Gentetic engineering Flashcards
1
Q
Define genetic engineering/ DNA technology
A
Aims to remove a desired gene and transfer it to another organism where it can be expressed. This means that the required protein can be synthesised within the new organism.
2
Q
Why do we use DNA technology/ genetic engineering?
A
Sometimes humans cant make certain proteins because of faulty genes like insulin so this technique allows for the mass production of a protein to treat medical conditions. It allows us to study gene function Allows us to treat genetic conditions/ diseases via gene therapy
3
Q
How is it possible that a gene from ONE organism can be transferred into another completely separate species and expressed in that organism (GMO
A
Genetic code in all organisms is universal The process of making proteins from the genetic code is universal- mechanisms of transcription and translation is the same in ALL organisms
4
Q
What are the 2 main ways of genetic cloning
A
- In vivo - cloning is done using a living organism – 5 stages (to be discussed) OR 2. In vitro – cloning does not involve a living organism – in another lesson
5
Q
What are the outcomes of gene cloning
A
-rDNA (recombinant DNA) -if it involves a living organism: GMO (genetically modified organism) This expresses the desired gene and will produce the protein
6
Q
What are the 6 stages of gene cloning
A
- Isolation of the DNA that contains the gene – 3 ways 2. Amplification – this step not taught at AQA 3. Insertion – inserting gene into plasmid (vector) 4. Transformation – inserting vector into a suitable host (bacteria) 5. Identification – seeing which bacteria express the gene of interest 6. Growth/cloning – mass production as the bacterial (host cells) reproduce
7
Q
STEP 1: ISOLATING THE GENE what are the 3 ways you can do this
A
- Using reverse transcriptase 2. Using restriction endonucleases (enzymes) 3. Creating the gene in a gene machine – based on KNOWN protein structures
8
Q
what are retroviruses?
A
A group of viruses e.g. HIV The coded genetic info in retroviruses is in the form of RNA
9
Q
Isolating target gene: method 1: Reverse Transcriptase DRAW A DIARGRAM/ DESCRIBE THIS PROCESS
A
Use a cell that express large amounts of your desired protein e.g. insulin (β cells of the pancreas) mRNA will be present which codes for insulin e.g. UGCUUA Use an enzyme called reverse transcriptase (from a retrovirus) to converts mRNA to cDNA (complementary DNA). mRNA acts as a template to build cDNA - ACGAAT Make a double stranded molecule by using DNA polymerase
10
Q
ISOLATING TARGET GENE STEP 1, METHOD 2: How do bacteria use restriction endonucleases
A
Bacteria are frequently infected with viruses – that inject their DNA into them. Bacteria then use restriction endonucleases to chop of the viral DNA and makes it non-functional.
11
Q
What makes each restriction endonuclease unique?
A
They all recognise a different recognition sequence so need different shaped active sites
12
Q
Method 2: Restriction enzymes/endonucleases Describe this process
A
Used to cut the gene out directly They each cut DNA at specific sites which is 4-6 bases long– called a recognition sequence They can cut straight or staggered ends Straight – cuts occur between 2 opposite base pairs, leaving blunt edges Staggered – cuts occur that are not opposite base pairs – sticky end
13
Q
What are restriction sites
A
The sites recognised by the endonucleases are called restriction sites and these are usually in the form of palindromes (reads the same both ways, like “Hannah” or “Madam“ - symmetrical)
14
Q
STEP 1, METHOD 2: Restriction endonucleases whats the difference between blunt and sticky ends
A
Enzymes may cut at the SAME place in BOTH strands creating blunt ends e.g HpaI or at DIFFERENT places in the 2 strands leaving so-called sticky ends (staggered unpaired bases) as in EcoR1 or HindIII. Sticky ends important for cloning
15
Q
STEP 1, METHOD 2: Restriction endonucleases What are sticky ends
A
Have exposed bases that will form hydrogen bonds with complementary sticky ends from other DNA molecules cut with the SAME enzyme
16
Q
STEP 1, METHOD 2: Restriction endonucleases What are blunt ends
A
Some restriction enzymes, such as Hpal, cut the DNA strands at positions directly opposite one another, giving blunt ends to the fragments
17
Q
Method 3: The Gene Machine Describe the process
A
- Determine the desired protein’s amino acid seq therefore mRNA seq, to determine complimentary DNA triplets.
