Video Transcript
In this video, we’ll learn about
some different molecular technologies. We’ll discuss bioinformatics and
how it can be used to look at evolutionary relationships, so- called gene machines,
to produce desired sections of synthetic DNA, as well as DNA hybridization. We’ll discuss the advantages of
these technologies and their applications.
We’ll begin by discussing an
important molecular technology known as bioinformatics. Humans and chimpanzees share a lot
of their DNA in common. In fact, about 99 percent of our
DNA is the same. So, how did we determine this? Well, we basically need to line up
the two sequences and then look for differences. The problem is, and it’s a big
problem, is that the sequence of the genome, or our total DNA and the chimpanzee, is
about three billion base pairs long. This is a huge number. It may be easy to write it out on
paper, but to give you an idea, one billion seconds is just over 31 years.
So, to make our lives a little
easier, we use one of humankind’s greatest tools, the computer. Computers can align these sequences
and check for mismatches a lot faster than any human or chimpanzee for that matter
is able to. This is an example of the field of
bioinformatics. It’s a combination of computer
science and biology to analyze large and complex biological data, like our DNA
sequence here. So, by using a computer, we can
line up the three billion or so base pairs in the genome of the human and
chimpanzee, and it will tell us how similar they are in a reasonable amount of
time.
Comparing DNA sequences can tell us
a lot about how closely or distantly related we are to other species. So, while humans and chimpanzees
share about 99 percent of their DNA, what about another species, like a chicken? Now, if we include some of the
sequence of the chicken’s DNA, we’ll see that there’s more differences. In the chimpanzee, only one base
pair was different from human DNA, which is now underlined in black, whereas in
chicken DNA, we can see five base pairs that are different. Because human and chicken DNA is
less similar compared to human and chimpanzee DNA, humans and chickens are said to
be more distantly related than humans and chimpanzees. This is because humans and
chimpanzees share a more recent common ancestor than humans and chickens do and is
why humans and chimpanzees have more DNA in common.
Besides looking at DNA sequences,
we can also compare the amino acid sequence in proteins to study evolutionary
relationships. So, here’s a section of the hormone
insulin in our three species, and similar to DNA, bioinformatics can help us figure
out the differences in these sequences. Bioinformatics isn’t just about
figuring out evolutionary relationships. We can also use it to study gene
expression or how proteins fold or how tens of thousands of proteins can interact
with each other. There’s a lot going on in a cell
that we want to understand, and a computer is the right tool for this job.
Suppose we wanted to study the
evolutionary relationship of insulin between these species in more detail. There’s another way we can do this,
but before we get into it, we first need to produce a copy of the DNA of the insulin
gene. The right tool for this job is a
gene machine. A gene machine is a laboratory tool
that synthesizes genes or sections of DNA from an input sequence on a computer. You can either put in the DNA
sequence directly, or if you only have the amino acid sequence of the protein, then
it can work out the DNA sequence from this. Let’s look at this process in more
detail.
You’ll recall that DNA is
transcribed to make an mRNA sequence, which is then translated to make the amino
acid sequence of a protein. So, let’s go through this. Here’s a section of the insulin
protein, and if we’re converting this amino acid alanine back into mRNA, we need to
look at a codon wheel to do it. So, alanine is located right here,
and the corresponding codon for alanine is GCU. The next amino acid is serine, and
this corresponds to the codon UCU. Filling in the rest of the codons
will give us this mRNA sequence.
Now, we can convert this into the
DNA sequence. You’ll recall that mRNA and DNA
have directionality, so let’s fill that in. Now, we’ll simply convert this
sequence in the DNA, remembering that thiamine in DNA replaces uracil in mRNA. So, there’s one strand. Now, let’s do the complementary
second strand. Great! So, there’s our DNA sequence for a
segment of the insulin protein. As you’ve seen, we can work this
all out by hand, but the gene machine can do this automatically using a
computer. Next, the gene machine needs to
synthesize this DNA sequence, and to do that, it uses something called an
oligonucleotide.
