THE INDIAN PUBLIC SCHOOL – SALEM

Grade: A-level Handout

Topic 19 : Genetic Technology
Genetic engineering is a technique used to deliberately modify a specific characteristic (or
characteristics) of an organism. The technique involves removing a gene (or genes) with the desired
characteristic from one organism and transferring the gene (using a vector) into another organism
where the desired gene is then expressed.
• In order for an organism to be genetically engineered the following steps must be taken:
• Identification of the desired gene
• Isolation of the desired gene by:
• Cutting from a chromosome using enzymes (restriction endonucleases)
• Using reverse transcriptase to make a single strand of complementary DNA
(cDNA) from mRNA
• Creating the gene artificially using nucleotides
• Multiplication of the gene (using polymerase chain reaction - PCR)
• Transfer into the organism using a vector (e.g. plasmids, viruses, liposomes)
• Identification of the cells with the new gene (by using a marker), which is then
cloned
Process:
Isolating the Desired Gene
• The gene with the specific characteristic that is required can be obtained in the following
ways:
• Extracting the gene from the DNA of a donor organism using enzymes (restriction
endonucleases)
• Using reverse transcriptase to synthesise a single strand of complementary DNA
(cDNA) from the mRNA of a donor organism
• Synthesising the gene artificially using nucleotides
Specific restriction enzymes cut at specific DNA sequences. For example:
• EcoRI is an enzyme that cuts at the following sequence: GAATTC
• EcoRI was discovered in E. coli bacteria.
• The resulting pieces of DNA are called “restriction fragments.”

mRNA & reverse transcriptase

• Another method to isolate the desired gene is to use the mRNA that was transcribed for that
gene
• Once isolated, the mRNA is then combined with a reverse transcriptase enzyme and
nucleotides to create a single strand of complementary DNA (cDNA)
• Reverse transcriptase enzymes are sourced from retroviruses and they catalyse the reaction
that reverses transcription. The mRNA is used as a template to make the cDNA
• DNA polymerase is then used to convert the single strand of cDNA into a double-stranded
DNA molecule which contains the desired code for the gene
 • This technique for isolating the desired gene is considered advantageous as it is easier for
scientists to find the gene because specialised cells will make very specific types of mRNA
(eg. β-cells of the pancreas produce many insulin mRNAs) and the mRNA (therefore the
cDNA) does not contain introns

Artificial synthesis

• As scientists are becoming more familiar with the base sequences for our proteins (proteome)
it is possible to synthesise genes artificially
• With the knowledge of the genetic code (that is, which amino acids are required)
scientists use computers to generate the nucleotide sequence (rather than an mRNA
template) to produce the gene
• Short fragments of DNA are first produced which are joined to make longer sequences of
nucleotides and then inserted into vectors (eg. plasmids)
• This method is being used to create novel genes being used to make vaccines and even to
synthesise new bacteria genomes

Genetic Engineering: Enzymes

• Genetic engineering is the deliberate modification of a specific characteristic (or

characteristics) of an organism. The technique involves removing a gene (or genes), with the
desired characteristic, from one organism and transferring the gene (using a vector) into
another organism where the desired gene is then expressed
• In order to genetically engineer an organism there are a number of enzymes required:
• Restriction endonucleases (enzymes) – cuts the DNA strands so that the desired gene
can be isolated or spliced (inserted) into a vector
• Reverse transcriptase – reverses transcription to produce a single-strand
complementary DNA (cDNA) from an mRNA strand with the code for the desired
gene
• DNA polymerase – used to convert the single-stranded cDNA into a double-stranded
DNA molecule of the desired gene
• DNA ligase – is used to splice (insert) the gene into the vector

