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19.1 Principles of genetic technology

Learning outcomes
Candidates should be able to:
1 define the term recombinant DNA
Recombinant DNA (rDNA): DNA made by artificially joining together pieces of DNA from two
or more different species

The organisms which expresses the new gene is known as a transgenic organism (DNA
from another source) or GMO (DNA changed in a way that does not occur naturally by
selective breeding)
Chapter 19 Genetic Technology
An overview of gene transfer:
1. The gene that is required is identified. It may be cut from a chromosome, made from
mRNA by reverse transcription or synthesised from nucleotides.
2. Multiple copies of the gene are made using a technique known as the polymerase chain
Biology A level (Macleans College) reaction (PCR).
3. The gene is inserted into a vector which delivers the gene to the cells of the organism.
Examples of vectors are plasmids, viruses and liposomes.
4. The vector takes the gene into the cells
5. The cells that have the new gene are identified and cloned.

2 explain that genetic engineering is the deliberate manipulation of genetic material to modify
specific characteristics of an organism and that this may involve transferring a gene into an
organism so that the gene is expressed
Scan to open on Studocu Genetic Engineering: Any procedure in which the genetic information in an organism is
changed by altering the base sequence of a gene or by introducing a gene from another
organism

3 explain that genes to be transferred into an organism may be:

• extracted from the DNA of a donor organism
• synthesised from the mRNA of a donor organism
• synthesised chemically from nucleotides

4 explain the roles of restriction endonucleases, DNA ligase, plasmids, DNA polymerase and
reverse transcriptase in the transfer of a gene into an organism
Plasmids:
small circles of double stranded DNA usually occurring naturally in bacteria
→ vector that can be used to take DNA into a bacterial cell (If DNA inserted)
Vector: A means of delivering genes into a cell used in genetic technology e.g. plasmids and
viruses

Restriction endonuclease (Restriction Enzymes) cut the sugar phosphate backbone of DNA
at specific sites, known as restriction sites
Restriction enzymes can cut in staggered fashion to give sticky ends, or cut straight across
the backbone to give blunt ends
● Sticky ends: Short lengths of unpaired bases; Pieces of DNA with sticky ends that are
complementary to each other can join together by forming hydrogen bonds.

