DNA libraries & generating cDNA | Biomolecules | MCAT | Khan Academy

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[shaki]

Main thing that he didn’t mention was” why we we have to make cDNA? ”
Because Prokaryotes don’t have introns in their genome, but we do, so we have to find a method to introduce our gene in a way to be able to produce protein of interest in prokaryotes. ( also since we do have the mechanism of Alternative splicing we need to do trail and error to find where to put Exon of interest into plasmid ). We work our way back to find insulin gene that bacteria can translate. Additionally we use an mRNA that’s is already spliced and mature from Yeast cell ( which is an eukaryote) and then reverse transcribe it). This video is not complete at all. Missing key information.

[omarmartinez]

Is no one baffled by this guy's suggestion that we can work backwards from A.A. to DNA sequence? Due to the degeneracy of the genetic code, we cannot go from A.A. sequence to DNA sequence since our synthetic sequence most likely will not match the true sequence found in organisms. We isolate the mRNA that encodes our protein of interest and work backwards from that mRNA to find the true DNA sequence. Wrong use of terminology throughout the video was also painful. "Infecting" bacteria with a cloning vector? Lord.

[kevinfernandes]

Two questions 1) If you are able to find out the mRNA sequence of an AA, then don't you automatically know the DNA sequence?
2) When determining the mRNA sequence of an AA, how do you account for the various permutations in the genetic code for a given AA. Ex. Leucine is CUU and CUC. Or does it not matter?

[Szabarmawi]

Oh My God .. Thanks for making such video

[bobl]

Thanks! That short lucid explanation contributed significantly to my understanding of such libraries.

[hoangngocdiep]

You try to give the video more brightness it will be great if you do

[Nuri-sust]

I must agree with others here. There is definitely some wrong and leftout information in this video. Which is surprising inmidst the other videos which are all well researched and done

[BarrettLeona]

Smith George Garcia Nancy Thompson Mary

[RidingAGrizzly]

Cannot work your way backwards. mRNA is mature, modified, has exons, not introns. Before modification the sequence is different from what is translated. Besides, exons and introns can be different, even if the mRNA results from the same DNA sequence, due to alternative splicing.

[zaynahmed]

why do you need to do step 2 if you already have the double stranded dna from step 1?
also what does "sequencing" mean? 
can't you sequence after you have the double stranded dna from step 1?

[ethan]

I gotta say, this video is super disappointing. Not very helpful at all.

[CrazyGamer-xirf]

How would we deduce the mRNA sequence from the Amino acid sequence?
Aren't there more than one codon for a single Amino acid (wobble)? How would we know which one??

[davidbahry]

This video misrepresents DNA libraries on multiple counts. The mistakes are too ingrained to correct with annotation; the video should be completely replaced.


- It ignores the distinction between genomic libraries (containing all the DNA of the sample) and cDNA libraries (containing only the expressed DNA of the sample), which is essential for understanding why proteins and mRNA are relevant at all.

- The video claims that in constructing a cDNA library you begin by using an AA sequence to infer the mRNA sequence. An annotation on the Khan Academy website version points out that this is impossible due to redundancy of the genetic code. But the annotation does not then replace the false claim with a true one; so the video leaves the actual first step completely unexplained.

- The video implies that the mRNA sequence is known, and that the point of the procedure is to learn the DNA sequence. But if the mRNA sequence were already known then the DNA sequence could be easily derived just from base-pairing rules, no reverse transcriptase needed. In fact the mRNA is physically extracted without its full sequence being known, and then used to synthesize the cDNA.