Restriction endonucleases

• The role of restriction endonucleases (or restriction enzymes) in the transfer of a gene into
an organism is to:
 • Isolate the desired gene
• Separate the DNA strands (at the same base sequence) in a vector so the desired gene
can be inserted
• There are many different restriction endonucleases because they bind to a specific restriction
site (specific sequences of bases) on DNA, eg. HindIII will always bind to the base sequence
AAGCTT
• Restriction endonucleases will separate the two strands of DNA at the specific base sequence
by ‘cutting’ the sugar-phosphate backbone in an uneven way to give sticky ends or
straight across to give blunt ends
• Sticky ends result in one strand of the DNA fragment being longer than the other strand
• The sticky ends make it easier to insert the desired gene into another organism's DNA or into
a vector as they can easily form hydrogen bonds with the complementary base sequences on
other pieces of DNA that have been cut with the same restriction endonucleases

Reverse transcriptase

• The role of reverse transcriptase in the transfer of a gene into an organism is to produce
a single-strand complementary DNA molecule (cDNA) that contains the code for the
desired characteristic, this will then be inserted into a vector (after being converted into a
double-stranded DNA molecule)
• Reverse transcriptase enzymes are sourced from retroviruses and they catalyse the reaction
that reverses transcription. The mRNA (with the genetic code for the desired gene) is used as
a template to synthesise a single strand of complementary DNA (cDNA)
• Reverse transcriptase enzymes are often used as it is easier for scientists to find mRNA with
the specific characteristic because specialised cells make very specific types of mRNA (eg. β-
cells of the pancreas produce many insulin mRNA) and mRNA does not contain introns

DNA polymerase

• DNA polymerase is used to convert the single strand of cDNA into a double-stranded
DNA molecule which contains the desired code for the gene
• The enzyme builds the second strand by pairing free nucleotides with the complementary
bases on the cDNA strand

DNA ligase

• DNA ligase catalyses the formation of phosphodiester bonds in the DNA sugar-phosphate
backbone
• This enzyme enables the isolated desired gene to be spliced into a vector (generally a
plasmid) so that it can be transferred to the new organism

Vectors

• Vectors are used to transfer the desired genes into a foreign cell
• Plasmids are the most commonly used vector but viruses and liposomes (a small vesicle with
a phospholipid layer) can also be used to transfer genes
Plasmids

• Plasmids are small, circular rings of double-stranded DNA

• They occur naturally in bacteria, but can also been found in archaea and eukaryotic organisms
(eg. yeast and fungi) and can contain genes for antibiotic resistance
• Plasmids are used as they can self replicate
• A plasmid is used to transfer the desired gene to a new organism
• To insert the desired gene into the circular DNA of the plasmid it is ‘cut’ open. The same
restriction endonuclease that was used to isolate the desired gene is used to ‘cut’ open the
plasmid. This results in the plasmid having complementary sticky ends to the sticky ends on
the desired gene fragment
• DNA ligase forms phosphodiester bonds between the sugar-phosphate backbone of the DNA
fragment and the plasmid to form a recombinant plasmid (a closed circle of double-stranded

DNA containing the desired gene)

• Scientists can modify bacterial plasmids or artificially produce them. One benefit of this is
that the plasmids can have one or more marker genes so that cells that have the recombinant
plasmids can be identified
• Plasmids are transferred into host cells (usually bacteria) by a process called transformation.
Only a small proportion of bacteria will become transformed and therefore markers are used
to identify these. Transformation can occur by:

Bathing the plasmids and bacteria in an ice-cold calcium chloride solution and then briefly
incubating at 40°C. This makes the bacteria membrane permeable

Electroporation - where the bacteria is given a small electrical shock making the membranes very
porous (this technique can be used to get DNA fragments into eukaryotic cells)

Viruses

• Viruses are commonly used as vectors in the process of gene therapy, which is currently used
to treat genetic diseases such as cystic fibrosis
• The viruses are genetically modified to carry non-mutated genes into host cells
• Different types of viruses have been used; retroviruses, lentiviruses and adeno-associated
viruses

Liposomes

• Liposomes are small spherical vesicles with a phospholipid layer

• These vesicles can also be used in gene therapy to carry non-mutated genes into host cells
• The advantage of using liposomes as a vector is that they can fuse with the cell surface
membrane