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● Blunt ends: Can join with any other pieces of DNA, does not need to be complementary
and has non-specific recognition sites 8 describe and explain the steps involved in the polymerase chain reaction (PCR) to clone
and amplify DNA, including the role of Taq polymerase
DNA Ligase Polymerase chain reaction (PCR)
● Links together sugar-phosphate backbone of the DNA molecule and the plasmid Makes very large numbers of copies of DNA from very small quantities (1. Denaturation 2.
● Produces a closed circle of double-stranded DNA with the new gene - recombinant DNA Annealing 3. extension)
● DNA is denatured by heating (around 95ºC), separating DNA molecule (double helix) into
Reverse Transcriptase two strands and bases exposed
● Instead of cutting out a gene from the DNA, single stranded cDNA can be synthesised ● Primer DNA is added after cooling → complementary base pairing; needed to initiate DNA
directly from mRNA by using the enzyme reverse transcriptase. (Comes from retroviruses) polymerase
(Hard to locate and isolate gene coding for human insulin from rest of DNA in human cell) - Primer - short length of DNA 20 base pairs long, has a base sequence complementary to
the start of the part of DNA strand to be copied → DNA polymerase continues to add
DNA Polymerase: nucleotides along rest of DNA strand
Converts these single stranded DNA molecules to double stranded DNA molecules by - Primer attaches to ends of single stranded DNA (process of annealing, requires
making a complementary strand → now have complete gene to insert into plasmids to form temperature of 65ºC)
GMO ● The enzyme DNA polymerase is then used to build new strands of DNA against the
exposed ones → using free nucleotides to synthesise complementary strands
5 explain why a promoter may have to be transferred into an organism as well as the desired - Known as elongation, temperature of around 72°C
gene ● Once copied (parts of two DNA molecules), mixture is heated again, separating two
Promoter - region of DNA to which RNA polymerase binds as it starts transcription. strands in DNA leaving them available for copying again
Promoters control the expression of genes (e.g in the lac operon) - Not all genes are ● Again, primers fix themselves to start of strand of unpaired nucleotides, and DNA
switched on at once polymerase makes complementary copies of them
● Promoter must be inserted alongside the gene because organisms will not transcribe and
express a gene unless there is a binding site for RNA polymerase. Heat-stable DNA polymerases are used in PCR:
● Promoters also ensure that RNA polymerase recognises which strand is the template ● Taq polymerase was the first heat-stable DNA polymerase to be used in PCR; isolated
strand of the double DNA strand from bacteria found in hot springs which has evolved to withstand hot environments
● In eukaryotes, proteins known as transcription factors are also required to bind to the ● It is not destroyed by the denaturation step, so it does not have to be replaced during each
promoter region or to RNA polymerase before transcription can begin cycle.
● Its high optimum temperature means that the temperature for the elongation step does not
E.g. Insulin production → insulin gene inserted next to gene that codes for enzyme have to be dropped below that of the annealing process, so efficiency is maximised.
β-galactosidase, promoter switches on the gene when the bacteria needs to metabolise
lactose (in lactose medium → no glucose, high lactose) 9 describe and explain how gel electrophoresis is used to separate DNA fragments of
different lengths
6 explain how gene expression may be confirmed by the use of marker genes coding for Gel electrophoresis: The separation of charged molecules (e.g. DNA) by differential
fluorescent products movement through a gel in an electric field; the degree of movement is dependent on the
mass of the fragments of DNA
(No more antibiotic resistant genes due to creating antibiotic resistant bacteria.
enzymes from jellyfish make protein GFP (green-fluorescent protein) --> fluoresces bright Steps:
green in UV light ● A mixture of molecules is placed into wells cut into agarose gel and an electric field is
Gene for enzyme inserted into plasmids --> shine UV light onto bacteria to identify those that applied
have taken up the plasmid [Link] prepared: dissolving powder in hot water. While still fluid, poured into an
electrophoresis tank with 'comb' placed at one end. When solidified, comb removed, leaving
wells --> where samples loaded
7 explain that gene editing is a form of genetic engineering involving the insertion, deletion or 2. When gel set, buffer solution poured into tank, covering gel --> gives constant pH
replacement of DNA at specific sites in the genome 3. Micropipette usage → transfer samples of DNA to wells. DNA samples contain tracking
Gene editing: a form of genetic engineering in which specific sites in the genome of an dye --> show how far it travelled across gel
organism can be changed by deleting, inserting or replacing a length of DNA using a method [Link] sample with DNA fragments of known lengths placed into well on one/both
such as the Crispr/Cas9 system sides --> DNA 'ladder' used to determine lengths of fragments of DNA in samples

