you are saving me. 5 minutes ago I was crying from not being able to understand my molecular cell biology textbook (first year med student), and now it all made sense. thank you. love your videos, please never stop
THANKOU SO MUCH SIR! only if my lecturers could explain as awesome as you... I love your content and videos it ALWAYS helped me through understanding fully . i hope wherever you are you be always healthy and create more awesome videos. God bless you.
i absolutely love your lectures! thank you so much for all the hard work you put in. you're saving our lives so we can become doctors and save many more lives!
Hey, really good video. One question. In another video about cDNA you said that to separate the DNA/RNA hybrid we have to increase the pH, because at a basic pH RNA gets hydrolysed but DNA not, so we will remain with the sDNA, here you say we heat them up to separate, which makes sense for me, but which of the two methods are right? Or can we use both? Has one more advantage?
Assuming you have determined the sequence of a certain enzyme/protein product, how will you identify its correct DNA sequence (*some codons are redundant or wobbled) using the cDNA libary? just asking.
do we need to place our double stranded cDNA inside a vector of a prokaryote? (ie plasmid/lambda phage) or can we just insert the cDNA molecule directly inside a prokaryote??
Thank you sir, vividly explained but i have a little worry. is there a possibility for the sscDNA to be re-transcribe into RNA? And given that DNA polymerase needs a free 3' OH to be able to form the complementary strand or betterstill it needs a primer for elongation to take place. what happens in this case? where does the sscDNA get its primer from?
If restriction enzymes cleaves by recognition of some specific sequence.. Why does it selectivly cleave genes differently? Isnt there a high chance of braking middle of genes?
Out of curiosity I believe at 6.08 there maybe an error, unless I'm not thinking correctly. All eukar dna consists of polyA tails so therefore in order to prepare a cDNA library a poly-T primer is created for complementarity??
eukaryotic DNA doesn't have a poly A tail, it is the mRNA that does, when the reverse transcription is happening the poly A tail is removed and DNA does not contain poly T tail, therefore you don't need a Poly T primer in order to prepare a cDNA library
For stuff that relies on alternative splicing (ex. antibodies) I think you'd only be able to make cDNA for one transcript variant that codes for one specific protein
No, because then it wouldn't generate the same mRNA transcript due the 5'-3' directionality of DNA polymerase. The cDNA generated is an antisense strand (non-coding) which binds to the sense strand, or coding DNA.
wonderfully explained. I just have one problem with your lectures though. I have to have my inhaler next to me because you don't breath in between your explanations and that gives me short breath. It's not a joke I'm serious please take a breath sometimes. please!!
Amazing video, but I think there may have been some errors. Please correct me if I am wrong. 1. On the bottom right, the cDNA is shown to be 5' to 3' and the mRNA is also shown to be 5' to 3', should the cDNA actually be 3' to 5' so that is is complementary to the mRNA? 2. In 8:35, you say that you can take the double stranded processed DNA and place it in the eukaryotic bacterial cell, did you mean prokaryotic or am I confusing the meaning of your sentence and that the eukaryotic refers to the DNA?
For the first note, on the bottom right, the cDNA is shown to be 5' to 3' because it has been rotated 180 degrees. Notice the flat back bone is on top instead of the bottom of the bases, while the bases (perpendicular lines) are facing down. Imagine the cDNA was attached to the mRNA but then it was separated AND rotated 180 degrees to be put side by side so that they both are shown as 5' to 3'. I think that's what he did, but I am not sure.
yes actually the dna strand synthesised first must b 3'-5' nd cdna 5'-3' so thst it becomes complementary to the parent strand and has same polarity as the mrna strand...nd actually he meant prokaryotic bacteria
One application where cDNA is used is RNA-Seq: "While direct sequencing of RNA molecules is possible, most RNA-Seq experiments are carried out on instruments that sequence DNA molecules due to the technical maturity of commercial instruments designed for DNA-based sequencing. Therefore, cDNA library preparation from RNA is a required step for RNA-Seq." - RNA-Seq methods for transcriptome analysis. Hope that helps.