Genetic Engineering: Promoter

 • The promoter (an example of a length of non-coding DNA that has a specific function) is
the region of DNA that determines which gene will be expressed. This is because it is
the site where RNA polymerase binds to in order to begin transcription
• Thus the promoter is used to regulate gene expression because only if it is present will
transcription and therefore the expression of the gene occur
• If genetic engineers want to ensure the desired gene is expressed when modifying the plasmid
they have to add an appropriate promoter

Genetic Engineering: Marker Genes

• A marker is a gene that is transferred with the desired gene to enable scientists
to identify which cells have been successfully altered and now contain recombinant DNA
• Antibiotic-resistant genes were once commonly used as marker genes. Scientists genetically
modified the bacteria so that the plasmid contained the desired gene along with a specific
antibiotic-resistant gene (and promoter) and then grew the bacteria on agar plates embedded
with that antibiotic. The bacteria that contained the recombinant plasmids could be identified
as these were the bacteria that grew
• Using antibiotic-resistant genes as marker genes concerns scientists as:
• There is a risk that the antibiotic-resistant genes could be accidentally transferred to
other bacteria including pathogenic strains creating pathogenic antibiotic-resistant
bacteria
• If the resistance spread to other bacteria this could make antibiotics less effective
• The spread of the antibiotic-resistant genes can occur due to the conjugation (the transfer of
genetic material from one bacterium to another) or due to transduction (the transfer of
genetic material from one bacterium to another via a virus)
• So genes that express proteins that are fluorescent are now commonly used as markers
• The fluorescence is due to the presence of a green fluorescent protein (GFP)
• The GFP gene along with the desired gene are linked to a specific promoter and once this
promoter is activated, and the protein is expressed, the recombinant bacteria are detected
when they glow green under exposure to ultraviolet light
• The use of fluorescent genes as markers is preferable because:
• They are easier to identify (all that is required is the ultraviolet light)
• More economical (do not need to grow the bacteria on plates of agar infused with
antibiotics)
• No risk of antibiotic resistance being passed onto other bacteria
• There are antibiotics that are no longer effective and therefore would not stop any
bacteria from growing

Gene Editing

• Gene genome editing (or editing) allows genetic engineers to alter the DNA of organisms
by inserting, deleting or replacing DNA at specific sites in the genome known to cause
disease.
• It is a form of genetic engineering where foreign DNA is not introduced into the genome
• Gene editing enables the scientists to be more accurate in their manipulation of the genome.

What is CRISPR-Cas9?
 • CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit
parts of the genome by removing, adding or altering sections of the DNA sequence.
• It is currently the simplest, most versatile and precise method of genetic manipulation and is
therefore causing a buzz in the science world.

How does it work?

• The CRISPR-Cas9 system consists of two key molecules that introduce a change (mutation)
into the DNA. These are:
• an enzyme called Cas9. This acts as a pair of ‘molecular scissors’ that can cut the two
strands of DNA at a specific location in the genome so that bits of DNA can then be
added or removed.
• a piece of RNA called guide RNA (gRNA). This consists of a small piece of pre-
designed RNA sequence (about 20 bases long) located within a longer RNA scaffold.
The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the
right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point
in the genome.
• The guide RNA is designed to find and bind to a specific sequence in the DNA. The
guide RNA has RNA bases that are complementary to those of the target DNA
sequence in the genome. This means that, at least in theory, the guide RNA will only
bind to the target sequence and no other regions of the genome.
• The Cas9 follows the guide RNA to the same location in the DNA sequence and
makes a cut across both strands of the DNA.
• At this stage the cell recognizes that the DNA is damaged and tries to repair it.
• Scientists can use the DNA repair machinery to introduce changes to one or
more genes in the genome of a cell of interest.
 CRISPR is one of the tools scientists can use to edit genes

What are the applications and implications?

• CRISPR-Cas9 has a lot of potential as a tool for treating a range of medical conditions that
have a genetic component, including cancer?, hepatitis B or even high cholesterol.
• Many of the proposed applications involve editing the genomes of somatic? (non-
reproductive) cells but there has been a lot of interest in and debate about the potential to
edit germline? (reproductive) cells.
• Because any changes made in germline cells will be passed on from generation to generation
it has important ethical implications.
• Carrying out gene editing in germline cells is currently illegal in the UK and most other
countries.
• By contrast, the use of CRISPR-Cas9 and other gene editing technologies in somatic cells is
uncontroversial. Indeed they have already been used to treat human disease on a small
number of exceptional and/or life-threatening cases.

