Download 8.2 Structure of DNA 4.4.3 State that gel

Genetic
Engineering
8.2 Structure
of DNA Review: Topic 4.4
4.4.1
Outline the
of polymerase chain reaction (PCR) to
8.2 Structure
ofuse
DNA
copy and amplify minute quantities of DNA
• PCR is a way of producing
large quantities of a specific
target sequence of DNA
• It is useful when only a small
amount of DNA is available
for testing
• E.g. crime scene samples of
blood, semen, tissue, hair, etc.
4.4.1
Outline the
of polymerase chain reaction (PCR) to
8.2 Structure
ofuse
DNA
copy and amplify minute quantities of DNA
PCR occurs in a thermal cycler and involves a repeat
procedure of 3 steps:
1. Denaturation: DNA sample is heated to separate it into
two strands
2. Annealing: DNA primers attach to opposite ends of the
target sequence
3. Elongation: A heat-tolerant DNA polymerase (Taq) copies
the strands
DNA strands
primer 1
polymerase
nucleotides
primer 2
4.4.1
Outline the
of polymerase chain reaction (PCR) to
8.2 Structure
ofuse
DNA
copy and amplify minute quantities of DNA
• One cycle of PCR yields two identical copies of the DNA
sequence
• A standard reaction of 30 cycles would yield 1,073,741,826
copies of DNA (230)
4.4.2
State that, in
electrophoresis, fragments of DNA can
8.2 Structure
ofgel
DNA
move in an electric field and are separated according to their size
• Gel electrophoresis is a technique which is used to
separate fragments of DNA according to size
4.4.2
State that, in
electrophoresis, fragments of DNA can
8.2 Structure
ofgel
DNA
move in an electric field and are separated according to their size
• Samples of fragmented DNA are placed in the wells of an
agarose gel
• The gel is placed in a buffering solution and an electrical
current is passed across the gel
• DNA, being negatively charged (due to phosphate),
moves to the positive terminus (anode)
• Smaller fragments are less impeded
by the gel matrix and move faster
through the gel
4.4.2
State that, in
electrophoresis, fragments of DNA can
8.2 Structure
ofgel
DNA
move in an electric field and are separated according to their size
• The fragments are thus separated according to size
• Size can be calculated (in kilobases) by comparing
against a known industry standard
4.4.3
State that of
gelDNA
electrophoresis of DNA is used in DNA
8.2 Structure
profiling
• DNA profiling is a technique by which individuals are identified
on the basis of their respective DNA profiles
• Within the non-coding region of an individual's genome, there
exists satellite DNA - long stretches of DNA made up of
repeating elements called short tandem repeats (STRs)
• These repeating sequences can be excised to form fragments,
by cutting with a variety of restriction endonucleases (which
cut DNA at specific sites)
4.4.3
State that of
gelDNA
electrophoresis of DNA is used in DNA
8.2 Structure
profiling
•As individuals all have a different number of repeats in a given
sequence of satellite DNA, they will all generate unique
fragment profiles
• These different profiles can be compared using gel
electrophoresis
http://www.pbs.org/wgbh/nova/education/body/c
reate-dna-fingerprint.html
4.4.4
Describe the
application of DNA profiling to determine
8.2 Structure
of DNA
paternity and also in forensic investigation
Two applications of DNA profiling are:
1) Paternity testing (comparing DNA of offspring against
potential fathers)
2) Forensic investigations (identifying suspects or victims
based on crime scene DNA)
on crime-scene DNA)
4.4.5
Analyze DNA
profiles to draw conclusions about
8.2 Structure
of DNA
paternity or forensic investigations
•Paternity Testing: Children inherit half of their alleles from
each parent and thus should possess a combination of
their parents alleles
•Forensic Investigation: Suspect DNA should be a complete
match with the sample taken from a crime scene if a
conviction is to occur
4.4.5
Analyze DNA
profiles to draw conclusions about
8.2 Structure
of DNA
paternity or forensic investigations
4.4.5
Analyze DNA
profiles to draw conclusions about
8.2 Structure
of DNA
paternity or forensic investigations
4.4.5
Analyze DNA
profiles to draw conclusions about
8.2 Structure
of DNA
paternity or forensic investigations
4.4.5
Analyze DNA
profiles to draw conclusions about
8.2 Structure
of DNA
paternity or forensic investigations
4.4.6
Outline three
outcomes of the sequencing of the
8.2 Structure
of DNA
complete human genome
The Human Genome Project (HGP) was an international
cooperative venture established to sequence the 3 billion
base pair (~25,000 genes) in the human genome.
