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Gen tech 1

Principles of Genetic Technology 
Recombinant DNA​ is DNA made by joining pieces from two or more different sources
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Multiple copies of the gene are made by ​PCR
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Vector ​transports ​the gene into the cells
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DNA is ​denatured ​to 95C
➢ This separates the DNA molecule into two strands
➢ By breaking the Hydrogen bonds
➢ So the bases are exposed
➢ To produce template strands for complementary pairing
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TAQ POLYMERASE joins nucleotides to primer, extending new strand
➢ Synthesises complementary DNA strands
➢ Taq pol used as it will not denature at high temps - heat stable
➢ Doesn’t have to be replaced after every cycle so process is more efficient (than using
DNA polymerase)
➢ High optimum temp means temp for elongation step does not have to drop below
that of annealing >> max efficiency
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Promoter initiates transcription/causes gene expression
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Otherwise gene has to be inserted near an existing promoter
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In eukaryotes, the precise position of the promoter is important
Genetic Markers
➢ Concern about using antibiotic resistance genes as marker
○ Can be spread to pathogenic bacteria
➢ Enzymes that produce fluorescent substances are used instead

Gen tech 5

○ Advantage: each enzyme molecule produces more fluorescent substance
(only a few molecules of GFP produced so not v bright)
○ Enzyme can be reused
➢ Eg GFP - green fluorescent protein (green in UV light)
○ Easy to identify the bacteria that have successfully taken up the plasmid as
the gene for the enzyme is inserted into the plasmid
➢ Eg enzyme 𝛽-glucuronidase
○ Cells containing this enzyme will transform into coloured products when
incubated with specific colourless substrates
Use of fluorescent/easily stained substances as markers
● Emits bright light when exposed to UV light
● Visible colour change
● Add marker gene to vector/plasmid
● Easy to identify transformed bacteria
● Gene of interest inserted close to marker gene
● Easy to identify cDNA
● Easy to identify transgenic organisms
● Eg GFP/beta galactosidase
● No known risk (eg compared to antibiotic resistance genes)
Why fluorescent markers are preferred over resistance genes:
● Easier to identify/screen
● More economical/time saving
● Resistance genes can be pass on to other bacteria
● Antibiotics no longer effective against these bacteria/development of new
antibiotics required
How marker genes (eg GFP) are used to show successful uptake of a gene for wanted
protein:
● Marker gene linked to gene for wanted protein
● With promoter
● GFP gene transcribed/expressed
● Producing GFP which fluoresces under UV light
Confirming presence of gene:
● Shine UV light - gene will fluoresce
● Comparison with reference piece of DNA
● Use of a DNA probe
● Single stranded/complementary base pairing

Gen tech 6

Hybridisation probe: ​fragment of single stranded DNA (or RNA) of variable length which can
be radioactively labelled; then used in DNA/RNA samples to detect the present of
nucleotide sequences (the DNA ​target​) that are complementary to the probe
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Restriction endonucleases
● Cut sugar-phosphate backbone at specific places in the DNA molecule
● Binds to specific target sites and cuts there
● Usually palindromic
● Can ​produce sticky ends
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Ligases
● Catalyses linkage of nucleotides during formation of a DNA molecule
● Links together sugar-phosphate backbone
Microarrays - ​set of DNA sequences representing the entire genome of an organism,
arranged in a grid pattern for use in genetic testing
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Eg gene sequencing + comparing/microarrays/amino acid sequencing/protein structure
Role of bioinformatics
● Particularly useful when an organism’s genome has been sequenced in comparing
different strains
● Close similarities indicate common ancestry
● Eg identifying genes in pathology
● Eg ​plasmodium​ - development of vaccine for malaria
Advantages of producing human proteins by recombinant DNA techniques
eg
● Now relatively easy to identify and extract the genes for different human proteins useful for research and mass production
● These genes can be inserted into bacteria easily eg using plasmids
● Plasmids can be easily modified - eg genes of interest
● Bacteria are easy to grow
○ Easy to provide ideal conditions/large quantities
● No ethical issues with manipulation/growth of bacteria
● Reduces risk of zoonoses (animal transmitted disease) or rejection of the protein
○ Eg extracting insulin from pig pancreas
○ HIV from blood transfusion
● Reliable supply to meet increasing demand
● Disadvantages:

