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[MUSIC].
Hi, I'm Rob DeSalle and
we're in the
Sackler Institute
for Comparative Genomics Labs here at the American Museum of Natural History.
What I want to do today is show you some
of the techniques and technology that are used in generating
genome sequences.
And the first step in generating any genome sequence is to obtain tissues.
And then, to isolate DNA from those tissues.
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So, the first thing to do is to get a tissue sample.
And this is just a little bit of salt in, in distilled water.
And I'm just going to put it in my mouth and swish it around in my mouth.
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>> And that's the end of the gross part.
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>> Now what I have in this tube is just a bunch of cells from my mouth.
And this'll include.
Cells from me, but also cells from anything that I've eaten for
breakfast or anything that I've eaten for lunch, and also
any of the 500 or so species of, of bacteria that live in my mouth.
The next step is to simply lice the cells in the, in the mouthwash.
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And we use a simple detergent called SDS to do that.
And you just simply add it, and you might see a little bit of clearing
of the solution, and that's because the at first, that's because the.
SDS is actually blowing the cells apart and
making this illusion a little bit more translucent and
now the final step is simply to add ethanol
and to precipitate the DNA away from the solution.
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Now this DNA is, after it's purified, is then
carried through a series of steps that allow us to
determine the sequence of a's, g's, t's, and c's
on the strands of DNA that are in this tube.
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And, each of the wells, on this plate, has a
DNA sample in it.
And again, we just need to deliver the sequencing chemistry to
each one of them, and, to do that we use a robot.
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Now we're moving to the next step of DNA
sequencing and we're doing what is called Sanger sequencing.
We've taken our plate of 100 samples and put it on this robot.
And the robot is simply delivering different
chemicals and different solutions to our DNA sample.
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And chemical reactions are going on in the plates,
and these chemical reactions are labeling the
DNA with fluorescent molecules One for each of
the different bases, g, a, t, c and the chemical reactions are splitting the DNA
up, making ladders of DNA so that we can then read the g's, a's, t's,
and c's in the order that they appear on the DNA fragments that we're sequencing.
And then allows us to move to the
third step of the procedure, which is to place them on the sequencing machine.
So, this is the plate with the chemical and biochemical reactions
that we did to do the dna sequencing, with the four
different fluorescences for g, a, t, and c And the fragments have been
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separated in the chemical reactions, and this machine, the ABI3730,
now will allow us to separate the fragments by size
and then, read the florescences from the fragments to give
us the sequence of g's, a's, t's and c's in
all of the wells that we see on this plate.
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Now once the sequence run has been done on the 3730 with your Sanger
sequences, you need to process them and
to look for a singular nucleotide polymorphisms.
So, in my sequence, you can see in this region right
here a black peak, a green peak, and a blue peak.
That's my sequence.
And then in, in this individual, it's a black peak, green peak, blue peak.
But in this individual, it's a black peak, green peak, red
peak.
This particular, sequence right here, where it's a c in,
in my sequence, a c in this other reference sequence.
And a t in this bottom sequence.
What this means is that we have a polymorphism in this position right here.
That is what we call a single nucleotide polymorphism.
And these single nucleotide polymorphisms, or snips, are what
we use in the genetic technology and the genetic techniques
that we described in lecture.
These chromatographs are relatively hard to look at,
so what the computer also does, the programs
also do, is they transform this peak information
into actual DNA sequence information, into the g's
t's a's and c's, so that this region right here that my finger is pointing to,
is the same region we talked about when
we were looking at the pigs in the chromatagraph.
So my sequence has a c. The middle reference sequence
has a c and the bottom reference sequence
has a t indicating a single nucleotide polymorphism which
is the main thing that you use in
doing genomic analysis that we talked about in lecture.
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So now were sitting next to a
next generation sequencing machine here, there 454
machine, and this machine can sequence DNA
fragments much more efficient than other methods.
and this machine, what it does is it uses small beads
where the DNA that you're trying to sequence are attached to.
And then those beads are placed onto this tiny plate.
Now if we could look at this plate under a microscope we would see that there are
hundreds of thousands of little depressions in the
plate and the beads fall into these little depressions.
On each of the beads, we'll do a sequencing reaction and
we do them in a highly parallel fashion, about 100,000 reactions at a time.
In this way, the process of DNA sequencing
has been sped up several orders of magnitude.
There are other next generation sequencing machines
that are even much more powerful than this.
That, use different technology.
but also generate thousands times more sequence than the previous
generation sequencers.
Rob DeSalle, Ph.D.Curator
David Randle, Ph.D.Senior Manager of Professional Development