4.4.6
Outline three
outcomes of the sequencing of the
8.2 Structure
of DNA
complete human genome
The outcomes of this project include:
• Mapping: We now know the number, location and basic
sequence of human genes
• Screening: This has allowed for the production of
specific gene probes to detect sufferers and carriers of
genetic disease conditions
• Medicine: With the discovery of new proteins and their
functions, we can develop improved treatments
(pharmacogenetics and rational drug design)
• Ancestry: It will give us improved insight into the origins,
evolution and historical migratory patterns of humans
4.4.6
Outline three
outcomes of the sequencing of the
8.2 Structure
of DNA
complete human genome
With the completion of the Human Genome Project in
2003, researcher have begun to sequence the genomes of
several non-human organisms.
4.4.7 State that, when genes are transferred between species, the
8.2
Structure
of DNA
amino
acid sequence
of polypeptides translated from them is
unchanged because the genetic code is universal
• The genetic code is universal, meaning that for every
living organism the same codons code for the same
amino acids (there are a few rare exceptions)
• This means that the genetic information from one
organism could be translated by another (i.e. it is
theoretically transferable)
4.4.8 Outline a basic technique used for gene transfer
8.2
Structure
of DNA
involving
plasmids,
a host cell (bacterium, yeast or other
cell), restriction enzymes (endonucleases) and DNA ligase
1. DNA Extraction
• A plasmid is removed from a bacterial cell (plasmids are
small, circular DNA molecules that can exist and replicate
autonomously)
• A gene of interest is removed from an organism's genome
using a restriction endonuclease which cut at specific
sequences of DNA
• The gene of interest and plasmid are both amplified using
PCR technology
4.4.8 Outline a basic technique used for gene transfer
8.2
Structure
of DNA
involving
plasmids,
a host cell (bacterium, yeast or other
cell), restriction enzymes (endonucleases) and DNA ligase
2. Digestion and Ligation
• Cutting with certain restriction enzymes may generate
“sticky ends” that allow the two DNA constructs to fit
together
• The gene of interest and plasmid are spliced together by
DNA ligase creating a recombinant plasmid
4.4.8 Outline a basic technique used for gene transfer
8.2
Structure
of DNA
involving
plasmids,
a host cell (bacterium, yeast or other
cell), restriction enzymes (endonucleases) and DNA ligase
3. Transfection and Expression
• The recombinant plasmid is inserted into the desired host
cells (this is called transfection for eukaryotic cells and
transformation for prokaryotic cells)
• The transgenic cells will hopefully produce the desired
trait encoded by the gene of interest (expression)
• The product may need to subsequently be isolated from
the host and purified in order to generate sufficient yield
4.4.8 Outline a basic technique used for gene transfer
8.2
Structure
of DNA
involving
plasmids,
a host cell (bacterium, yeast or other
cell), restriction enzymes (endonucleases) and DNA ligase
Treating Hemophilia via the Isolation of
Human Factor IX Clotting Protein
from Transgenic Sheep Milk
4.4.8 Outline a basic technique used for gene transfer
8.2
Structure
of DNA
involving
plasmids,
a host cell (bacterium, yeast or other
cell), restriction enzymes (endonucleases) and DNA ligase
http://webecoist.momtastic.com/2010/02/23/animal-hybrids-the-half-lives-of-10-curious-creatures
/ (animal hybrids)
4.4.9
State two of
examples
8.2 Structure
DNA of current uses of genetically
modified crops or animals
Crops
1. Engineering crops to extend shelf life of fresh produce:
Tomatoes have been engineered to have an
extended keeping quality by switching off the gene for
ripening and thus delaying the natural process of softening
of fruit
2. Engineering of crops to provide protection from insects:
Maize crops (Bt corn) have been engineered to be toxic to
the corn borer by introducing a toxin gene from a bacterium
(Bacillus thuringiensis)
4.4.9
State two of
examples
8.2 Structure
DNA of current uses of genetically
modified crops or animals
Example: Maize introduced with a bacterial gene encoding a
toxin to the European Corn Borer (i.e. Bt Corn)
