Lecture 18
Good morning.
It can't go without at least some acknowledgement and mention.
If you should ever find yourself in life in a situation where you have or are about to give up all hope, you think things are utterly impossible and there's no way, you will remember this week that nothing is impossible.
It is possible to come back three Lecture 18
Good morning.
It can't go without at least some acknowledgement and mention.
If you should ever find yourself in life in a situation where you have or are about to give up all hope, you think things are utterly impossible and there's no way, you will remember this week that nothing is impossible.
It is possible to come back three games down in the bottom of the ninth inning, you've got to believe you can do it, and remember to have Dave Roberts pinch run.
Just a general bit of good advice. What an amazing week, just absolutely amazing week. Wow. There are lessons in life to be taken from it. Please do take them. You know, there really are. I mean I'd given up hope by that point, I confess. I wish I could say oh, I knew they were going to pull it out, but I didn't. And, boy, they pulled it out one at a time. So, all of you think good thoughts this week. This could be a historic week, you know, you were here.
Anyway, onward.
We were talking last time about how to analyze your clone. The notion of cloning random pieces of DNA, identifying your clone within a library, purifying the DNA from a clone, doing some preliminary analysis by maybe cutting with a restriction enzyme, then sequencing it using these techniques that I'd described would allow you to take the clone that was, say, able to rescue the yeast that couldn't grow without arginine and figure out what its DNA sequence was.
You could take the clone that you had obtained by hybridizing with the DNA sequence corresponding to the protein sequence for beta-globin and sequence it and see the beta-globin gene sequence perhaps.
This is very powerful. I want to take a brief moment, we'll come back to it in more detail in a subsequent lecture, but I really described how you would sequence one clone. I just want to make a note, because someone asked about it last time, about how you would sequence an entire genome. Someone asked about this.
Remember before we pulled out our clone, we sequenced it, we got its DNA sequence. What if I wanted to sequence the entirety of a genome? Yeah. Do a lot of this, right, basically if I got a whole genome. Well, somebody asked could I put a primer here and just sequence?
It would take a very long time. And it turns out that it wouldn't work because the separation that you can achieve through gels is a function, the separation between N and N plus 1 in length goes like the logarithm of the ratio. So, it turns out that when N and N plus 1 get to like about a thousand, you can achieve very little physical separation between them. And so, DNA sequencing runs cannot go much past the thousand bases.
So, the problem with sequencing a genome by putting down a primer on an extraordinarily long piece of DNA, a hundred million bases, is you cannot separate the little fragments like that. So, what you do is you break up your genome into lots of pieces. One strategy, break it up into a library of some very big pieces. It turns out you can make pieces at random of a hundred thousand base pairs.
Cloning these in bacterial artificial chromosomes, as we talked about before. Take a library of bacterial artificial chromosomes and then begin sequencing them. And take any given bacterial artificial chromosome and break it up into a whole lot of pieces that are maybe a thousand bases long, and you could sequence all of those. How do you arrange to get just a perfect overlapping set of thousand base pair clones that perfectly tile across the sequence with no redundancy?
