Learning aim D: Explore basic DNA techniques and the use of genetic
engineering technologies. last assignment of unit 11, I was awarded a distinction for this assignment.
DNA extraction: A pestle is used to lyse (break open) cells, or they can be blended. The cell
membranes are then lysed with a buffered detergent. DNA and organelles are dissolved. Salt
precipitation and centrifugation may be used to remove large proteins and debris. Since DNA is
insoluble in ethanol and would precipitate out, ice cold ethanol is applied. Real life example
of how it is used: DNA extraction is the first step in the production of many pharmaceuticals.
The hepatitis B vaccine and human growth hormone are two examples of recombinant
genetics-based pharmaceuticals (hGh). Insulin is one of the most used hormones developed
using DNA extraction, in addition to many other hormones. Why is it used: DNA can be used
for molecular analysis such as PCR, electrophoresis, sequencing, fingerprinting, and cloning
once it has been extracted. It's also required for forensic science, genome sequencing,
detecting bacteria and viruses in the environment, and paternity determination.
DNA can be used for molecular analysis such as PCR, electrophoresis, sequencing, cloning and
fingerprinting once it has been removed.
Benefits to medicine or industry& effectiveness and cost … The ability to extract DNA is critical
for researching the genetic causes of disease and developing diagnostics and medications. By
experimenting with the DNA that has been removed, scientists may be able to find cures for
these diseases.
Gel electrophoresis: When electrophoresis is used to analyse DNA, restriction enzymes are
used to break the DNA into pieces, resulting in DNA fragments of various lengths. To make the
DNA solution denser, a dye such as bromophenol is added after the DNA has been sliced. In
the meantime, the electrophoresis tank has been set up with a liquid agarose gel, which is a
polysaccharide polymer that allows DNA molecules to pass through it while its in the tank .
There are combs in place, which will be extracted after the gel has formed, leaving wells in one
end of the gel. A buffer solution is poured into the tanks, fully covering the gel. A well is filled
with the DNA samples that will be analysed or compared. This must be done with caution. The
dyed solution is permitted to fall into the well while the micropipette is placed above it. A
cathode and anode, both electrodes, are positioned at either end of the tank, the anode at the
far end away from the wells and positively charged, and the cathode at the far end near the
wells and negatively charged. The electrodes are attached to a power supply and left for two
hours. Since DNA has many phosphate groups in its sugar phosphate backbone, it has an
overall negative charge, and DNA fragments can migrate through the gel toward the positively
charged anode. Shorter DNA fragments migrate faster than longer DNA fragments. The power
supply is turned off after two hours, and the buffer solution is poured away. A dye is applied,
which stains the DNA fragments, revealing the banding pattern. Real life example of how it
is used: In forensics, suspects can be removed if their DNA pattern does not fit the pattern of
DNA molecules found at the crime scene, and gel electrophoresis can be used for DNA
fingerprinting to examine crime scenes. Other people can become suspects if their DNA
matches the pattern of the person who committed the crime. Gel electrophoresis may also be
used to look for genes linked to a specific disease. The technique of DNA electrophoresis can
be used to classify mutations in particular diseases. A restriction fragment of different size may
be used to detect deletion mutants. Gel electrophoresis is a laboratory technique for separating
DNA, RNA, and protein mixtures based on their molecular size. Why is it used: Gel
1
BTEC Assignment Brief v1.1
BTEC Internal Assessment QDAM January 2015
, electrophoresis is a laboratory technique used for separating charged molecules such as DNA,
RNA, and proteins based on their size. Electrophoresis can be used to determine how many
different DNA fragments are present in a sample and how large they are in relation to one
another. Benefits to medicine or industry: Gel electrophoresis allows researchers to assess
the antibiotic's concentration, allowing for more precise dose. It is also used to check vaccine
purity and concentration. Researchers use electrophoresis to perform experiments to find the
best available form of a single vaccine by testing variations of vaccines with various amounts
and forms of antibodies. Protein and antibody detection is another important application for
electrophoresis. Immunoelectrophoresis is the type of electrophoresis used in this application,
and it helps researchers to study the interactions between proteins and antibodies.
Immunoelectrophoresis can be used to screen samples from medical patients for a variety of
immune disorders, including kidney failure and multiple sclerosis. Researchers will also look at
how different antibodies interact with irregular proteins in these samples to see if there are
any new therapies or even cures for autoimmune diseases. The gel is relatively inexpensive
and simple to use, which is beneficial to the healthcare industry. Separating nucleic acids with
agarose gel electrophoresis has proven to be an effective and efficient process. The high gel
strength of agarose allows for the separation of large DNA fragments on low percentage gels.
DNA Amplification:
Method of PCR
Denaturation: To use PCR to amplify a fragment of DNA, the sample is heated first, causing
the DNA to denature, or split into two pieces of single-stranded DNA. The reaction is heated to
94-98°C in this process. This stage denatures your DNA and primers, allowing them to anneal
to one another in the following step. The strands split, revealing the target DNA and
nucleotides on either side.
Annealing: The temperature is rapidly lowered to 50-64°C in the second stage. Low enough
temperature for the oligonucleotide primers to base pair with the complementary sequence on
the exposed DNA strands. The concentration of primers is greater than the separated single
DNA strands to prevent them from recombining into a double stranded DNA. It is also crucial
that the various primers do not have complementary sequences to avoid primer hybridization.
Extension: The annealed oligonucleotides serve as primers for DNA synthesis in the third
stage by providing a free 3' hydroxyl group for DNA polymerase. This enzyme, which comes
from the bacterium Thermus aquaticus, synthesises and creates two new strands of DNA while
using the original strands as templates and extending the primers. This process causes the
original DNA to be duplicated, with one old and one new strand of DNA in each of the new
molecules. The reaction is rapidly heated to 72-80°C in this process. This is when the
polymerase starts reading (in the 5-3 direction) and copying (in the 3-5 direction) the
template DNA. Since the temperature is higher during this process, non-specific
primer/template DNA interactions are reduced, raising the specificity of your reaction.
Real life example of how it is used: Applications of the technique include DNA cloning for
sequencing, gene cloning and manipulation, gene mutagenesis; construction of DNA-based
phylogenies, or functional analysis of genes; diagnosis and monitoring of hereditary diseases.
Why it is used: PCR is used to amplify a specific region of DNA. The amplified DNA can be
used for many purposes, such as identifying different genes and species of bacteria. These
nucleic acid amplification methods can create millions of identical copies of a DNA or RNA
"target" sequence in a matter of hours. These can then be used in the identification and
detection of infectious diseases . Benefits to medicine and industry: Genetic disorders are
caused by mutations that range from simple changes in the base sequence of the DNA double
helix through to changes in large DNA sequences and even whole chromosomes. PCR helps
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