You don’t know what you’ve got til you mutate it! Today I want to tell you more about how I make SPECIFIC mutations in proteins by changing their DNA instructions with SITE-DIRECTED MUTAGENESIS. This lets me see what different parts of proteins do – and I want to tell all of you!

We looked at how the sequence of DNA letters that get copied into RNA letters determines the sequence of proteins letters (amino acids) in a protein. And because those different amino acids have different properties (size, charge, water-likingness), they affect how the protein folds (structure) & how it acts (function). So if we change the gene, we can change the protein and potentially its functioning

Structure & function are intimately connected (think about a spoon versus a knife). The same holds true for biochemical “utensils” like proteins, and we can exploit this relationship to learn about function from structure and structure from function by making specific changes to specific parts using site-directed mutagenesis, then testing to see if those changes affected the protein’s activity. 

With site-directed mutagenesis you can do things like make

  • insertions (add some DNA lettttters)
  • deletions (remove some DNA ltrs)
  • substitutions (change some DNA lteters)

Some notations we use to describe what we’ve done to it 👇

🔹 For substitutions, we put the original letter first & what it’s changed to afterwards. So, say you changed the 91st amino acid from a glycine (G) to an alanine (A). You could write this as G91A.

🔹 And, to indicate taking out stretches of amino acids, we use the delta sign, Δ. So if we removed amino acids 80-100, we would write Δ80-100

I can introduce specific mutations to the genetic instructions I put into cells to make protein for me – basically, you can make a copy of a gene for a protein you want to study and stick it into a circular piece of DNA called a plasmid vector and stick that into cells (often bacteria) to get them to make the corresponding protein. We call this RECOMBINANT protein expression.

I make the copies using PCR (polymerase chain reaction) which uses the original gene as a template for making the copies & you provide short strings of DNA called primers that match where you want DNA Pol to start and stop copying. The primers “bookend” the coped parts so If you put mutations in the primers, the copies will include them. And since you design the primers, you can custom design mutations.

You might be wondering – does that mean you can only mutate the starts of genes? No! I use this technique called SLIC where you basically just have to make 2 “parts” with other lapping ends. And once the gene’s in the plasmid you just have a circle you can divide however you want – so you can have 1 “half” start smack dab in the middle of your gene if you want to put a mutation there (this probably makes more sense in the pics)

And the “halves” don’t have to be halves. They can be different lengths (though you should account for that when you mix them since longer pieces you’ll need more mass-wise to get the same mol-wise (each strand is heavier since it’s longer so the same # of strands will weigh more). And you can piece together multiple pieces.

I like SLIC, but another common way is QuikChange which I’ll talk about further down in the post.

So – SITE & LIGATION INDEPENDENT CLONING (SLIC). With SLIC cloning we use polymerase chain reaction (PCR) to make lots of copies of (amplify) “gene pieces” & stick them into bacteria to piece them together.

In PCR you amplify specific stretches of DNA from longer pieces of DNA by designing short pieces of DNA (PRIMERS) that specify the “start & stop” sites of that region -> tell DNA Polymerase (DNA Pol) where to start laying down nucleotide “tracks” so we can make our pieces. more here: http://bit.ly/2FiBXsl

If you put some extra DNA letters at the start of those primers, those letters will get stuck onto the thing you’re copying (kinda like adding a generic letterhead & footer)- and if those letters you add complement letters on a different piece of DNA they can stick together. the letters of DNA like to bind their complementary letters, regardless of where they come from – it can be the second strand of the “same piece” or the opposite strand of the “other piece”

We design the gene piece to have bits of the different piece at the ends, so that when DNA Pol starts copying PIECE1, it adds on a bit of PIECE2 at the beginning (kinda like adding a few words from the page you want to come before it)- specifically it adds the part of the plasmid that’s flanking where your gene will go

_

the PCR reaction gives us double-stranded DNA so the complementarity is “hidden” by the second strand. You have to chew back one of the strands a bit (with an exonuclease) to generate single-stranded “sticky ends” – only the end is chewed & this is the part that matches the vector, so it exposes vector-matching single-stranded DNA that can stick to the vector DNA. The exonuclease chewing is much less precise than endonuclease cleavage, so you’ll get overhangs of different lengths and when you combine them & they stick together, they’ll leave gaps – but this is ok because the bacteria can fill them in

But in order for the bacteria to fill it in properly, they need the right template sequence – the original gene doesn’t “know” the sequence of the other piece – so if you cut off some of the sequence you loose those instructions & the bacteria don’t know how to fill it in -> BUT if you add enough of the other piece’s sequence to the ends, that’s what gets chewed back – that part will match the other part so you’ll get sticking, & the other piece will be there to provide complete info. So you design primers so that: 1: the ends match & when you chew them back  they’re long enough that you don’t loose the “unique” information (you want your primers to have ~20bp overlap between the end of thing 1 & the beginning of thing 2).

So if your thing is a small mutation, you want to make sure to have ~20 bp on the other sides so the mutation doesn’t get chewed out. 

Another common site-directed mutagenesis method is “Quikchange” – I used this in undergrad. “Quikchange” is the trademark name but you can do it without a kit. Unlike SLIC, which amplifies pieceS – plural – Quikchange makes a single piece. And in QuikChange the product of one reaction is NEVER used as a template for the next, so you don’t get exponential amplification – no pyramid-scheme-like growth. Instead of PCR, it’s “primer extension”

Why? You can’t amplify the first product because it’s nicked. it’s kinda like you have 

bbbbbbbbbbbaaaaaaaaaaX

and 

Xbbbbbbbbbbbaaaaaaaaaa

you want to make 

aaaaaaaaaaXbbbbbbbbbbb

but there’s nothing “to the right” of X in the first reaction to read until you stitch them together. So you have to put in a lot of the template

And with QuikChange the polymerase has to get all the way around the plasmid – that can be a long way to go without making mistakes so you want a polymerase that is highly processive (can add lots of bases without falling off) and high-fidelity (isn’t prone to making tpyos – you only want the mutations you’re meaning to put in!)

With QuikChange your primers are at the “Same spot” but different strands – so you have 1 primer making a mutated copy of each strand. For substitutions, you center the mutation in the middle of the primers so that there’s enough on either side that it’ll still bind as if it really did match

In both methods you add an enzyme called DpnI. This chops up the parent plasmid. But not the new one. This is because the parent will be methylated but the new one won’t be. More here: http://bit.ly/2tZ17FJ

It’s really important to remove the parent cuz it’ll be circular already and have a major transformation (getting into cells) advantage. 

Quick note: As we saw the other day when we were talking about radio labeling, when you order primers they usually come without the 5’ phosphate(s) that DNA normally has. And since the primers form the new ends of your new products, your new products will have unphosphorylated ends. 

This isn’t a problem with these methods that rely on bacteria to stitch them back together – the bacteria has the equipment needed to patch it up. but it IS a problem if you’re using a method which relies on DNA ligase stitching the break together in a test tube BEFORE you stick it in the bacteria. So when you add the ligase you also have to add a kinase to add on those phosphates

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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