- Feed desired DNA seq to computer to check for biosafety,biosecurity and so it meets teh standards and ethical requirements
- Computer designs oligonucleotides to be assembled into a gene
- Gene replicated with PCR to make sticky ends
- Check for sticky ends
18
Q
what are advantages to gene machine?
A
Any gene can be made in a short amount of time- about 10 days Very accurate Artificial genes are free of introns and other non-coding DNA and so can be transcribed and translated by prokaryotic cells
19
Q
IN VIVO Transformation: Describe the process
A
Complementary sticky ends
created by cutting the DNA
from each species with the
same restriction enzyme
Complementary bases on the sticky ends of the DNA from the different species are attracted to one another
Hydrogen bonds
form between the
bases and the
enzyme DNA ligase
seals the sugar-phosphate backbone
of the DNA molecule
20
Q
whats the next main step in recombinant DNA technology, after forming DNA fragments and what ways can you do this?
A
You need to amplify the DNA fragments so that there are more.
- In vitro: PCR (polymerase chain reaction)
- In Vivo
21
Q
Describe in vivo- transformation
A
1) DNA cut using restriction endonucleases to create fragments with sticky ends
2) A promoter and terminator region are added to allow transcription
3) The same restriction endonuclease is used to cut open the plasmid (DNA loop in bacteria, which adds as a vector) to allow complem., sticky ends
4) The enzyme DNA ligase used to incorporate the DNA fragments into the plasmid-recombinant DNA is formed
5) Recomb. plasmid transferred into bacteria via heat shock or calcium ions to increase membrane permeability
22
Q
what are the problems with in vivo- transformation and how do solve these
A
- not all plasmids take up the foreign DNA fragments
- not all recombinant plasmids will be taken up by the bacterial cells
-Can identify which bacterial cells have taken up the recombinant plasmids through marker genes
23
Q
how are marker genes involved in e.g. identifying if a plasmid is taken up
ANTIBIOTIC RESISTANCE MARKER GENES
A
- plasmids contain antibiotic resistance genes
- bacteria are grown on agar plates containing antibiotics
- if bacteria have taken up the plasmid they’ll survive as they will contain anibiotic resistance genes
- bacteria will die if they haven’t taken up the plasmid as they don’t contain antibiotic resistant genes
24
Q
how can marker genes be used in e.g. identifying if the plasmids are recombinant
FLUORESCENCE
A
- DNA fragments inserted in the middle of a marker gene e.g. GFP gene which encodes fluorescence
- GFP will no longer be made so the plasmids that don’t fluoresce are recombinant and those that do are non. so haven’t taken up the foreign DNA frag.
25
what is gene therapy and describe the process
mechnanism by which genetic disorders can be cured or treated by masking the effect of the faulty allele with the insertion of the functional allele.
