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In doing so, the majority lose the ability to divide by mitosis
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Polypeptides are made by ribosomes that assemble amino acids in a
sequence that is prescribed by the base sequence of a gene
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Whereas all the DNA of prokaryotes encodes polypeptides, much of the DNA of
eukaryotes does not encode polypeptides
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It contains the genome, the
complete set of chromosomes that are characteristic of the species and gives rise to all the somatic
cells of that animal or plant
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Factors Affecting Gene Expression:
There are many ways in which the transcription or translation of genes can be regulated in
eukaryotes, with the main five being,
•
•
•
•
•

DNA regulatory sequences and transcription factors
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Destruction of mRNA
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Histone acetylation
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Regulatory sequences and transcription factors:
Only about 2% of the DNA in the nuclei of human cells form genes, the other 98% is non-coding,
which only has the purpose of regulating the switching on and off of genes
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There are two
types of regulatory sequence
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They initiate
transcription by enabling RNA polymerase to bind to the gene they regulate
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They
stimulate promoters causing an increase in the rate of transcription of the genes they
regulate
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In order for a gene to be transcribed, RNA polymerase must
be able to attach to it, and if it cannot, the gene will not be transcribed, and the polypeptide it codes
for will not be produced
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One or more specific proteins called transcription

factors bind to the promoter
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Post-Transcriptional modification of mRNA:
A single gene can give rise to more than one polypeptide
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These organisms then process these multigenic transcripts to generate two or more different
polypeptides
...

The genes of eukaryotic cells contain base sequences that do encode mRNA, called exons, and
sections that do not, called introns
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This is then
spliced, and the introns are removed, and the
exons re-joined, to form mature mRNA
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The diagram shows alternative splicing, which
is common in humans
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Alternative splicing of pre-mRNA produced from a single gene can result in the
production of completely different polypeptides
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Destruction of mRNA:
RNA molecules have been discovered that were thought to be too small to have any important
function
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The destruction of mRNA involves these short RNA molecules binding to a protein to form an RNAinduced silencing complex (RISC)
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Small inhibitory RNA (siRNA) – Short, double stranded RNA
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Each is a long, hairpin-shaped precursor molecule
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Each miRNA molecule binds to a protein to form a RISC
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The two mRNA molecules bind by the formation of hydrogen bonds
between base pairs
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These prevent the mRNA from being
translated
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A RNA hydrolase, called a dicer,
hydrolyses these dsRNA molecules into lengths of about 20 base pairs
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Like miRNA, these molecules bind with a protein complex to form a RISC
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This single-stranded antisense
RNA binds to the target mRNA
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Finally, the bound mRNA is hydrolysed by yet another RNA hydrolase
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Each nucleotide contains the pentose
deoxyribose, a phosphate group, and a purine or pyrimidine base
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This means a methyl group can be added to one of their carbon atoms
...
Most are found where the
cytosine is linked by a phosphodiester bond to a guanine residue, represented as CpG
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The presence of methylated DNA
near promotors prevents the activation of the RNA polymerase
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Thus, before any gene that is silenced can be transcribed,
its promoter must be demethylated
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A thread of DNA is wound around ‘beads’ of histone
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This residue can be acetylated, where an acetyl group can be
transferred onto it from acetyl coenzyme A
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This means that transcription factors and RNA polymerase
can now gain access to it
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The
stabilisation of these patterns is called gene imprinting, which is important in both inheritance and
cell differentiation
...
The human zygote can form any human cell,
including those of the placenta and umbilical cord, and therefore it is totipotent
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As they do, their ability to divide and produce different types
of progeny cells becomes restricted
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Zygote
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Cell from inner mass of
blastocyst
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Haematopoietic cells in
bone marrow that give rise
to red blood cells, white
blood cells, and platelets
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Cells in the germinal
epithelium of the skin
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This presents a
problem in diseases that involve the death of cells
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Types of Stem Cell and their uses:
Stem cells in the bone marrow of a donor can be successfully used to replace those in the bone
marrow of a recipient
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Embryonic Stem Cells:
The best type of stem cell to use in transplants would be pluripotent cells, present in the inner cell
mass of a blastocyst, making them easy to harvest
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However, this
process destroys the embryo, which many take issue with due to it’s potential to become a human
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The unused embryos are donated specifically for this purpose by both members
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The Human Fertilisation and Embryology Authority regulates the donation of embryos
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These are adult stem cells and are usually unipotent or multipotent
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Induced Pluripotent Stem Cells:
Fibroblasts are unspecialised cells in connective tissue, capable of differentiating into adipose tissue,
bone, cartilage and smooth muscle
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This
creates cells as useful as embryonic stem cells, but without the ethical issues
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g
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To generate new types of cell that can be used in the laboratory to test the safety and
effectiveness of new drugs, decreasing the need for human and animal experimentation
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Two of the genes transforming fibroblasts (cMyc and KLf4) have cancer-causing properties
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Growing cells for therapies requires specialist systems and research centres
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7
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Although very
weak, the huge number of hydrogen bonds between complementary base pairs makes the doublestranded helix structure very strong
...
In eukaryotes, this sequence is then
edited to form mRNA
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A few techniques remain at the heart of the ever-changing methods of gene technology
...
One, called the polymerase chain reaction (PCR),
was originally carried out in glassware (in vitro), the other involves the use of living cells (in vivo) to
replicate fragments of DNA that have been inserted into their genomes
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Adding salty detergent to the chopped tissue, followed by incubation at 60’C for 15 minutes,
then moving the mixture to an ice bath, which releases the DNA from nuclei by degrading
the cell walls and membranes
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There is then enzymic breakdown of proteins of membranes and of scaffolds to
chromosomes, by the addition of protease solution followed by incubation
...