An oligonucleotide is a short piece
of synthetic DNA or RNA that is often single stranded. The gene machine assembles two
oligonucleotides. The first one corresponds to one
part of our sequence on one strand, while the second one corresponds to another part
of our target sequence on the opposing strand. You’ll notice that we now have two
oligonucleotides that span the length of our target DNA sequence, a portion of the
insulin gene. What’s now missing are these two
gaps in the sequence at either end. So, how do we fill these in? You’ll notice here we have an
overlapping section where the two oligonucleotides meet. This is a region of double-stranded
DNA and can act as a binding site for the enzyme Taq polymerase.
Taq polymerase is a type of DNA
polymerase, the enzyme that plays a role in replicating DNA in preparation for cell
division. We use this enzyme because it’s
able to fill in these gaps and can also be used for PCR, or polymerase chain
reaction. This is a technique that makes
multiple copies of this target sequence, and it’s helpful because we want to have a
lot of it so we can extract it and study it. So, Taq polymerase is able to fill
in the gap on the top strand by using the opposite strand as a template to add
complementary nucleotides, where it can then fall off and then reattach, where it
can now fill in the gap on the opposing strand.
We now have our completed DNA
sequence synthesized using our gene machine. An obvious advantage of using a
gene machine to synthesize genes or sections of DNA is how easy it is. You simply need to input the
protein sequence of your favorite protein and it synthesizes the gene for it. Another advantage is that the
synthesized DNA is free of introns. You’ll recall that genes often
contain introns, which are sections of noncoding DNA that need to be spliced out
from the mRNA in order to assemble the protein coding sequence. Since we’re inputting the protein
sequence directly into the gene machine, we don’t need to worry about noncoding
sequences.
Now that our insulin DNA has been
synthesized, we’re ready to study more about the evolutionary relationship between
our human, chimpanzee, and chicken insulin. To do this, we’ll look at a
molecular technology called hybridization. Hybridization is the process of
combining complementary single-stranded DNA or RNA molecules. The two DNA or RNA molecules do not
need to be from the same source. So, we could hybridize our human
insulin DNA with our chicken DNA to see how similar they are to each other. Let’s go over how this works. So, here is our human insulin DNA
segment, and in between the two strands are hydrogen bonds that hold the two strands
together.
By heating our DNA typically to 95
or 100 degrees Celsius, we can weaken and ultimately break these bonds, which causes
the two strands to separate into single-stranded DNA. And now, if we lower the
temperature rapidly to about five to 10 degrees Celsius, the complementary bases
will pair with each other again to reform the hydrogen bonds. This pairing of complementary
single-stranded DNA or RNA molecules is called annealing. What’s interesting is that these
two sources of DNA or RNA don’t need to be the same. So, if we had the corresponding DNA
for the chimpanzee insulin gene, we could hybridize these two DNA molecules
together. So, we mix these two DNA molecules
together then heat the sample to break the hydrogen bond and form single-stranded
DNA. And now, we’re going to cool the
sample so the single-stranded DNA molecules can anneal.
So, the two single-stranded
molecules of human DNA and chimpanzee DNA can come back together and form their
original DNA molecule. But sometimes, one of the strands
of chimpanzee DNA will anneal to the complementary strand of human DNA. So, let’s see what the
hybridization of these two strands looks like. So, here in this human- chimpanzee
hybrid of a section of the insulin gene, we can see that most of these bases are
complementary to each other and form hydrogen bonds except for one. This one base is different in the
chimpanzee DNA, and so this won’t hydrogen-bond. We can see a more dramatic
demonstration of this if we hybridize the human DNA to the more distantly related
chicken DNA.
Here we can see that there’s many
more mismatches. Because there’s fewer hydrogen
bonds holding the human and chicken hybrid together, it will take less energy or a
lower temperature to separate the two strands compared to the human-chimpanzee
hybrid, which has more hydrogen bonding and will therefore require more energy or a
higher temperature to separate the two strands. In this way, we can use
hybridization to show how similar two sequences of DNA are to each other, which can
be used to establish evolutionary relationships between species.