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5. battery pack connected to electrodes, negative electrode at same end as wells with DNA Microarrays are used in two ways:
6. When dye has moved across most of the gel, battery must be disconnected To analyse the presence or absence of genes in different genomes
7. Buffer solution poured away and suitable stain added to gel. Stain rinsed away to reveal ● DNA is collected from each species and cut up into fragments and denatured to give
bands across gel --> positions of DNA fragments. OR Probes labelled with fluorescent stain single-stranded DNA
--> only visible when gels are viewed in UV light. Lengths of DNA fragments determined by ● The DNA is labelled with fluorescent tags so that e.g DNA from one species is labelled
comparison with DNA ladder at side of gel with green tags and DNA from other species labelled with red tags.
● Hybridisation - labelled cDNA are applied to the probes of the chip, tagged cDNA will only
● The movement of charged molecules within the gel depends on: bind with its complementary probe (rest washed off)
- Net charge: highly charged molecules move faster (negative to anode (+) and positive to DNA hybridisation: binding together of two molecules of single-stranded DNA by
cathode (-) complementary base pairing
- Size: smaller molecules move through the gel faster Inspection under UV light causes tags to fluorescence → thus we know hybridisation has
- Composition of the gel: common gels are polyacrylamide gel for proteins (small fragments occurred (DNA fragments complementary probes)
of DNA that differ as little as one nucleotide in length) and agarose gel for DNA (fragments ● Where DNA from both species hybridise with a probe, a yellow colour is seen; the two
between 100 and 50000 base pairs in length); the size of the ‘pores’ within the gel species share the same gene
determines the speed of proteins/DNA fragments
Detect mRNA from cells to find the genes being expressed at any one time
Electrophoresis of DNA ● The mRNA from the two types of cell is collected and reverse transcriptase is used to
DNA fragments carry a small negative charge due to phosphate groups convert mRNA to cDNA (if cDNA too small use PCR)
Therefore in DNA electrophoresis, these fragments move through the gel towards the anode ● cDNA is labelled with fluorescent tags, denatured to give single-stranded DNA and allowed
(+) , the smaller the fragments, the faster they move (thus the further it travels through the to hybridise with probes on the microarray.
gel) ● Spots on the microarray that fluoresce indicate the genes that were being transcribed in
the cell.
Use of genetic profiling (fingerprinting) in forensic science: ● A high intensity of light indicates that many mRNA molecules were present in the sample,
● A region of DNA that is known to vary between different people is chosen. These regions while a low intensity indicates that there were very few (Results therefore show which genes
often contain variable numbers of repeated DNA sequences and are known as variable are active but also their level of activity → from intensity of light emitted)
number tandem repeats (VNTRs)
● DNA is cut close to the VNTR regions by restriction enzymes. 11 outline the benefits of using databases that provide information about nucleotide
When current turned off, DNA fragments separated but not visible sequences of genes and genomes, and amino acid sequences of proteins and protein
To make visible -> transferred onto absorbent paper → which is placed on top of the gel. structures
Paper heated just enough to make two stands in each DNA molecule separate from one Bioinformatics: Collection, processing and analysis of biological information and data using
another computer software
● Short sequences of single-stranded DNA called probes are added; they have base Associated with databases are tools for selection and retrieval of information.
sequences complementary to the VNTR regions. ● Search tool: BLAST algorithm (basic local alignment search tool) - compare primary
● The probes also contain a radioactive phosphorus isotope that allow DNA to be seen biological sequence information - to find similarities between sequence being studied
(when placed onto X-ray film → radiation emitted make film go dark) against those in databases
● Comparisons can be made with other known genomes and similarities calculated by
10 outline how microarrays are used in the analysis of genomes and in detecting mRNA in using DNA sequencing or primary structure of proteins
studies of gene expression - Close similarities indicate recent common ancestry
● Reading the gene sequences in plasmodium is providing valuable information in the
DNA is composed of both coding and non-coding sequences whereas cDNA only contains development of vaccines for malaria
the coding sequences - Finding new methods of controlling the parasite
● Identification of genes searching for start and stop codons and also by comparing
A microarray (DNA chip) contains thousands of DNA probes sequences against sequences of bases in mRNA from same organism
● DNA probes: single-stranded DNA used to detect the presence of complementary nucleic ● provide information that people beyond the research community can use e.g. info on
acid sequences by having a complementary base sequence. all 412 variants of the CFTR gene
● Each spot on it has many copies of single stranded DNA probes all complementary to one ● Human genes may be found in other organisms e.g Drosophila, making it a useful
gene (ie each spot is for one gene) model for investigating the way in which such genes have their effect

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● The sequencing of genomes in many organisms are known and stored in databases
e.g humans, fruit fly, parasite plasmodium carried by anopheles - Can be compared
to the human genome

19.2 Genetic technology applied to medicine

Learning outcomes
Candidates should be able to:
1 explain the advantages of using recombinant human proteins to treat disease, using the
examples insulin, factor VIII and adenosine deaminase
Advantages of producing human proteins by GMO bacteria, yeasts and cultures of
mammalian cells
● Cells have simple nutritional requirements
● Large volumes of products produced
● Production facilities don’t require much space
● Few practical and ethical problems - no need to extract from animals or collecting blood,
less spread of disease

The disadvantage of using bacteria is that they do not modify their protein in the same way
that eukaryotes do, therefore it is better to use eukaryotic cells.

