Question 1

At :28 he says that we can determine the mRNA sequence from looking at the codons used to form each Amino Acids. If multiple codon sequences code for the same AA, how can we be sure which one is used in each case?

Accepted Answer

You can't go from an amino acid sequence to mRNA but you can extract mRNA from a cell (through column purification),  convert it into DNA with reverse transcriptase and DNA polymerase, sequence, and then insert the DNA into a bacteria to determine the protein (find protein through the use of translational fusion). There is a lot of extra information but nice to know and clears things up.

Question 2

But isn't there many possible codons that can code the same amino acid? So then you couldn't really say the exact order of DNA's nucleic acids but a few possibilities for it?

Accepted Answer

It is not possible to go from an amino acid sequence to a specific mRNA sequence. According to Wikipedia, the process of cDNA generation begins with the purification of the mRNA strand rather than from the amino acid sequence.

Question 3

At about 3:00, he talks about how double-stranded DNA can be injected into a cloning vector (plasmid/virus), which can then be injected into a bacteria to make the desired protein. But wouldn't bacterial restriction enzymes recognize the foreign DNA (virus has injected its DNA into the bacteria again!) and cut it all out with restriction enzymes? (as mentioned in the last video) If someone could explain this to me quickly, that would be great!

Accepted Answer

I believe as he mentioned in the last video when we add the dna fragment (circular to straight and then addition of the portion of the DNA that we want), the cell recognizes it as self and does not attack it - restrict it- if it was a whole set of unique dna floating around by itself without any methy group then yes definitely but now it is part of the cells' DNA and newly synthesized DNA will be methylated just like the cell DNA and the replication process proceeds. Hope that helped!

Question 4

I am confused. At 1:32 the video says, "It's not possible to infer mRNA sequence from protein sequence, because multiple codons can code for the same amino acid. mRNA sequence can be inferred from DNA sequence." Isn't the whole point of this process to determine the DNA sequence of the protein. If you already know the DNA and thus can interpret the mRNA sequence why do you need to do this? Wouldn't attempting to create a cDNA library this way give you the possibility of multiple different, possibly incorrect, DNA sequences since multiple RNA codons can code for the same amino acid? It doesn't seem like this would work accurately if you are trying to determine the true DNA sequence of your protein.

Accepted Answer

This is a good point and the video vastly oversimplifies the process of deriving the mRNA sequence from the amino-acid sequence.  It actually can be done, but it's a bit of a pain.  In order to do it you will need to generate a probe that is complementary to some portion of the target mRNA sequence, and then do an RNA extraction on cells actively expressing your protein. How do you select the right probe without knowing which codons are actually coding for the aminos? By making several. Take a small section of the amino sequence (say 7 residues) and infer all the different 21 base sequences that would code for that 7 amino acid sequence, and generate a probe for each. Then conduct Northern blot analyses using each probe and the RNA extract and see if any bands correspond to mRNA of the right length (3 times the length of the amino sequence, plus a couple hundred extra bases for the poly-A tail). If so, you isolate the mRNA and then can carry on with the rest of the steps described in the video. It's labor intensive, but not strictly impossible.

Question 5

if we can look into an mrna table to find out the sequence of the dna, then why are we making all these proteins just find the same information (the dna sequence)

Accepted Answer

he's referring to the table that determines what amino acid is made of what codons, thus determine the according mRNA sequence that can be translated into that amino acid. The table doesn't include information about DNA.

Question 6

Are we using PCR or Bacteria to amplify our sequence? Which one would be better & why?

Accepted Answer

Typically libraries will be made in bacteria.

While PCR is very useful for rapidly making many copies of a sequence, there are a number of reasons why you might want to clone into a vector rather than just doing PCR:

0) Vectors can be used to do different things with the DNA.  A common example of this would be an expression vector — this causes the DNA to be transcribed and translated and would allow you to examine the protein encoded in the cloned DNA.

1) PCR is somewhat error prone, so if you need a lot of DNA having a bacterium make copies of it for you is much less likely to introduce mutations.

2) PCR is also more expensive if you want to make large amounts of DNA.

3) DNA is more stable as a circular plasmid, rather than a linear piece of DNA — this means to store DNA for any length of time you probably want to clone it into a vector anyway.

4) Long term storage — you can make a frozen (-80°C) stock of the bacteria containing the plasmid bearing the piece of DNA and you or someone else who wants to reuse that DNA can access it decades later by simply adding the bacteria to media and growing up as much as you want.

In fact, we will often use PCR to create enough of a specific fragment to put into a vector, which then gets transformed into _Escherichia coli_ (often abbreviated to _E. coli_).   We will then often sequence the cloned fragment to make sure we got what we really wanted and that it doesn't have any mutations.  This cloned DNA can now be stored and manipulated in whatever ways are necessary for our experiments.

Does that help?

MCAT

Course: MCAT > Unit 5

DNA libraries & generating cDNA

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Video transcript