What’s the future of CRISPR-Cas9?

• It is likely to be many years before CRISPR-Cas9 is used routinely in humans.

• Much research is still focusing on its use in animal models or isolated human cells, with the
aim to eventually use the technology to routinely treat diseases in humans.
• There is a lot of work focusing on eliminating ‘off-target’ effects, where the CRISPR-Cas9
system cuts at a different gene to the one that was intended to be edited.

Polymerase Chain Reaction (PCR)

 Polymerase chain reaction (PCR) is a common molecular biology technique used in most
applications of gene technology
 It can be described as the in vitro method of DNA amplification
 It is used to produce large quantities of specific fragments of DNA or RNA from very small
quantities (even just one molecule of DNA or RNA). By using PCR scientists can have
billions of identical copies of the DNA or RNA sample within a few hours
 The PCR process involves three key stages per cycle. In each cycle the DNA is doubled so in
a standard run of 20 cycles a million DNA molecules are produced. The three stages are
undertaken in a PCR instrument (or thermal cycler) which automatically provides
the optimal temperature for each stage and controls the length of time spent at each stage
 Each PCR reaction requires:
o Target DNA or RNA being amplified
o Primers (forward and reverse) – these are short sequences of single-stranded DNA
that have base sequences complementary to the 3’ end of the DNA or RNA being
copied. They define the region that is to be amplified by identifying to the DNA
polymerase where to begin building the new strands
 o DNA polymerase – is the enzyme used to build the new DNA or RNA strand. The
most commonly used polymerase is Taq polymerase as it comes from a thermophilic
bacterium Thermus aquaticus which means it does not denature at the high
temperature involved during the first stage of the PCR reaction and secondly, its
optimum temperature is high enough to prevent annealing of the DNA strands that
have not been copied yet
o Free nucleotides – used in the construction of the DNA or RNA strands
o Buffer solution – to provide the optimum pH for the reactions to occur in
 The three stages are:
o Denaturation – the double-stranded DNA is heated to 95°C which breaks the
hydrogen bonds that bond the two DNA strands together
o Annealing – the temperature is decreased to between 50 - 60°C so that primers
(forward and reverse ones) can anneal to the ends of the single strands of DNA
o Elongation / Extension – the temperature is increased to 72°C for at least a minute, as
this is the optimum temperature for Taq polymerase to build the complementary
strands of DNA to produce the new identical double-stranded DNA molecules

Gel Electrophoresis

• Gel electrophoresis is a technique used widely in the analysis of DNA, RNA and proteins.
During electrophoresis the molecules are separated according to their size / mass and
their net (overall) charge
• The separation occurs because:
• Of the electrical charge molecules carry – positively charged molecules will move
towards the cathode (negative pole) whereas negatively charged molecules will move
towards the anode (positive pole) eg. DNA is negatively charged due to
the phosphate groups and thus when placed in an electric field the molecules move
towards the anode
• Different sized molecules move through the gel (agarose for DNA and polyacrylamide
– PAGE for proteins) at different rates. The tiny pores in the gel result in smaller
molecules moving quickly, whereas larger molecules move slowly
• Of the type of gel – different gels have different sized pores which affects the speed
the molecules can move through them

DNA separation

• DNA can be collected from almost anywhere on the body, e.g. the root of a hair or saliva
from a cup. After collection DNA must be prepared for gel electrophoresis so that
the DNA can be sequenced or analysed for genetic profiling (fingerprinting)
• To prepare the fragments scientists must first increase (amplify) the number of DNA
molecules by the polymerase chain reaction (PCR). Then restriction endonucleases (enzymes)
are used to cut the DNA into fragments
• Different restriction enzymes cut the DNA at different base sequences. Therefore scientists
use enzymes that will cut close to the variable number tandem repeat (VNTR) regions
• Variable number tandem repeats (VNTRs) are regions found in the non-coding part of
DNA. They contain variable numbers of repeated DNA sequences and are known to vary
 between different people (except for identical twins). These VNTR may be referred to as
‘satellite’ or ‘microsatellite’ DNA