1) Healthy allele isolated and inserted into cells using vectors
2) If mutant allele is recessive , a dominant allele is inserted and if the mutated allele is dom, DNA is inserted into middle to silence it
26
what are the 2 main types of gene therapy
somatic and germline
somatic= alleles in body cells are altered and isn't passed on to offspring
germline= alleles in sex cells are altered and are passed on to offspring but is considered unethical due to concern over designer babies or safety of gene therapy
27
what is genetic fingerprinting
- method used to produce a specific pattern of DNA bands from an individual's genome
- they use VNTRs
28
what are VNTRs
- variable number tandems repeats
- short, repeating sequences of DNA in non-coding regions
- in every individual, they vary in length and in the number of repeats so the probability of 2 individuals having the same VNTRs is v low
29
Describe the process of DNA fingerprinting
) extraction of the DNA of interest and amplification of PCR
2) digestion using the specific restriction endonucleases into DNA fragments
3) separation of DNA fragments by gel electrophoresis
- DNA fragments move towards the negative electrode
- smaller fragments move further down the gel
- different sized fragments are separated into bands
4) VNTRs are hybridised at specific complemen. base seq. with DNA probes
5) Gel develops- pattern of bands can be visualised by putting gel on xray film as probes emit radiation which reveals the positions
30
how can we use bands from gel electrophoresis for DNA profiling
- can determine genetic relationships by looking at how similar the banding patterns are
- can use in forensics for comparing DNA profile of suspect with those found at the crime scene
- can use in medical diagnosis- identify individuals at risk of developing particular diseases e.g Huntingtons
- Can use in plant/animal breeding- used to prevent inbreeding by not breeding those with similar patterns
31
what are DNA probes and why are they used
- DNA probes are short sections of DNA that are complementary to a known DNA seq. e.g. mutated allele
- labelled with a fluorescent or radioactive tag
- mainly used for genetic screening: study of an individual's DNA to identify whether they possess a mutated allele that causes a particular disease
32
how do DNA probes work
- the labelled DNA probe is mixed with denatured DNA samples from an individual
- if the individual has the mutant allele, the probe will bind to the complementary base seq. in one DNA strand- hybridisation
- the hybridised DNA is detected using the radiation and fluorescence
33
describe what DNA hybridisation is
- DNA seq. from different species that have complem. base pairs are mixed together and therefore hybridise
- the more closely related the species are, the higher the temperature is takes to break hydrogen bonds between the 2 strands
34
how can DNA probes be used
- genetic screening to see if an individual is the carrier of a recessive mutation or to evaluate their risk of developing diseases such as cancer
- allows for personalised medicine-medicines that are specifically tailored to an individual's genotype
- genetic counselling to provide info and support about the results of the screening and how you can lower your risk of getting a particular disease
35
What does PCR do?
Amplifies tiny quantities od DNA for scientific analysis and has revolutionised medicine etc
It copies a DNA template
2n( n= number of cycles)
It used DNA polymerase (TAQ) which comes from bacteria in hot springs which is good coz PCR uses high temp.
36
-What things are needed for PCR?
DNA fragment to be copied, all 4 nucleotides, DNA polymerase to join nucleotides by forming phosphodiester bonds between adjacent nucleotides, and finally primers.
Primers are just short sequences of nucleotides which are complimentary to the ends od the DNA fragment you want copied
Thermocylcer (automated machine).
37
Why does PCR need primers
-Primers are responsible for preventing DNA re-annealing, acting as the starting point for DNA polymerase to bind and marking sections of DNA toe eb copied.
38
Describe the process of PCR
1) Denaturing (separating – making double stranded DNA single stranded) 95 degrees. Hydrogen bonds broken.
2) Mixture is cooled to 55 degree.Involves getting primers to anneal (bind) to the their complementary bases at the END of the denatured DNA fragment (binds at 3’ end of DNA). The primers provide the starting sequence for the DNA polymerase to copy the DNA. Primers STOP the denatured stand rejoining.
3) Raise the temperature to 72 degrees. Optimum for DNA polymerase. This is the extension cycle where DNA polymerase adds the complementary nucleotides and copies the 2 strands of DNA (extension). Sugar phosphate formed. Continues until reaches end of the chain. So rapid replication
39
What is PCR used in?
Gene cloning, paternity testing, virus testing, forensics and research, genetic fingerprinting
40
Advantages to In vivo cloning
- Useful when you want to introduce a gene into another organism.
- No risk of contamination because the gene cut with the same restriction endonuclease can match the sticky ends of the opened up plasmid and no contaminant DNA is taken up. In vitro needs a pure sample :(
- very accurate
- cuts out specific genes
- Produces transformed bacteria that can be used to produce large quantities of gene products so good for medical use like insulin.
41
Advantages to In vivo cloning AND in vitro cloning
- VERY RAPID so is really good for crime investigations. This can be quickly increased PCR so that there is less time wasted. BUT it also increases the chances of contamination of DNA at the scene.