In Vitro amplification- The Polymerase Chain Reaction:
PCR is used to amplify target DNA
sequences present within a DNA
source, producing multiple copies
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The reagents
include:

•
•
•

Primers- Short lengths of single-stranded DNA complementary to the base sequence of the
3-prime ends of the strands of target DNA
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Thermostable DNA polymerase

Double stranded DNA with known end sequences is heated to 95 degrees to separate the strands
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Heat tolerant taq polymerase and nucleotides
are added and heated to 72 degrees
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PCR used in DNA profiling:
The term DNA profiling refers to the general use of DNA tests to establish the identity of an
individual or the relationship between individuals
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Within some non-coding sequences between
genes, there is a short sequence of bases repeated many times, which are called short tandem
repeats, but often referred to as ‘satellite’ regions of DNA
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Microsatellites of 2-5 bases, repeated 5-15 times
...
It is the microsatellites that we use as the genetic markers in DNA
profiling
...
Using PCR to amplify the desired DNA:
• A sample of DNA is obtained from cells, and PCR amplifies the sections known to
contain the desired satellite regions
...

Since the satellite regions will have different numbers of tandem repeats, the
amplified sections will be of different lengths
...
Separating DNA fragments by electrophoresis:
• Electrophoresis is used to separate the amplified sections, and the result is
converted into a pattern of bands similar to a bar code
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The major component is a tank, and each end has an electrode connected to a
power source
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The samples containing
DNA fragments are placed in wells cut into a
sheet of gel, supported by a glass plate
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•

When migrating from the cathode to the anode through the gel, small molecules
move faster, and thus travel further
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3
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To carry out a Southern
blot, the gel with the separated
DNA fragments is placed on a
blotting paper ‘wick’ fed by alkaline
buffer solution
...

• As the buffer solution is drawn up
through the gel, contact between
the alkali and the DNA fragments breaks the hydrogen bonds between
complementary bases, so the fragments become single-stranded
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• The nylon membrane is removed, and DNA fragments are detected by the
application of selected radioactive DNA probes (a short length of DNA produced
with a base sequence complementary to that within a DNA fragment you wish to
locate)
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Uses of DNA Profiling:
DNA profiles result in a unique binding pattern for every individual
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Identification of Family Relationships in Paternity Testing:
This is most commonly used at the request of an immigration officer to decide whether a child
should be granted immigration status
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Similar DNA profiling also has applications in studies of wild animals, for example, to ensure that
only unrelated animals are used in captive breeding programmes of animals in danger of extinction
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After amplification, a tiny amount of bodily fluid or hair roots will provide
sufficient DNA for profiling
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Using Gene Sequencing:
Gene sequencing involves finding the sequence of bases in a DNA molecule
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DNA is able to be screened of:
•
•
•
•

Potential parents to find if one is a carrier of an inherited disorder
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Embryos prepared for IVF, so those with faulty genes can be rejected
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Each of these techniques raises question about the ethics and cost-effectiveness of these
procedures
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Replication is stopped
when particular nucleotides are incorporated, in a process known as termination sequencing
...
A
nucleotide containing dideoxyribose can pair with a nucleotide that has a complementary base, but
as it lacks the OH group on carbon three, DNA polymerase cannot add another nucleotide to it
...

