Genetic Engineering
4.4.1
Brief Summary of the Unit
• Playing roles as Forensic Evidence Specialists,
students are asked to use numerous science
skills to solve a crime. First, they must
understand the importance, structure, and
function of DNA. Students will be able to
apply this knowledge and understanding to
identify individuals. Also students will be
able to relate the limitations of this
technique.
Students will understand
• Students will understand DNA structure and
function, Sterile technique, that each living thing
has unique DNA, and applications that exploit
this difference (DNA fingerprinting and its
limitations)
• Advances in technology can change the process
of natural human reproduction. The pros and
cons of these advances can be debated.
Essential Questions
• Why is DNA important?
• What are common misconceptions about
DNA?
• What are the strengths and weaknesses of
using DNA fingerprinting in court cases?
• Students will know
• Structure and function of DNA
• How DNA unifies all living things yet is
different in all living things.
Students will be able
• to view differences among using gel
electrophoresis
• To compare and analyze DNA profiles
4.4.1 Outline the use polymerase chain reaction (PCR) to copy
and amplify minute quantities of DNA.
Exploring DNA
• In the past few decades astounding genetic techniques have been
developed, which enable scientists to explore and manipulate
DNA. These include
• Copying DNA in a laboratory – the polymerase chain reaction
(PCR);
• Using DNA to reveal its owner’s identity –DNA profiling;
• Mapping DNA by finding every A, T, C and G is – the Human
Genome Project.
• Cutting and pasting genes to make new organisms – gene transfer.
• Cloning cells and animals.
• These techniques offer new hope for obtaining treatments and
vaccines for diseases; for creating new plants for farmers; for
freeing wrongly convicted people from prisons.
Heated Debate
• Techniques such as gene transfer and cloning
have sparked heated debate.
• Is it morally and ethically acceptable to
manipulate nature in this way?
• Are the big biotech companies investing huge
sums of money into this research to help their
fellow citizen or are they just in it for economic
profit?
• With regard to cloning and stem cell research, is
it morally and ethically acceptable to create
human embryos solely for scientific research?
• Part of being a responsible citizen is making
informed decisions relating to these difficult
questions. It is not technical complexity that
makes these questions difficult, it is also
because humans have never had to face
them before.
Polymerase Chain Reaction (PCR)
• PCR is a laboratory technique which takes a very
small quantity of DNA and copies all the nucleic
acids in it to make millions of copies of the DNA.
• This is very useful when very small quantities of
DNA are found in a sample and larger amounts
are needed for analysis. DNA from very small
samples of semen, blood or other tissues or
even from long-dead specimens can be
amplified using PCR.
• http://www.youtube.com/watch?v=_YgXcJ4nkQ
• PCR is used to solve a very simple problem: how
to get enough DNA to be able to analyse it.
• Analysis is impossible without the DNA from just
one or a few cells. When collecting DNA from
the scene of a crime or from a cheek smear,
often only a limited number of cells are
available. By using PCR, forensics experts or
research technicians can obtain millions of
copies of the DNA in just a few hours. Such large
quantities are large enough to get results from,
notably using gel electrophoresis.
PCR
PCR was developed in 1983 by Kary Mulis who received the Nobel
prize for chemistry in 1993 for his work.
PCR is carried out in a thermal cycler
http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
Good Animation
Steps involved in PCR
• PCR is carried out at high temperatures using a DNA polymerase
enzyme from Themus aquaticus, a bacterium that lives in hot
springs.
• This enzyme is able to survive the heating stages of each cycle.
• DNA amplification by PCR
• In this technique, any specific target segment within a DNA
molecule can be quickly amplified (copied many times) in a test
tube. Starting with a single DNA molecule, automated PCR can
generate 100 billion similar molecules in a few hours.
• In principle PCR is simple. A DNA sample is mixed with the DNA
replication enzyme DNA polymerase, nucleotide monomers, and a
few other ingredients.
• The solution is then exposed to cycles of heating (to separate the
DNA strands) and cooling.
• During each cycle, the DNA is replicated, doubling the amount of
DNA.
• The key to automating PCR was the discovery of an unusual heatstable DNA polymerase, first isolated from prokaryotes living in hot
springs. This enzyme can withstand the heat at the start of each
cycle.
• Devised in 1985, PCR has had a major impact on biological research
and biotechnology.