To separate the DNA fragments in gel electrophoresis the scientists:

• Create an agarose gel plate in a tank. Wells (a series of groves) are cut into the gel at one
end
• Submerge the gel in an electrolyte solution (a salt solution that conducts electricity) in the
tank
• Load (insert) the fragments into the wells using a micropipette
• Apply an electrical current to the tank. The negative electrode must be connected to the
end of the plate with the wells as the DNA fragments will then move towards the anode
(positive pole) due to the attraction between the negatively charged phosphates of DNA
and the anode
• The smaller mass / shorter pieces of DNA fragments will move faster and further from the
wells than the larger fragments
• The fragments are not visible so must be transferred onto absorbent paper or nitrocellulose
which is then heated to separate the two DNA strands. Probes are then added, after which an
X-ray image is taken or UV-light is shone onto the paper producing a pattern of bands which
is generally compared to a control fragment of DNA
• Probes are single-stranded DNA sequences that are complementary to the VNTR regions
sought by the scientists. The probes also contain a means by which to be identified. This can
either be:
• A radioactive label (eg. a phosphorus isotope) which causes the probes to emit radiation that
makes the X-ray film go dark, creating a pattern of dark bands
• A fluorescent stain / dye (eg. ethidium bromide) which fluoresces (shines) when exposed to
ultraviolet (UV) light, creating a pattern of coloured bands

Protein separation
 • The different amino acids (because of the different R groups) determine the charge of
proteins. The charge of the R groups depends on the pH and therefore buffer solutions are
used during the separation of proteins to keep the pH constant
• Gel electrophoresis is used to separate polypeptide chains produced by different alleles eg. the
haemoglobin variants (α-globin, β-globin and the sickle cell anaemia variant of β-globin)

Microarrays

• Microarrays are laboratory tools used to detect the expression of thousands of genes at the
same time and to identify the genes present in an organism’s genome
• Microarrays are used in medical diagnosis and treatment (e.g. comparison between healthy
cells and diseased cells to find the characteristics of the disease), biotechnology (eg. in
agriculture to identify insect pests), as well as crime (forensic analysis)
• As large numbers of genes can be studied in a short period of time microarrays have been
very valuable to scientists
• The microarray consists of a small (usually 2cm 2) piece of glass, plastic or silicon (also
known as chips) that have probes attached to a spot (called a gene spot) in a grid pattern.
There can be 10 000 or more spots per cm2
• Probes are short lengths of single-stranded DNA (oligonucleotides) or RNA which are
synthesised to be complementary for a specific base sequence (this sequence depends on the
purpose of the microarray)
• Microarrays can be used to detect whether a gene is being expressed (a method used to
research cancerous vs non-cancerous cells) by detecting the quantity of mRNA present

Procedure for microarray

• When a microarray is used to analyse genomes:

• DNA is collected from the species going to be compared
• Restriction enzymes are used to cut the DNA into fragments
• These fragments are denatured to create single-stranded DNA molecules
• These DNA fragments are labelled using fluorescent tags (the fragments from the
different sources are tagged different colours, usually red and green)
• Once these fragments are mixed together they are then allowed to hybridise with the
probes on the microarray
• After a set period of time any DNA that did not hybridise with the probes is washed
off
• The microarray is then examined using ultraviolet light (which causes the tags to
fluoresce) or scanned (colours are detected by the computer and the information is
analysed and stored)
• The presence of the colour indicates where hybridisation has occurred, as the DNA
fragment is complementary to the probe. If red and green fluorescent spots appear
then only one species of DNA has hybridised, however, if the spot is yellow then both
species have hybridised with that DNA fragment, which suggests that both species
have that gene in common
• If a spot lacks colour that indicates the gene is not present in either species

