mutations are on chromosomes or if an individual
has that particular mutation.
Another thing we can use to identify pieces
of DNA. So yes, we have chopped them up into pieces
and we have perhaps sequenced them, but where
are these pieces? How do we organize them?
And we can determine how we can organize them
by using sequence tag sites or STSs. Sequence
tag sites can provide a sort of a scaffolding
of how the pieces in the genome go together.
We are getting a little bit more granular
perhaps we got to Colorado. We are now looking
at a map of the whole of the Colorado. We
can't see the streets in Denver yet. When
we look at sequence tag sites, we will be
investigating locations of known DNA sequences
on a chromosome. Now there are libraries of
sequence tag sites, databases available online.
The sequence tag sites are 200 to 500 base
pairs long, pieces of sequence that have a
single occurrence in the human genome. We
have the sequence of the human genome and
these are tag sites that come up only once
and we know their specific location from previous
research. DNA fragments from our DNA
libraries can be
cut up with restriction enzymes just like we have
seen before. Common things here. They run
through a gel with electrophoresis separating
pieces by size as we have seen before. Now
from each of the clones that we had in the
libraries we have different pieces of DNA.
And because of the sequence tag sites, we
can align these pieces of DNA. One of the
other pieces that come into here is polymerase
chain reaction. You will recall. Previously
we use polymerase chain reaction to amplify
DNA in a PCR machine. We separate DNA and
replicate DNA and stick it back together and
replicate and stick it back together. Now
we can identify these sequence tag sites using
PCR and probes. The probes will attach when
the DNA separates and then we can locate those
probes by using visualization techniques.
So we know whether that STS exists on that
particular piece of DNA and we can put those
pieces together to make a sequence. All of
these DNA sequencing or physical mapping techniques
really involved taking DNA from libraries
chopping those chopped up pieces into more
chopped up pieces and then trying to reassemble
the puzzle once we have marked what is on
them. Again here we are at the Colorado kind
of level map looking at where Denver might
be. STSs might tell us that sort of location.
So now, let us think about how we can put these two
pieces together. Often it will take a physical
map and a genetic map and impose them upon
each other to form a greater idea of where
these genes are. We may know where the genes
are based on phenotypes and recombination
frequencies, but then we can actually use
some of these markers that might be associated
with the gene in order to figure out precisely
how far away they are. The ultimate physical
map is provided by the exact DNA sequence
on a chromosome. Here we have moved from our
state level map to our map of downtown Denver
and specifically where a restaurant is that
we want to visit. We can say exactly where
the gene is on a chromosome using DNA sequencing.
How do we do DNA sequencing?
First of all, let us go back to the beginning
of our technologies we were using restriction
enzymes to cut up all of the DNA in some things
genome. Let us say it is the human genome.
And when we chop up all that DNA, we are going
to store it in a DNA library and we have inserted
them into plasmid factors. We ask these plasmid
factors to be taken up by transformation and
stored in bacteria. Here we have our volume
of books. We call that a DNA library, it's not
like a book library because each volume has a
overlapping fragments. We have looked at few
of the ways that we might line up these overlapping
fragments, but now let us look at specifically
how we sequence the DNA, then we will move
into how we put these sequences back together
some modes that are used for large genomes.
Before we move in to how sequencing works.