Text of Biotechnology Genetic Research and Biotechnology
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Biotechnology Genetic Research and Biotechnology
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Restriction Enzymes These enzymes are isolated from bacteria
and cut a DNA strand at a specific base pair sequence using a
hydrolysis reaction. Depending on which restriction enzyme is used,
the resulting ends may be one of two types: 1. Sticky ends ends of
DNA fragments with single- stranded overhangs. 2. Blunt ends ends
of DNA fragments that are perfectly paired.
Plasmids Sometimes we want to excise a gene from a source DNA
and express it in a different organism.
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This is accomplished through bacterial machinery know as
plasmids. Plasmids are small, circular double-stranded DNA
molecules. They exist in the bacterial cytoplasm but are not a part
of the chromosome. They range in size from 1000-200 000 bp.
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Plasmid
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Although plasmids are not part of the bacterial chromosome,
they do offer benefits to the bacteria. They often carry genes for
antibiotic resistance as well as resistance to toxic heavy metals
and some herbicides.
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How are they used in biotech? Restriction endonucleases are
used to cut the plasmid in one area only so that it becomes linear.
The foreign gene is cut with the same endonuclease and so, contains
complementary ends. The foreign DNA is placed in solution with the
linear plasmid. The foreign DNA fragment will anneal to the plasmid
and permanently become a part of it with the help of DNA ligase
forming the phosphodiester bonds.
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Now the plasmid is considered recombinant DNA and can be
inserted into a bacterial cell. The cell will express the genes
contained on the plasmid, including the foreign gene, making many
copies. In this way, the gene will be cloned.
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Transformation The process of introducing recombinant DNA into
host bacterial cells is called transformation. The plasmid is
called a vector because it can carry genes from one source to
another. CaCl 2 at 0 degrees C is used to stabilize the bacterial
cell membrane. The solution of bacteria and plasmid is then
subjected to a quick increase of heat which creates a draft that
physically sweeps the plasmid into the cell.
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The cells are then returned to a comfortable temperature of 37
degrees C where they can then grow and reproduce. Not all cells
will take up the plasmid so there must be a way to determine which
ones contain the desired gene. Selective plating is used for this
purpose. The plasmid also contains a gene for antibiotic
resistance.
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If a bacteria takes up the plasmid, it will also be resistance
to an antibiotic. If the nutrient agar on which the bacteria is
being grown contains an antibiotic, then only those cells with
resistance will grow. Others will die. In this way, we can select
for the transformed bacteria. To further ensure that the
transformed bacteria has the desired gene, it can be subjected to
the original restriction enzyme, isolated and run on a gel to
confirm the expected pattern of bands. Read page 293-294 to learn
about how this process is used in medical biotech.
Polymerase Chain Reaction (PCR) PCR is a more efficient and
direct way of making copies of a DNA sequence without using
plasmids. The process of PCR is similar to DNA replication. 1.
Strands of DNA are separated using heat (94- 96 degrees C) 2. DNA
primers that are complementary to the target sequence are added,
one on each strand, but on opposite ends because the strands are
anti-parallel.
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3. Taq polymerase is a DNA polymerase isolated from a bacteria
that lives in hot springs. This is used rather than DNA polymerase
III to build the strands because the process takes place at 72
degrees C. Once the strands have been built, the cycle repeats
itself, doubling the number of double- stranded copies of the
target DNA.
DNA Fingerprinting or Profiling The DNA of every individual is
unique. This fact can be used to accurately identify an individual
based only on a sample of DNA which can be obtained from skin,
hair, bodily fluids, etc. Biotechnology is used in this
process.
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The basic process DNA is a double helix composed of millions of
nucleotide base pairs. The specific sequence of these base pairs is
unique from one individual to the next. In order to identify the
specific sequences, the DNA must be cut into smaller pieces.
Restriction endonucleases (restriction enzymes) cut the DNA at a
particular sequence.
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This creates DNA sections of different lengths. Every
individual will have a different combination of segment lengths
Since DNA is so small, a process is needed to visually see the
different DNA segments.
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Gel Electrophoresis is used to visualize the unique pattern of
a persons DNA. The DNA sample is placed on a gel plate. The gel
allows the DNA to travel through from one end to the other. The
different DNA segments separate on the gel according to size;
shorter segments travel farther.
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A DNA marker with known sizes of segments is run alongside the
sample so the samples segment sizes can be estimated. No two people
will have exactly the same pattern of DNA fragments on the gel
except for identical twins.
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Gel Electrophoresis This process separates DNA fragments based
on size. The differences in size exist because each fragment (after
being cut by a restriction enzyme) has a different number of base
pairs. The DNA travels through the gel according to size. The gel
is porous and acts like a sieve with smaller segments being able to
navigate around the pores easier than larger segments. For this
reason, smaller segments will travel farther than larger
segments.
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The DNA travels through the gel because it is negatively
charged because of its phosphate group which gives it a net -1
charge. An electrical current is run through the gel with the
negative end closest to the samples and the positive end farthest
away. The negative DNA is repelled by the closer negative end and
travels toward the positive end.
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A DNA marker is loaded along with the sample and can be
visualized as it migrates toward the other end. Once
electrophoresis is complete, the gel is stained (used to be
ethidium bromide but not so much anymore) so the sample can be
seen. The pattern is then compared to the marker and the fragment
sizes estimated. Each persons pattern will be unique.
DNA Sequencing The Human Genome Project (HGP), initiated in
1990, had as one of its goals to determine the DNA sequence of all
3 billion base pairs of the human genome. It has since been
completed but the process was very slow at first and it cost
billions of dollars and numerous researchers.
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While the HGP used computer technology to read the sequence,
this technology was made possible because of the lab techniques
developed during the 70s and 80s. Since the completion of this
project, sequencing has improved even more and is now more
efficient and less costly.
Next-generation Automated sequencing Current methods are
greatly improving the speed of sequencing. This is beneficial
because medical science is working towards the use a persons DNA
sequence to diagnose and treat various diseases such as cancer. For
example, a cancerous tumour can be sequenced to determine the exact
nature of the mutation and determine a course of treatment.