To compare which genes are being expressed using microarrays the following steps occur:
 • mRNA is collected from both types of cells and reverse transcriptase is used to
convert mRNA into cDNA
• PCR may be used to increase the quantity of cDNA (this occurs for all samples to
remain proportional so a comparison can be made when analysis occurs)
• Fluorescent tags are added to the cDNA
• The cDNA is then denatured to produce single-stranded DNA
• The single-stranded DNA molecules are allowed to hybridise with the probes on the
microarray
• When the ultraviolet light is shone on the microarray the spots that fluoresce indicate
that gene was transcribed (expressed) and the intensity of the light emitting from the
spots indicates the quantity of mRNA produced (i.e. how active the gene is). If the
light being emitted is of high intensity then many mRNA were present, while a low
intensity emission indicates few mRNA are present

Bioinformatics

• The various technologies (eg. microarrays and gene sequencing) being used today to analyse
genes and proteins generate enormous quantities of data
• The data being collected ranges from the sequences of genomes, when genes are being
expressed during an organism’s life to the structure (amino acid sequence) and functions of
proteins
• To analyse all of this data scientists are using bioinformatics
• Bioinformatics is an interdisciplinary science (incorporating biology with computer
technology and statistics) where biological data is collected, organised, manipulated, analysed
and stored
• The European Molecular Biology Laboratory – Nucleotide sequence database
• ArrayExpress – a microarray database with the level and types of mRNA expressed
in different cells
• Protein Data Bank at Europe – Protein sequence searches
• BLAST (Basic Local Alignment Search Tool) – used by researchers to find
similarities between sequences they are studying with those already in the database

Recombinant Human Proteins

• DNA that has been altered by introducing nucleotides from another source is
called recombinant DNA (rDNA)
• If the organism contains nucleotides from a different species it is called
a transgenic organism
• Any organism that has introduced genetic material is a genetically modified
organism (GMO)
• Recombinant DNA has been used to produce recombinant proteins (RP), thus recombinant
proteins are manipulated forms of the original protein

The advantages of genetic engineering organisms to produce recombinant human proteins are:

• More cost-effective to produce large volumes (i.e. there is an unlimited availability)

• Simpler (with regards to using prokaryotic cells)
 • Faster to produce many proteins
• Reliable supply available
• The proteins are engineered to be identical to human proteins or
have modifications that are beneficial
• It can solve the issue for people who have moral or ethical or religious concerns
against using cow or pork produced proteins

1. Insulin

• In 1982, insulin was the first recombinant human protein to be approved for use
in diabetes treatment
• Bacteria plasmids are modified to include the human insulin gene
• Restriction endonucleases are used to cut open plasmids and DNA ligase is used to
splice the plasmid and human DNA together
• These recombinant plasmids are then inserted into Escherichia coli by transformation (bath of
calcium ions and then heat or electric shock)
• Once the transgenic bacteria are identified (by the markers), they are isolated, purified and
placed into fermenters that provide optimal conditions
• The transgenic bacteria multiply by binary fission, and express the human protein - insulin,
which is eventually extracted and purified

The advantages for scientists to use recombinant insulin are:

• It is identical to human insulin, unless modified to have different properties (eg. act
faster, which is useful for taking immediately after a meal or to act more slowly)
• There is a reliable supply available to meet demand (no need to depend on
availability of meat stock)
• Fewer ethical, moral or religious concerns (proteins are not extracted from cows or
pigs)
• Fewer rejection problems or side effects or allergic reactions
• Cheaper to produce in large volumes
• That it is useful for people who have animal insulin tolerance

2. Factor VIII

• Factor VIII is a blood-clotting protein that haemophiliacs cannot produce

• Kidney and ovary hamster cells have been genetically modified to produce Factor VIII
• Once modified these recombinant cells are placed into a fermenter and cultured
• Due to the optimal conditions in the fermenter, the hamster cells constantly express Factor
VIII which can then be extracted and purified, and used as an injectable treatment for
haemophilia
• The advantages for scientists to use recombinant Factor VIII are:
• Fewer ethical, moral or religious concerns (proteins are not extracted from human
blood)
• Less risk of transmitting infection (eg. HIV) or disease
 • Greater production rate