- Does not require living cells so no complex culturing techniques, time or effort is needed.
42
How can you calculate the number of DNA fragments prod by a given number of cycles
2(X)
X= number of cycles
43
1. What is gene therapy and how does it work
* Involves altering (by replacing) the ‘faulty’ genes inside a persons cells to treat a genetic disorder/cancer. The ‘healthy’ gene is expressed!
* If disease is caused by a recessive allele – add a ‘functional’ dominant to mask the recessive
* If caused by a dominant allele – add DNA into the gene to silence the allele
*
44
How are genes delivered to target cells?
* Gene therapy involves adding DNA to a persons DNA – just like rDNA to add the additional piece of DNA you need a vector
* This can be via viruses (infect and target specific cells), plasmids or liposomes (molecules of fat that can easily cross the phospholipid bilayer of cells)
45
46
How could it help a person with a genetic condition e.g. CF?
-Cystic fibrosis is a genetic disorder that affects the respiratory system so you’d use somatic therapy in order to target the epithelial cells in the lungs (it can’t spread around the body).
47
* . What are the positives and negatives to gene therapy treatment (using rDNA technology)
Pros
* Helps to temporarily cure disease
* Better quality of life
* Prolongs life
Cons
* Not permanent solution – short lived
* Will need frequent/multiple treatments
* Virus could cause disease
* Expensive
* Unknown long-term effects – impact on other genes
48
What causes cystic fibrosis?
A deletion mutation where 3 nucleotides are deleted so 1 amino acid is missing therefore the CFTR gene can’t bind to chloride ions and transport them across the epithelial membrane.
49
How do membranes in ppl with CF differ to ppl without?
* -The membranes of non-affected people are moist.
* In people with CF the protein is not made or does not function properly. The membranes are dry and the mucus is sticky and viscous.
* Water is retained in cells
50
* Are there any ethical considerations to gene therapy?
* Worries about the procedure being used in ways other than for medical treatment e.g. cosmetic effects of aging
* Worries there is more harm than good done e.g. producing too much of the missing protein
51
Give examples of genetically modified micro-organisms
1. Production of insulin treat diabetes (medicinal)
2. Bacteria can produce rennin (an enzyme) – used in cheese making. Means cheese can be made cheaply and in large quantities without killing cows. This cheese is suitable for vegetarians (industry – food)
3. Microorganisms that produce antibiotics, hormones and enzymes (medicinal)
4. Micro-organisms designed to control pollution
52
Give examples of genetically modified plants
1. Golden rice – modified to produce beta-carotene – which helps in production of Vit A. Used in areas to reduce Vit A deficiency. (Asia, Africa) – prevent blindness (agricultural)
2. Crops manipulated to be resistant to pests, droughts – this means less money spent of pesticides, thus reducing environmental effects too. Crops can grow is area where water is in short supply (agricultural)
53
Give examples of genetically modified animals
1Goat which produce spider silk – the silk can be used for parachutes, artificial ligaments (strong) – industry
2. Faster growing salmon and salmon that can grow all year round regardless of the temperature of the water (industry)
3. Cows that produce less methane
4. Featherless chickens (industry) – less time plucking! Saving time.
5. Drug production, antibiotics, hormones and enzymes
6. Gene therapy – replacing defective genes in humans, (medicinal)
54
What are some negatives of genetic engineering?
1. If farmers only grow ONE type of plant – whole crop is vulnerable to disease - concerns with reducing biodiversity if plants all genetically identical
2. Concerns over superweeds - if transformed cross breed with wild plants – leading to the spread of rDNA with unknown consequences (unknown ecological consequence releasing GMO organisms into the environment)