Examples:
● GM hamster cells used to make factor VIII protein which is essential for blood clotting (lack
of factor VIII causes haemophilia)
- Human gene for VIII protein inserted into hamster kidney cells then cultured in fermenters
which constantly produces factor VIII
- Used to come from donated blood which carried risks of infection
● High yields of enzyme adenosine deaminase (ADA), used to treat severe combined
immunodeficiency disease (SCID), are made by genetically modified insect larva of cabbage
looper moth caterpillar
- The enzyme is administered to patients while they are waiting for gene therapy or when
gene therapy is not possible

Insulin production
Before insulin from GM bacteria became available, people with diabetes were treated with
insulin extracted from the pancreases of pigs or cattle. Now it is produced by GM bacteria.
● There is now a reliable supply available to meet the increasing demand
● Supplies are not dependent on factors such as availability through the meat trade 2 outline the advantages of genetic screening, using the examples of breast cancer (BRCA1
and BRCA2), Huntington’s disease and cystic fibrosis
To insert the human insulin gene into bacteria: Genetic screening is the analysis of a person’s DNA to check for their presence of any faulty
● mRNA for insulin was extracted from pancreatic β cells, the only cells to express this gene alleles that can cause disease
● The mRNA was incubated with reverse transcriptase to make single stranded cDNA ● Done at different stages: adult, fetus/embryo in uterus, embryo made by in vitro fertilisation
● Single-stranded DNA was then converted to double-stranded DNA using DNA polymerase
● The insulin genes were inserted into plasmids to transform the bacterium Escherichia coli An adult woman (usually with a family history of breast cancer) may be screened for faulty
alleles of genes Brca-1 and Brca-2, which increases the chance of developing breast cancer.
If positive woman may have frequent tests for first signs of cancer of decide to have
treatment with drugs such as Tamoxifen -> blocking the action of oestrogen on breast tissue.
OR have breasts removed before any cancer appears

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If a person from a family with Huntington's disease begins to develop symptoms of the Genetic screening
disease --> advised with a genetic test to confirm diagnosis. People who are free of Social benefits:
symptoms (e.g. family members offered test) of a disease, the test is called genetic People at risk of developing disease (e.g. breast cancer) can be identified before developing
screening. (but test is same). Involving PCR to amplify gene HTT and electrophoresis to any symptoms and offered preventative treatments/advice with lifestyle changes to reduce
determine lengths of two alleles and count CAG repeats. Genetic counsellors may help --> risk of developing condition.
make a decision on how to act on this information Economic benefits: Saved money on health services from long-term treatments
Economic drawback: resources spent on providing expensive genetic technology for the rich
If a couple finds that they are both heterozygous for faulty alleles of CFTR, then a genetic could be spent on providing basic health care for poor
counsellor may suggest that they undergo IVF (In vitro fertilisation) treatment and have an Ethical issues: principles that determine people's behaviour and decisions they make over
embryo biopsy carried out on the embryos so that the implanted embryos are those that will personal/professional lives. E.g. Genetic screening for Huntington’s
develop into people free of CF → late-onset disease (symptoms do not usually appear until middle age → usually already
had children → no cure for disease (treatments only alleviate symptoms) → ethical dilemma
3 outline how genetic diseases can be treated with gene therapy, using the examples severe → people in families with HTT should have genetic test to find out whether they have
combined immunodeficiency (SCID) and inherited eye diseases dominant allele for disease? Either told that you are at high risk of developing disease (even
Gene therapy: treatment of a genetic disorder by inserting genetically corrected cells into the though nothing can be done) or live with uncertainty of not knowing. Start family? Even more
body or introducing functioning genes directly into affected cells difficult decisions: possibility that a person with the dominant allele for Huntington’s may live
Common vectors used to carry normal alleles into host cells are: viruses, liposomes, or their whole life completely free of the disorder as it sometimes does not develop
inserting DNA directly into cells (naked DNA)
Parents having a child to find a tissue match for an elder sibling
Severe combined immunodeficiency (SCID) ● In 2004, UK, baby produced that was a tissue match with an elder sibling → purpose was
● Inability to make enzyme adenosine deaminase (ADA), vital for functioning of immune to use cells from umbilical cord as transplant into the sick elder child (Child purpose in life?)
system
● T lymphocytes were removed and introduced with normal alleles of ADA gene using a PGD & prenatal testing abused? (IVF procedure: mixture of father’s sperm with mother’s
virus as a vector, then the cells were replaced. eggs (oocytes) in a dish + at eight cell stage, one cell from embryo removed where DNA
● Not a permanent cure, regular transfusions necessary to keep the immune system analysed) → terminate pregnancies if not sex they want or use PGD to select sex
functioning (unbalance sex ratio)
● Later, gene therapy using stem cells harvested from bone marrow was successful (DNA
with correct base sequence of ADA inserted into adeno-associated virus, which then inserts Gene therapy:
normal allele of ADA into stem cells → the stem cells with normal allele then are injected into Dangerous side effects: immune system stimulated by viral vectors or protein that is coded
blood which divide and differentiate to T-lymphocytes that have functioning ADA), but use of by gene inserted.
retrovirus as vector caused leukaemia due to random insertion of genes into the host -Huge cost of money - very few success or just medical trials. Limited benefits to society →
genome only minority have benefitted
Gene therapy using stem cells - Ethical: Changing genes ethical? Eugenics → Nazism?
Risk of cancer - retrovirus insert the gene randomly IVF - Germ cell therapy through inserting ‘normal’ alleles into the harvested oocyte through
IVF is illegal in humans
Leber congenital amaurosis (hereditary eye disease)
● Disease where retinal cells die off gradually from an early age 19.3 Genetically modified organisms in agriculture
Allele inserted into cells of people who are homozygous recessive for gene RPE65 Learning outcomes
Eye - good organ to develop gene therapy Candidates should be able to:
Small & easy target 1 explain that genetic engineering may help to solve the global demand for food by
Genes can be delivered precisely to retina. improving the quality and productivity of farmed animals and crop plants, using the examples
Can be monitored of GM salmon, herbicide resistance in soybean and insect resistance in cotton
Little activity of immune cells in the eye. Herbicide resistant crops:
The herbicide glyphosate inhibits an enzyme involved in synthesis of three amino acids:
Genome of adenoviruses: cannot cause infections, has correct allele added and promoter. phenylalanine, tyrosine and tryptophan → amino acids necessary to produce essential
proteins for growth. Glyphosate absorbed by plant’s leaves and then transported to growing
4 discuss the social and ethical considerations of using genetic screening and gene therapy tips. Without production of protein in growth areas, plant dies.
in medicine