3. Adenosine deaminase

• Adenosine deaminase (ADA) is an enzyme used to treat the inherited condition

called Adenosine Deaminase Deficiency
• ADA Deficiency is a common cause of Severe Combined Immunodeficiency (SCID)
• This is because the immune system is damaged
• The larva of the cabbage looper moth has been genetically modified (using a virus vector) to
produce the enzyme adenosine deaminase so that it can be used as a treatment whilst the
patients wait for gene therapy or when gene therapy is not possible
• The advantages for scientists to use recombinant adenosine deaminase are:
• Fewer ethical, moral or religious concerns (proteins are not extracted from cows)
• Less risk of transmitting infection or disease (from cows)
• More reliable production of enzyme
• Faster to produce many proteins

4. Genetic Screening

• In certain circumstances (eg. in the pregnancy in an older woman, or pregnancy where there is
a family history of a genetic disease) may require individuals to determine if they have a
particular allele present in their genome. This can be determined by genetic screening
• Genetic screening can help identify individuals who are carrying an allele at a gene locus for
a particular disorder
• Genetic screening is the testing of an embryo, fetus or adult to analyse the DNA
• The sample of DNA to be analysed can be obtained by:
• Taking tissue samples from adults or embryos produced by in-vitro fertilisation
• Chorionic villus sampling or amniocentesis of embryos and fetuses in the uterus
• As genetic screening can leave future parents with many questions, genetic counsellors are
available to help. The counsellors will read the results and explain them. Counsellors can also
be seen before screening has occurred. They may discuss the following with the prospective
parents:
• The chances of the couple having a child with a certain disease
• Termination of the pregnancy
• Therapeutic treatments possible for the child
• Financial implications of having the child
• Effect on existing siblings
• Ethical issues

5. Breast cancer (BRCA1 and BRCA2)

• BRCA1 and BRCA2 are genes that produce tumour suppressor proteins and thus they play an
important role in regulating cell growth
• Faulty alleles of these particular genes exist which increase the risk of an individual
developing breast and ovarian cancers during their lifetime
• Faulty BRCA1 and BRCA2 alleles can be inherited from either parent
 • The advantages of genetic screening for an adult who has a family history
of BRCA1 and BRCA2 gene mutations are:
• That the person may decide to take preventative measures (e.g. by having an elective
mastectomy – breast removal – to reduce the risk of developing cancer)
• Screening for breast cancer may begin from an earlier age or more frequently, and
the individual (if female) will have more frequent clinical examinations of the ovaries
• That it enables the person to participate in research and clinical trials

6. Huntington’s disease

• Huntington’s is a progressive (gets worse with time) inherited disease that affects the brain
• Signs of the disease typically appear in affected individuals after reaching their 40’s and
include uncontrolled movements, lower cognitive (thinking) ability and emotional
problems
• There is no cure for the Huntingdon’s disease, with treatments available only alleviating the
symptoms but not curing it
• Huntington’s is an autosomal dominant disease (therefore if the person has an allele for
Huntington’s they will get the disease)
• The advantage of genetic screening for Huntington’s is it enables:
• People to plan for the future (how they will live and be cared for)
• Couples to make informed reproductive decisions (as the risk that their children may
inherit the disease is 50%)
• People to participate in research and clinical trials

7. Cystic fibrosis

• Cystic fibrosis is an autosomal recessive genetic disorder that is caused by a mutation of the
gene that codes for a transported protein called CFTR
• It is a progressive disease that causes mucus in various organs (lungs, pancreas, lungs) to
become thick and sticky. This is because the faulty CFTR protein no longer transports
chloride ions across the cell plasma membrane and therefore water does not move by osmosis
across the membrane either (the presence of water would normally make the mucus thinner
enabling cilia to remove it)
• There is no cure for cystic fibrosis, although there are many different treatments that help
alleviate symptoms. The common cause of death is bacterial infection in the lungs
• The advantage of genetic screening for cystic fibrosis is:
• It enables couples to make informed reproductive decisions (as both may be carriers
and therefore not display any symptoms)
• That people can participate in research and clinical trials