4.4.9
State two of
examples
8.2 Structure
DNA of current uses of genetically
modified crops or animals
4.4.9
State two of
examples
8.2 Structure
DNA of current uses of genetically
modified crops or animals
Animals
1. Engineering animals to enhance production:
Sheep produce more wool when engineered with the gene
for the enzyme responsible for the production of cysteine the main amino acid in the keratin protein of wool
2. Engineering animals to produce desired products:
Sheep engineered to produce human alpha-1-antitrypsin in
their milk can be used to help treat individuals suffering
from hereditary emphysema
4.4.10
Discuss the
potential benefits and potential harmful
8.2 Structure
of DNA
effects of one example of genetic modification
Potential Benefits
• Allows for the introduction of a characteristic that wasn't
present within the gene pool (selective breeding could not
have produced desired phenotype)
• Results in increased food production (requires less land for
comparable yield)
• Less use of chemical pesticides, reducing the economic
cost of farming
• Can now grow in regions that, previously, may not have
been viable (reduces need for deforestation)
4.4.10
Discuss the
potential benefits and potential harmful
8.2 Structure
of DNA
effects of one example of genetic modification
Potential Harmful Effects
• Could have currently unknown harmful effects (e.g. toxin
may cause allergic reactions in a percentage of the
population)
• Accidental release of transgenic organism into the
environment may result in competition with native plant
species
• Possibility of cross pollination (if gene crosses the species
barrier and is introduced to weeds, may have a hard time
controlling weed growth)
• Reduces genetic variation / biodiversity (corn borer may
play a crucial role in local ecosystem)
8.2
Structure
of DNA
4.4.11
Define clone
A clone is a group of genetically identical organisms or a
group of cells derived from a single parent cell
4.4.12
Outline aof
technique
8.2 Structure
DNA for cloning using differentiated
animal cells
Somatic Cell Nuclear Transfer (SCNT) is a method of
reproductive cloning using differentiated animal cells.
• A female animal (e.g. sheep) is treated with hormones
(such as FSH) to stimulate the development of eggs
• The nucleus from an egg cell is removed (enucleated),
thereby removing the genetic information from the cell
• The egg cell is fused with the nucleus from a somatic
(body) cell of another sheep, making the egg cell diploid
4.4.12
Outline aof
technique
8.2 Structure
DNA for cloning using differentiated
animal cells
• An electric shock is delivered to stimulate the egg to
divide, and once this process has begun the egg is
implanted into the uterus of a surrogate
• The developing embryo will have the same genetic
material as the sheep that contributed the diploid
nucleus, and thus be a clone
4.4.12
Outline aof
technique
8.2 Structure
DNA for cloning using differentiated
animal cells
4.4.13
Discuss the
8.2 Structure
ofethical
DNA issues of therapeutic cloning in humans
Different Uses of Cloning
4.4.13
Discuss the
8.2 Structure
ofethical
DNA issues of therapeutic cloning in humans
Arguments for Therapeutic Cloning
• May be used to cure serious diseases or disabilities with
cell therapy (replacing bad cells with good ones)
• Stem cell research may pave the way for future discoveries
and beneficial technologies that would not have occurred if
their use had been banned
• Stem cells can be taken from embryos that have stopped
developing and would have died anyway (e.g. abortions)
• Cells are taken at a stage when the embryo has no nervous
system and can arguably feel no pain
4.4.13
Discuss the
8.2 Structure
ofethical
DNA issues of therapeutic cloning in humans
Arguments Against Therapeutic Cloning
• Involves the creation and destruction of human embryos (at
what point do we afford the right to life?)
• Embryonic stem cells are capable of continued division and
may develop into cancerous cells and cause tumors
• More embryos are generally produced than are needed, so
excess embryos are killed
• With additional cost and effort, alternative technologies may
fulfill similar roles (e.g. nuclear reprogramming of
differentiated cell lines)