DNA libraries & generating cDNA | Biomolecules | MCAT | Khan Academy
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- Опубліковано 21 сер 2024
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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.
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.
Omar Martinez He should have specified, but you can essentially get the likely different mRNA sequences from the aa sequence. In fact it can usually be pretty accurate if you know the organism you're working with and what codons it typically uses for each aa. Even if you don't know that, you can work with degenerated primers to get your cDNA, basically a mix of all the primers that could work. Not as efficient nor easy as he makes it sound but certainly possible.
Omar Martinez 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
does it still sound baffling now mrna vaccines are out ?
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?
+Kevin Fernandes No, because there is a degree of promiscuity when it comes to AA and the Codon table. You have multiple codons for a single AA.
Just to add, lambda phage genomic library contributes to various applications, especially for antigen discovery and immune response investigations. By constructing genome libraries derived from specific pathogens (i.e. virus, bacteria or organ) and screening against the whole antibody repertoire of infected individuals, efficient identification of a large panel of antigenic regions can be achieved. Reference: www.creative-biolabs.com/genome-library-construction-service-via-lambda-phage.html
Kevin, sequencing requires an abundance of DNA so it can be visually seen using an electrophoresis or similar method. Electrophoresis separates a sample according to size and concentrates them into visible lines. Sanger sequencing (a specific form of electrophoresis) may be a good place for you to get a grasp of how we visualize DNA sequence. in essence, we cannot sequence one (s/d) strand of DNA accurately, it's too small. to solve this, we make lots and lots so we can see it.
you bring up a great point with your second question. if your goal is to remake more of this protein, it shouldn't matter, but you cannot claim your DNA library is necessarily the same as the actual DNA which coded for the protein in that organism. However, it can provide you information to create primers to find the actual sequence in the organism's DNA (a primer which has a CUC and another which has a CUU in the same spots. hopefully you can strategically choose primers to reduce the number of these primers necessary). you can then use those primers to perform a southern blot and identify where in the DNA those sequences are. hopefully only one primer will actually bind , otherwise it's back to the lab.. Then you can cut/amplify that DNA, sequence it to verify any other potential codon discrepencies, and record your findings.
Oh My God .. Thanks for making such video
Thank you so much
thanks!
Thanks! That short lucid explanation contributed significantly to my understanding of such libraries.
THE BEST
Couldnt u amplify with pcr too ?
From my understanding you can, although naked DNA will deteriorate over time. Cloning the DNA in a vector stabilizes the DNA for later retrieval and probing.
You try to give the video more brightness it will be great if you do
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
Here after Rashad Evans comment.
Oh my Gooooooood
thanks
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.
Ravenclaw Phoenix absolutely! These videos are misleading !
then it would mean the mrna vaccines out now are useless and ineffective .
@@kakaboo no, this is not what it means. An mRNA codes for a very specific protein. It is made such from the DNA blueprint. The vaccines are that mRNA. Inside our cells it codes for the antigen that's specific to the virus and helps the immune system make memory cells
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?
is DNA library same as CDNA library?
there are 2 DNA libraries, cDNA and genomic libraries.
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??
if your goal is to remake more of this protein, it shouldn't matter, but you cannot claim your DNA library is necessarily the same as the actual DNA which coded for the protein in that organism. However, it can provide you information to create primers to find the actual sequence in the organism's DNA (a primer which has a CUC and another which has a CUU in the same spots. hopefully you can strategically choose primers to reduce the number of these primers necessary). you can then use those primers to perform a southern blot and identify where in the DNA those sequences are. hopefully only one primer will actually bind , otherwise it's back to the lab.. Then you can cut/amplify that DNA, sequence it to verify any other potential codon discrepencies, and record your findings.
Brendy Fountaine great explanation thank youuuuu
This video misrepresents DNA libraries on multiple counts. The mistakes are too ingrained to correct with annotation; the video should be completely replaced.
- It conflates "DNA library" with "computerized gene sequence database". In fact a DNA library is a physical collection of actual DNA fragments, generally stored in a population of microorganisms (e.g. a population of E coli bacteria or yeast cells that have been transformed with recombinant plasmids). en.wikipedia.org/wiki/Library_(biology)
- 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.
A correct, brief explanation is here: ua-cam.com/video/SvjeCxVu2dI/v-deo.html
I gotta say, this video is super disappointing. Not very helpful at all.