Gene Therapy

• Gene therapy involves using various mechanisms to alter a person's genetic material to
treat, or cure, diseases
• As scientists gain a better understanding of the human genome and therefore the location of
genes that cause genetic disorders, the possibilities of gene therapy being able to replace a
faulty gene, inactivate a faulty gene or insert a new gene are growing
• Experimental techniques are being used to treat and research treatments for genetic diseases
such as severe combined immunodeficiency (SCID), Leber congenital amaurosis – a rare
form of blindness, β-thalassaemia and haemophilia B
• Most gene therapies are still in the clinical trial stage because scientists are having difficulty
finding delivery systems that can transfer normal alleles into a person’s cells and how to
ensure the gene is correctly expressed once there
• Finding an appropriate delivery system has been one of the problems. Vectors are currently
used as the delivery system, with viruses being the most commonly used, but non-viral
vectors are also being researched (eg. liposomes and ‘naked’ DNA)
• Viruses (eg. retroviruses and lentiviruses) are the most commonly used vectors as they have
the mechanisms needed to recognise cells, and deliver the genetic material into them
• Changes in genetic material are targeted to specific cells and so will not be inherited by
future generations (as somatic gene therapy does not target the gametes)
• Often the effects of changing the somatic cells are short-lived
• There are two types of somatic gene therapy:
• Ex vivo – the new gene is inserted via a virus vector into the cell outside the body.
Blood or bone marrow cells are extracted and exposed to the virus which inserts the
gene into these cells. These cells are then grown in the laboratory and returned to the
person by an injection into a vein
• In vivo – the new gene is inserted via a vector into cells inside the body
• There is the potential for new genetic material to be inserted into germ cells (cells involved in
sexual reproduction eg. gametes or an early embryo)
• However, this is illegal in humans as any changes made to the genetic material of these cells
is potentially permanent and could therefore be inherited by future generations
• The two types of somatic gene therapy - ex vivo and in vivo. Two of the methods scientists
are using to alter a patient’s genetic material to treat or cure diseases
Severe combined immunodeficiency (SCID)

• Severe combined immunodeficiency (SCID) is caused by the body's inability to

produce adenosine deaminase (ADA), an enzyme that is key to the functioning of the
immune system. Without this enzyme children can die from common infections and
therefore need to be keep isolated often inside plastic ‘bubbles’
• To treat SCID scientists have used ex vivo somatic gene therapy. During this therapy, a
virus transfers a normal allele for ADA into T-lymphocytes removed from the patient and
the cells are then returned via an injection
• This is not a permanent cure as the T-lymphocytes are replaced by the body over time and
therefore the patient requires regular transfusions every three to five months to keep their
immune systems functioning
• Originally retroviruses were used as the vectors, however these viruses insert their genes
randomly into a host’s genome which means they could insert the gene into another gene or
into a regulatory sequence of a gene (which could result in cancer)
• Initial treatments did cause cases of leukaemia in children, so researchers switched to using
lentiviruses or adeno-associated viruses as vectors. Lentiviruses also randomly insert their
genes into the host genome however they can be modified to not replicate, whereas adeno-
associated viruses do not insert their genes into the host genome and therefore the genes are
not passed onto the daughter cells when a cell divides. This is an issue with short-lived cells
like lymphocytes but has not been a problem when used with longer living cells such as liver
cells

Inherited eye diseases

• An example of a group of inherited eye diseases that causes blindness due to damage to the
light receptors in the retina are Leber Congenital Amaurosis. It begins to affect children
from birth and by their 20s or 30s the person is totally blind. There is no cure for these
diseases
• Using in vivo somatic gene therapy, doctors injected into the retina adeno-associated
viruses that contained the normal alleles of one of the genes that caused damage to the
photoreceptors (there are at least 18 known mutated genes causing this group of diseases). All
patients that have had the injections have shown improvement in their eyesight