Chapter 12 Recombinant DNA Technology

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Chapter 12Recombinant DNA Technology
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This chapter is an introduction to biotechnology.
I'm going to skip around the chapter and some
sections won't be covered at all. The best way
to determine which sections to read is to look
for these figures in the text and read that
section. For example the first figure is from
section 12.5 so read that section.
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12.5- Bacteria produce enzymes that cut DNA,
that is not their own DNA, into small fragments.
This protects them to a degree from viruses
called bacteriophages that infect them. In the
sixties several scientists were studying these
enzymes and realized that they could be used like
scissors to cut large chromosomes into small,
easily handled fragments that could then be
inserted into molecules called plasmids, that
bacteria would replicate. Once the bacteria made
many copies of the cloned fragment it could be
used for any number of things including
expressing the genes that were on the fragment.
Figure 12.5 shows how these enzymes are
used.Fortunately, most of them leave extensions
when they cut DNA which makes the ends "sticky".
When these ends find each other they stick long
enough that another enzyme called DNA ligase can
reform the phosphodiester bond and glue the
pieces together.
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12.6- Figure 12.6 shows how that can be done
with a fragment of human DNA, but I do not have
an electronic version of the figure. The figure
below is a similar figure but missing a large
portion of the important stuff. Basically, if
you cut a fragment from the human DNA with a gene
of interest on it, you can insert it into a
bacterial plasmid and clone the gene. This type
of cloning in not related to the cloning of whole
organisms.
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12.4- Figure 12.4 shows how cloning can be used
to make Bacteria that can do what we want them to
do by giving them the genes that allows them to
make certain proteins. In the examples we are
using the bacteria to produce proteins that cause
ice crystals (snow) to form at temperatures above
freezing and to make pharmaceutically important
proteins. On the left the bacteria are using
genes to make proteins that allow the cell to
break down crude oil to form organic molecules.
Also shown is a bacterium that can transfer DNA
to a plant and in that way genetically engineer
the plant. (We will discuss the plant
engineering at the end of the lecture.
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12.8- There is a problem with cloning genes from
Eucaryotic organisms into Prokaryotes for
expression. As we discussed in chapter 11
messenger RNA (mRNA) is translate from the AUG
start codon to a distant stop codon, one codon
after another. In prokaryotes that is no problem
but eukaryotic genes have big regions within the
gene that are not part of the coding sequence.
These are called introns and introns must be
spliced out of the pre-mRNA before it can be
translated to make protein. The problem in
cloning these genes is that bacteria, being
prokaryotes, do not have the mechanisms
responsible for splicing the transcripts into
mRNA. So if you cut chromosomal DNA from a human
or other eukaryotic chromosome it will not be
expressed in a bacterium.
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The problem can be remedied by getting the mature
mRNA molecule that has been spliced and making a
DNA copy of it. This can be done with an enzyme
called reverse transcriptase. It uses a RNA
template to make DNA molecule. That can then be
cloned and expression in a bacterium to make the
protein that the gene encodes.
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12.11- There are many other uses for
biotechnology that do not necessarily include
cloning and expressing genes. One of the most
important advances has been it the identification
of markers that can be used to detect alleles
that result in disease. The next four figures
shows the theory and method.
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Figure12.11a shows two homologous sequences that
are different in that one has an extra cut-site
for a particular restriction enzyme. If one of
these sections for example has a gene that
results in cystic fibrosis or some other disease
you can now tell which have the bad allele and
which chromosomes have the good allele by looking
for the extra cutsite. Lets say that here that
the bad cystic fibrosis gene is located in the
region shown below and that there is an extra
cutsite, very close to the allele on the
chromosome. If we can come up with a way to
detect the cut-site we can tell who is carrying
the gene.
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We detect the cutsites by looking at the sizes of
the fragments that are generated by cutting the
DNA. This is done by a process called
electrophoresis using an agarose or
polyacrylamide gel. Basically you cut the DNA
and load it on one end of the gel. You then pass
a current through the gel and the DNA will move
toward the positive electrode since the
phosphates in the backbone make the DNA a
negatively charged molecule. The fragments
separate according to their sizes since small
fragments can move through the gel matrix faster
than larger ones. The gel would look like this
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The actual experiments look more like the figure
below. When you cut a human chromosome you have
lots of fragments and they all go into the gel.
You then treat the DNA to separate the strands
and then add a single-stranded fragment that is
radioactive and will base pair with the sequences
that you are looking for. When the gel is then
exposed to x-ray film the bands that pair with
the "probe" will be the only ones you see.
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12.16- Genetic engineering can be done in plants
using Agribacterium and its plasmid called the Ti
plasmid. The Ti plasmid is transferred from the
bacterium to an infected plant and intergrates
into it's DNA. If you clone a gene into these
plasmids it will be taken into the chromosome of
infected plant cells where they can be expressed
to form proteins. This process has been used to
make soy beans that are Roundup resistant. So a
soybean field could be treated with Roundup to
kill the weeds, but the soybeans are fine.
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