Besides using hybridization to
study evolution, it has other applications as well, one of which is called a
microarray chip. So, suppose you work with blood
donation and thousands of people have donated their blood. Your job is to screen these samples
for infectious diseases like HIV or hepatitis. Handling this task one by one would
take a very long time. A microarray chip is a little chip
that could fit in the palm of your hand and contains thousands of tiny wells that
are like little test tubes for hybridization experiments.
Let’s take a closer look at one
now. Attached to the bottom of each well
are oligonucleotides with a specific sequence. Since we’re talking about
infectious diseases, these can be specific to hepatitis, HIV, or any kind of
pathogen that causes that disease. So, our blood sample is processed
to extract all the DNA or RNA present in the donor blood, which potentially includes
the DNA or RNA of our pathogen that we want to detect. This is then labeled with a marker
shown here in green.
If the sample contained a pathogen
that we’re screening for, then its DNA or RNA will anneal to the complementary
oligonucleotide attached to the chip. This will fluoresce and cause the
corresponding well in the microarray to light up. We can see the results over here,
with each dot representing a different donor, with positive samples being indicated
by a green dot. These donors would not be suitable
for blood donation.
Now, let’s look at a practice
question and apply what we’ve learned in this video.
DNA from different sources can be
combined, or hybridized, in a series of steps. Firstly, the double-stranded DNA is
broken into single strands. After that, how are the single
strands of DNA from different organisms annealed to each other? (A) The enzyme DNA ligase is used
to catalyze the formation of peptide bonds. (B) The enzyme DNase is used to
repair the broken covalent bonds between bases. (C) The temperature is rapidly
increased to provide the energy required for hydrogen bonds between bases to
form. (D) The strands are physically
forced together until they bind. Or (E) the temperature is cooled so
hydrogen bonds between complementary bases can form.
This question is asking us about
the steps involved in DNA hybridization. Let’s clear these answer choices so
we can have more room to work with. DNA hybridization is the process of
combining two complementary single-stranded DNA or RNA molecules and has many useful
applications, for instance, in determining evolutionary relationships between
organisms.
Suppose a new species of ape was
discovered, we’ll call it big foot because of its impressively large feet, and we
want to see how closely related we are. We happen to have a bit of their
DNA, a portion of which is shown here, and we want to compare the two sequences. One way we can do this is with DNA
hybridization. You’ll notice that both strands are
indicated, one being the five prime the three prime strand and the other being the
three prime the five prime strand. You’ll also notice that these two
strands are being held together by hydrogen bonds shown here as these black
lines. These hydrogen bonds are holding
the two strands together with a certain amount of energy.
By increasing the temperature, we
can break these bonds. This then allows a double-stranded
DNA molecule to separate into two single-stranded DNA molecules. This process can be reversed by
lowering the temperature. This allows the hydrogen bonds to
reform between the two strands, and they can anneal or come back together to form
the double-stranded molecule. In this case, both of the original
strands came back together in the human and big foot DNA. This does happen, but sometimes the
big foot DNA will come together with the human DNA too. Because the two sequences aren’t
identical, not all of the bases will pair.
Now, if we were to isolate this
human-big foot hybrid and increased temperature, because of these mismatches, the
energy or temperature required to break these strands apart can be used to estimate
how similar the sequences are, which can be used to describe an evolutionary
relationship. During DNA hybridization, no
enzymes are used, only changes in temperature. And by lowering the temperature,
the two single strands of DNA can anneal to each other to form hydrogen bonds.
Now, let’s go over some of the key
points that we covered in this video. There’re many different molecular
technologies that are commonly used. Bioinformatics uses computers to
evaluate biological data like DNA sequences and can be used to help establish
evolutionary relationships. Gene machines synthesize genes, or
segments of DNA, from protein sequences. And not only does this make
obtaining sequences of DNA easy, but these sequences are free of noncoding
regions. DNA hybridization can be used to
anneal DNA or RNA from two different sources. And this can be used to help
establish evolutionary relationships between species or to perform specialized
techniques such as a DNA microarray.