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Resistance: Version of enzyme (that synthesises these three amino acids) that are not
affected by glyphosate → came from Agrobacterium

Concerns:
GM plants become weed if it is grown on other fields of crops.
Cross pollination with wild relatives - invasive hybrid weeds
Herbicide resistant weeds are selected/evolve

Insect resistance:
Cotton protected against pests such as boll worm.
Bt toxin is lethal to insects but harmless to other animals (activated in gut of insect but not in
acidic conditions of human gut). Taken from Bacillus thuringiensis, different strains produce
different toxins used against different insect species.

Disadvantages:
Risk of insect resistance - accelerate evolution
Evolution of resistance in pest insects
Damaging effect on other species of insect
Transfer of the gene to other species of plant.

Benefits:
Less insecticide use

Genetically modified animals:

GM Atlantic salmon: growth-hormone regulating gene from a Pacific Chinook salmon and a
promoter from ocean pout injected into the fertilised egg of the salmon
● Now, salmon are able to grow all year by producing growth hormone throughout the year
- Reaches market size in 18 months compared to 3 years
● Goes to the same size as a non-GM salmon but attains the maximum size more quickly
Characteristics of GM salmon reduce ability to compete with wild salmon in natural
environment, therefore safe and has minimum effect on environment.

2 discuss the ethical and social implications of using genetically modified organisms (GMOs)
in food production
Advantages:
Higher yields
Less clearing of more land
Avoid heavy crop loss - insect resistant /herbicide resistant crop

Disadvantages
Modified crop - can become weed/invasive species
Introduced gene to wild relatives - more invasive
Introduced gene transferred by pollen to organic crops
Direct hazard to humans/animals: toxic/allergies
Herbicide can leave residue on GM foods
Growers need to buy seed every year.
Danger of losing traditional variety.

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