Gen tech 8

○ Bacteria do not modify proteins in the same way eukaryotes do
Advantages of genetic screening for conditions
● Analysis of a person’s DNA to check for the presence of a particular allele
○ Breast cancer genes brca1 and brca2 - double mastectomy
○ Haemophilia, sick cell anaemia, Huntingdon’s disease, CF
● Even if not curable:
○ Treatment of symptoms
○ Improve quality of life
○ Allows for regular checkups eg mammograms
Insulin
Inserting a insulin gene into ​E
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Isolate gene
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Use restriction endonucleases to form complementary sticky ends
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coli
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Use DNA ligase to seal sugar-phosphate backbone
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Screen for and obtain successfully transformed cells
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Difficulties of treating diseases with gene therapy
● Difficult to insert working copy of gene into every cell in the body
● Difficult to get it to function properly when it is there
● Have to choose an appropriate vector (viruses - retroviruses/liposomes/naked DNA)
● Successes:
○ Eyesight of young men with hereditary blindness
What makes a condition good for gene therapy?
● Caused by a single gene
● Caused by a recessive allele
● So delivery of normal/dominant allele could correct condition
● Only need to get allele into a few cells eg eye conditions
● Ease of access to affected area
● Serious so worth the risk

Gen tech 9

● No surgery needed
Eg
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and disadvantages:
● Effects only last a few days
● Low uptake by target cells
● Only targets specific cells (eg lung)
● Side effects eg from virus
Social and ethical considerations for gene testing/therapy:
● Embryo can be chosen if it doesn’t have an allele for a genetic disease/if it has
correct tissue type for a sick sibling - transfer
● Embryo can’t be genetically modified (germ cell therapy)/have an allele added
● Foetus can be tested in utero for genetic disease by amniocentesis or chorionic villus
sampling
Use of PCR/DNA testing in forensic medicine/criminal investigations
● PCR used in forensic science to amplify DNA samples from smallest tissue samples
left at scene of a crime
○ Use gel electrophoresis to analyse DNA

Gen tech 11

○ Region of DNA that varies between people is chosen - contains variable
numbers of repeated DNA sequences (VNTRs)
○ DNA extracted from root of hair/spot of blood/semen/saliva
○ Quantity amplified by PCR
○ DNA cut into fragments using restriction endonucleases known to cut near
VNTR regions
○ Gel electrophoresis
○ Southern blotting
Ethical and social considerations of gene testing embryos for genetic diseases
➢ PROS
○ Can avoid having offspring with serious/genetic disease
○ Can avoid late abortions
○ Allows couples to have children who would otherwise choose not to (due to
risk of genetic disease)
➢ CONS
○ Variable embryo(s) discarded
○ Use of healthcare resources by couple that can conceive naturally
○ May conflict with religious beliefs
○ Could lead to selection based on gender or specific traits
○ Genetic disease may not develop
Advantages of screening for genetic conditions
● Information about person having increased risk of genetic conditions
● Eg breast cancer BRCA gene
● Allows people to prepare for late onset of conditions
● Eg Alzheimer’s, Huntingdon’s
● Identify is foetuses will develop a genetic condition
● Allows early treatment at birth
● Allows parents to prepare for birth of a child who will need long-term/lifelong
treatment
● Identifies carriers of genetic conditions
● Helps to provide early diagnosis
● Allows couples who are both carriers of a genetic condition make decisions about
having children/IVF
● Avoids late abortions
Screening for BRCA allele
● Advantages
○ If present, enables lifestyle change/early treatment/regular check ups
○ If not present, removes worry
○ Preventative treatment may be cheaper than treating disease itself

Gen tech 12

● Disadvantages
○ If present may cause worry
○ Person may not develop cancer if present
○ Test is expensive
○ May have implications for life insurance
○ May decide not to have children OR may be tested after they have children

GMOs in Agriculture
Significance of genetic engineering in improving the quality/yield of crops/livestock - solving
global food demand
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ananatis)​
And daffodils/maize
Inserted into plasmids - plasmids used as vector
Promoters added
Plasmids put into ​Agrobacterium tumefaciens
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and economic:
● Problem with competition between crops and herbicide-resistant weeds
● Contamination of organic crops/accidental mixing of GM and non-GM crops and
financial consequences of this eg compensation
● High cost of GM seeds/plants - poor farmers can’t afford them
● Cost of herbicide

Gen tech 14

● Cost of pollution caused by agriculture
● Cost of human health problems eg allergic reactions/residue from herbicides
Maize: protected against corn borer (Bt maize)
Cotton: protect against boll weevil
Tobacco: tobacco budworm resistant plants exist, but not yet grown commercially; also
resistant to sulfonylurea
Oil seed rape: resistant to herbicide glyphosate - inhibits enzyme involved in synthesis for
amino acids
Benefits of GM foods…
● Nutritional value of foods can be improved
○ Eg introducing proteins/vitamins/vaccines
● Crops can be produced which lack known allergens
● Crops can be grown in arid conditions for better yield
○ Eg by introducing drought resistant genes
○ Less competition as fewer other plants can survive in tough environment
● GM crops can produce herbicides to kill pests
● Improve food supply/agriculture in less developed countries (can be engineered for
better yield)
● GM crops may have longer shelf lives (less spoil)
● Economic costs and carbon footprint can be reduced
○ Less need for land clearing/pesticide use

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