The reagents used include a heat-resistant DNA polymerase, short primers, with base sequences
complementary to the start of the section of DNA to be copied, and an excess of nucleotides
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Each dideoxynucleotide is labelled with a different fluorescent dye
...

DNA polymerase attaches to the primer and begins to produce a complementary strand,
adding nucleotides with bases complementary to the bases on the template strand
...
You can
deduce that in this case, thymine must occur at the positions on the template strand
...

The fragments of different lengths are then separated by electrophoresis, but instead of gel
in a tank, gel within a capillary tube is used
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By compiling these results from each of the four bases, an automated sequencer can be
used to determine the entire base sequence of the original DNA fragment
...

Larger fragments can be sequenced by the use of overlapping fragments
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In-vivo Amplification of DNA:
DNA can be amplified by inserting fragments into the genomes of living cells
...

The DNA is extracted from tissue samples and can then be cut into fragments by the addition of
restriction endonucleases/restriction enzymes
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The specific base
sequence is called the recognition sequence of that endonuclease
...
Like all enzymes, restriction
endonucleases are specific in the substrates to which
they bind
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They also produce one of
two types of ‘cut,’ with some making staggered cuts, producing fragments with overhanging ‘sticky’
ends, and others making straight cuts, producing fragments with ‘blunt’ ends
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Producing Recombinant DNA:
Before a fragment can be cloned using in vivo technology, it must be
added to a vector, which will be used to transfer it into living cells
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One such vector is commonly found in bacteria
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Plasmids are easily isolated from a bacterial cell to be reintroduced later, and therefore they are
useful as DNA vectors
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A plasmid is cut open using a restriction endonuclease
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If not, complementary sticky ends are added to the
blunt ends of both the plasmid and DNA fragment
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Ligase occurs naturally in the nuclei of eukaryotic cells,
where it repairs DNA damaged by replication
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Complementary base pairing leads to the formation of hydrogen bonds, and the formation of the
sugar-phosphate backbone is catalysed by ligase
...
Bacteria that have taken up the plasmid are
‘transformed,’ and if the plasmid contained one or more entire genes, they are also ‘transgenic
...
It is necessary to know which bacteria are transformed, so that
only transformed bacteria can be grown in a culture medium
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One way is to culture the mixture of bacteria onto agar plates
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We can use the example of a plasmid containing a gene for the resistance
to an antibiotic ‘A,’ and having the point where it is cut, and the other gene is inserted being in the
middle of a gene responsible for the resistance to antibiotic ‘B
...
We can repeat this process, pressing the filter paper onto the surface of an agar plate in which the
agar contains antibiotic B
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The bacteria that successfully took up the plasmid and
contained a recombinant plasmid, would grow on the replica plate for A, but would not grow on the
plate for B
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We can then transfer samples of the successful colonies from the original agar plate to sterile
nutrient medium and allow them to increase in number
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The virus
must be weakened to do this so that it does not cause the death of host cells
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The lipid
coating allows the droplet to cross cell-surface membranes
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The DNA can be fired
directly into an organ or tissue culture
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The affected cells can then able to take up naked recombinant DNA and
incorporate it
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The transgenic organisms formed are referred to as genetically modified
(GM) organisms, and their production is now a major industry
...
Each modification
involves the introduction of recombinant DNA containing a gene that changes the properties of the
plants
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Resistance to pets, including fungi, nematode worms, and insects
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In other cases, the gene changes the biochemical components of the soya beans, for example by
changing the balance of the fatty acids in order to reduce trans fats when cooked, and causing the
production of active pharmacological ingredients
...
They do this by adding a piece of foreign DNA into the genome that disrupts the transcription
of a gene
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By knocking out the activity of a gene, the function of that
gene can be investigated
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However, they are not always
helpful, as often the effect of a particular gene in a mouse will be different to that of a human with
the same gene
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Reassurances

Potential Dangers

The nutritional value of foods can be
quickly increased
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Genes conferring to resistance to
adverse conditions mean yields in
more hostile environments increase
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GM organisms are tested before
commercial growth
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People can choose to avoid GM
products
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In humans inherited conditions can
be avoided
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Only one gene is involved in the
transfer, so there is minimal effect
on the genome
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