• PCR has been used to amplify DNA from a wide variety of sources:
fragments of DNA from a 40,000-year-old frozen woolly mammoth;
DNA from fingerprints or from tiny amounts of blood, tissue, or
semen found at crime scenes, DNA from single embryonic cells for
rapid prenatal diagnosis of genetic disorders, and DNA of viral genes
from cells infected with such difficult to detect viruses as HIV.
4.4.2 state that, in gel electrophoresis, fragments of
DNA move in an electric field and are separated
according to their size
• Electrophoresis is a technique used to
separate large molecule (nucleic acids or
proteins) based on their different rates of
movement in an electric field caused by a
combination of their charge and their size.
Electrophoresis
Gel electrophoresis can
separate nucleic acids that
differ in size. DNA fragments
created by restriction enzymes
are separated based on their
rate of movement through a
gel in an electric field.
How far a DNA molecule
travels is inversely
proportional to its length.
After the current is turned off,
a dye is added; this reveals the
separated bands by fluorescing
in ultraviolet light.
http://media.pearsoncmg.com/bc/bc_campbell_biol
ogy_7/media/activities/load.html?20&D
• DNA may be analyzed by gel electrophoresis. In this
process DNA molecules migrate to the positive pole as
current passes through the gel.
• Making the gel is the first step in gel electrophoresis. A
common material used for gel electrophoresis of DNA
is agarose. Agarose gels are made by first boiling a
mixture of powdered agarose and buffer. When the
mixture is cooled to about 65oC, the solution is poured
into a gel mold. When further cooled to room
temperature, the agarose solidifies to produce the gel
with indentations called wells.
Steps
• The agarose gel is placed in an electrophoresis
apparatus and buffer is added to cover the gel.
• DNA is loaded into the wells using a micropipettor.
Each DNA sample has tracking dyes added to it (usually
blue) and either sucrose or glycerol to make the
solution dense and "sink" into the well.
• After the wells are filled, the power supply is turned on
and the DNA moves towards the positive pole. At this
stage the DNA band(s) cannot be seen, but the tracking
dye (dark blue) allows the progess of the
electrophoresis to be followed. The smallest DNA
fragments typically follow the blue dye down the gel.
Steps
• The DNA is visualized by staining, for example
with ethidium bromide, and then destaining in
water. The ethidium bromide binds to the
DNA and makes it fluoresce orange under UV
light.
• A photograph of the results is taken and used
for analysis and for a permanent record of the
experiment.
Electrophoresis
Useful tips
• Larger molecules will move more slowly
through the gel than smaller molecules.
• If a spot of the unknown sample has travelled
exactly the same distance as one of the known
molecules, they are likely to be the same.
• PCR can be used to increase the size of the
sample and then gel electrophoresis can be
used to compare the sections of DNA with the
DNA of various suspects in an investigation
Review and Fun
• Watch this animation
• http://learn.genetics.utah.edu/content/labs/g
el/
• Learn Genetic: Super Flashy Animation
4.4.3 state that gel electrophoresis of DNA is
used in DNA profiling
• The process of matching an unknown sample of DNA
with a know sample to see if they correspond is called
DNA profiling. This is also referred to as DNA
fingerprinting because of some of the similarities with
fingerprints but the techniques are very different.
• If, after separation by gel electrophoresis, the pattern
of bands formed by two samples of DNA fragments are
identical, it means that both most certainly came from
the same individual.
• If the patterns are similar, it means that the two
individuals are probably related.
DNA Profiling
• DNA profiling is also known as DNA fingerprinting. It is a technique that
compares DNA from different sources without mapping the entire
genome since that would take a long time.
• DNA profiling is used to compare the DNA of a suspect with, for
example, blood found at a crime scene or to compare the DNA of a child
with that of an adult who could be the parent.
• When a small amount of DNA is found, for example, in a spot of blood on
a crime scene, the PCR can be used to increase the amount of DNA. The
two strands of DNA double helix are separated. Restriction enzymes or
endonucleases, which will cut between specific sequences of organic
bases are used to cut the DNA into sections. The sections are separated
using gel electrophoresis.
• The gel electrophoresis will separate the sections of DNA according to
size and charge. This creates a pattern of stripes and bands, determined
by the sequence of the organic bases. As every person’s DNA is unique, it
would be highly unlikely that two different people would produce the
exact same pattern of DNA sections on the gel.
Interpreting DNA Profile
Paternity testing is a form of DNA profiling. The DNA of the man who may
be the father of a child is compared to the child's DNA and to that of the
mother.
An example of the DNA bands from a paternity test is shown below. The
standard band provides a control or comparison with which to compare the
samples.
