two consecutive nucleotides will show higher peak but on their specific site on X Axis But I think of you see 4:15 in the video, ma'am also said that we are adding nucleotides one by one. So their will be no confusion of getting intensity of two different nucleotides
@ChitraK16
Жыл бұрын
@@kishanjilka8820 just clearly read the graph and their axis ( mentioned points on x Axis) , that's all you need to understand during interpretation.
@kishanjilka8820
Жыл бұрын
@@ChitraK16 okay okay I will try to get more clarity on your point... Thank you for showing interest on my question 🙂
Ma'am next which Unit you're going to start on unacademy?
@BaaYo
Жыл бұрын
unit 13
How to get this pdf?
mam please make a video on chromosome walking and jumping method in dna sequencing
Hello mam, im Dr Aamer , mam aap ka o porana teaching method tik ta , jab aap white board py para rahe t ,
How will we know the sequence of primer ma'am as this is an unknown sequence for us
@sushmitakushwaha8172
Жыл бұрын
In order to synthesis primer we use known sequence of adaptor. The sequence of adaptor is know to which the primer and ssdna is attached.
@user-mr5yt5ls1i
7 ай бұрын
@@sushmitakushwaha8172sare sequencing methods me template ka primer adaptor add kar k hi hota hai? Jo b template hai usse adaptor ko attach karna and adaptor k sequence k hisab se primer dalna? Sahi samjha Maine? Ya phir vector me b clone Kiya jata hai template (ssdna fragment to b amplified)
Kitna acha pdhaya mam. thank you so much 💕
@BaaYo
Жыл бұрын
Thank You Deepika :) please share this channel with your classmates also :)
Maam plz upload transcription and translation lecture
@BaaYo
Жыл бұрын
soon
Sangar sequencing main cloning hota hai q ki temlet unknown hai but pyrosequencing main kese primer use karsakte hain
@user-mr5yt5ls1i
7 ай бұрын
Adaptor use krte hai Attach adaptor to the template and adaptor k hisab se primer add Krna. Kisine comments me bola hai ye.
Mam please add sequencing by ligation and by synthesis, ion.... 13 sept ko exam hai and kuch samajh nhi arha hai.
@BaaYo
Жыл бұрын
13 sep to gyi.
Mam when we already have ppi then why we need to have ppi again after so many reactions
@BaaYo
11 ай бұрын
Didn't get ur question.. we don't have ppi.. it releases when a dnpt incorporated into the chain..
@muskanjain8532
11 ай бұрын
Mam I want to ask that the ppi is their in the 1st few steps but then also why we has so many components in the test tute to again produce ppi and light
@BaaYo
11 ай бұрын
No we are not producing ppi again.. all other reagents are to produce some light .. so that detector can detect it
Ma'am mujhe bas yahi samjh nahi ata ki template ka sequence hume nahi pata to kaise hum primer add krte hai? Sare methods me mujhe yahi ek confusion rehta hai
@BaaYo
7 ай бұрын
Pyrosequencing me hum emulsion pcr krte hai... yani apne template ko adapter se ligate krte hai.. adapter bead se attached hota hai.. adaptor ke complementary primer use krte hai.. i hope u got it now
@user-mr5yt5ls1i
7 ай бұрын
@@BaaYo yes ma'am..now clear 👌 thanks for quick reply 🫂
Fuck
आप खड़े होकर समझाते हैं तो एकाग्रता बनी रहती है मैडम आरती जी , इस तरीक़े से आप समझा अलग रहे हैं और पॉइंटर कहीं रहता है , पॉइंटर आप की कलाम या पेन सही रहता है जी , मेरा अपना सुझाव है कि आप पहले की जैसे खड़े होकर समझाएँ ।❤
Thank u mam ❤
Fuck