Is the man the father of the child?
a) True b) False
What is DNA Profiling?
• Even though each person’s DNA is unique, with
current technology it is not practical to look at each
difference. Currently in the laboratory we look at 10
different areas of DNA, which are known to vary
widely between people. These 10 areas contain short
repeating sequences known as Short Tandem Repeats
(STR). The number of these repeating sequences
varies between individuals. An additional area is also
provided which indicates whether the person is male
or female. The technique of DNA profiling is centred
on analysing and measuring these differences in
length.
4.4.4 Describe the application of DNA profiling to
determine paternity and also in forensic
investigations.
• DNA Use in Forensic Cases
• Biological fluids such as blood, semen, hair and saliva can play a
role in shedding light on various types of criminal cases. In the
case of sexual assault cases, semen found on vaginal swabs and
/or clothing of a victim can be compared to the DNA profile of a
nominated suspect. Similarly in murder/assault cases blood found
on the clothing of a nominated suspect can be compared to the
DNA profile of the victim.
• Contrary to a number of years ago the smallest of traces left
behind in criminal offences can now suffice for a DNA
analysis. With the currently applied DNA techniques, a profile can
be constructed, even of old and (partially) decayed biological
traces.
• Forensic DNA analysis can also establish the origin of a biological
sample with an extremely high degree of probability.
Forensic Investigations
• Compare DNA from the suspect with DNA from the
crime scene sample
• This technique breaks down DNA into sections which
are separated by gel electrophoresis. If the DNA bands
of a suspect are a match with those found in a sample
(e.g. blood, semen, hairs) at the crime scene, then the
suspect probably was at the scene although it is not
always this sample.
• http://en.wikipedia.org/wili/Genetic_fingerprinting
• Problems that arise when using DNA profiling as
evidence
Paternity Profiling
• DNA profiling can be used in paternity suits
when the identity of someone’s biological father
must be known for legal reasons.
DNA profiling to determine paternity uses the
concept that all of the child’s DNA comes from its
parents. Each band shown on the DNA profile of a
a child must correspond with a band in the profile
of the father or mother.
• DNA technology is used in courts of law
• http://in.youtube.com/watch?v=veYl0vigfLk
Use a relative’s DNA to determine the identity of
a victim
• This technique has also been used to determine the
identity of the remains of dead people. People claimed
that the Tsar of Russia and his family were shot dead
during the Russian Revolution and bodies were shown
to prove this. However, not everyone believed that
they were really the remains of the Romanovs. The
identity of these bodies could not be proven until DNA
fingerprinting was brought in. by taking blood samples
of relatives of the Romanovs, DNA patterns could be
established. Samples from the bodies showed similar
DNA patterns and the conclusion was that the bodies
were likely to be the Romanov family.
TOK DNA Profiles
• Blood typing was used in paternity and forensic testing
before the advent of DNA profiling. The outcome of tests
using blood typing is so broad that it is only useful in
excluding an individual (that is, it can say that the
individual dos not have the same blood type as the one
searched for. To say that they do have the same blood type
would not be useful unless the pool of possible suspects
was very small and all with different blood types.
• DNA profiling, on the other hand, compares parts of non
coding DNA that are unique to each individual (except
identical twins). Profiles constructed using appropriate
procedures have statistically extremely high reliability rate
and as such, are widely used in paternity and forensic cases
with great success
4.4.5 Analyse DNA Profiles to draw conclusions
about paternity or forensic investigations
• Forensic Investigation
• A crime has been committed and two suspects are under
investigation. DNA profiling has been carried out and the
results are shown in the figure (Page No. 66 IB Biology By
Minca Peters) it can be seen that the two bands visible in the
“sperm DNA” match the DNA bands of suspect 1 but not
Suspect 2
Paternity Investigation
• When the paternity of a child is investigated, the band found in the
child’s DNA are compared to those of the mother and the alleged father.
Since all of the child’s DNA comes from its parents, all bands should be
found in either parent. Of course, a parent only gives half of its DNA to
the child, so not all the parent’s bands can be found in the child
• Refer to paternity investigation in the text book IB Biology by Mica and
also IB Biology By Heinemann Page number 102
Activity: Page Number 130 IB
Biology By C.J Clegg
Reference/Resource
• http://i-biology.net/ibdpbio/04-genetics-andgenetic-engineering/genetic-engineering-andbiotechnology/
Stephen Taylor
click 4 Biology
IB Biology By Heinemann
IB Biology By C.J. Clegg, IB Biology By Minca Peters
Biology By CampBell Reece