3. GM bacteria could pass antibiotic resistance genes to other bacteria
4. IS the money spent on rDNA technology justified? Should it not be spent on other things e.g poverty? Hunger?
5. Is it right to modify genes of an organism? Should allow nature take its course naturally
55
-SUMMARISE HOW GENETIC FINGERPRINTING IS CARRIED OUT
* 1, DNA extracted from sample;
* 2 DNA cut / hydrolysed into segments using restriction endonucleases;
3 must leave minisatellites / required core sequences intact;
* 4 DNA fragments separated using _electrophoresis_; separates fragments based on size
5 detail of process e.g. mixture put into wells on gel _and_ electric current passed through;
* 6 immerse gel in alkaline solution / two strands of DNA separated;
* 7 Southern blotting / cover with nylon filter / absorbent paper (to absorb DNA);
8 DNA fixed to nylon / membrane using uv light
* 9 radioactive marker / probe added / complementary to VNTR;
* 10 (areas with probe) identified using X-ray film / autoradiography;
* 11 pattern unique to each individual - - based on VNTR
56
-What are the uses for genetic fingerprinting?
* Genetic relationships and variability (paternity testing)
* Forensic Science
* Medical diagnosis
* Plant and animal breeding- i.e prevent inbreeding, for predigrees
57
-Describe the process of interpreting DNA fingerprinting results
NA fingerprints from 2 samples are usually **visually checked**
e.g. blood at a crime scene and suspect
If they appear a match the DNA fragments produced on the GF are individually passed through a machine to calculate the length exactly of each fragment – to see how close a match they are really.
58
5. Dsecribe development
5.Development
X ray film is placed over the nylon membrane and left for a few hours.
The film is exposed to the radiation from the probe. A unique series of bars are revealed.
59
Desrcibe 4. Hybridisation
A radioactive (or fluorescent) DNA probe is used to bind to the VNTR
The DNA probe is single stranded and complementary to the VNTR.
More than one probe can be used to bind to other core sequences (repeat sequences) (although only one probe can be done at one time. XS probe is washed off before the next applied)
Then you flood the nylon membrane with more copies of probe. Probe DNA will attach to complementary sections of profile DNA.
You can also use other types of probe which would clearly highlight more DNA fragments
60
-3.5 SOUTHERN BLOTTING TECHNIQUE TO TRANSFER DNA ONTO A NYLON MEMBRANE
Why?
Why?
Because the bands are invisible and nylon is durable.
1. Add a probe which will only bind to single stranded DNA. Then you place gel in alkaline solution to separate the double DNA strands
2. A thin nylon membrane is laid over the gel
3. Membrane is then covered with several sheets of absorbent paper.
4. This acts to draw up the DNA onto the membrane by capillary action which transfers the DNA fragments to the nylon membrane in precisely the same position that they were on the gel.
5. Then U.V. light ‘fixes’ DNA fragments to the membrane
61
Describe 3. Separation via electrophoresis
The DNA fragments are separated according to their length (size) by **gel electrophoresis** using electrical voltage.
At pH 7 the fragments are negatively charged. DNA is negatively charged and so moves to the positive electrode.
When an electric current is applied the fragments migrate to the positive electrode, with smaller fragments travelling further.
3.5 SOUTHERN BLOTTING TECHNIQUE TO TRANSFER DNA ONTO A MEMBRANE
62
Describe 2. DNA digestion
Use **restriction endonucleases** to cut DNA into fragments **just outside** the core sequence leaving the VNTRs intact, producing _various DNA fragments of different sizes_
-Sizes depend on:
how many times the core sequence is present in genome
the number VNTRs in each core sequence
63
Describe 1. DNA extraction of G.F
-Collect tiny sample of tissue containing cells with nuclei and extract the DNA from the rest of the cell. Quantity will likely be small therefore we can increase this by DNA digestion
64
-What is the function of VNTRS?
The number of repeats in a VNTR varies among individuals and so its unlikely that 2 ppl will have the same no. VNTRS in the same place in their genome. So we can use this for comparing ppl at crime scenes via genetic fingerprinting.
65
-What are VNTRS?
VNTRS are sections of DNA found throughout the genome.
Sequences of VNTRS consist of a series of bases that are repeated in the same sequence many times (e.g CCGACCGACCGA) which can vary.
66
-What is genetic fingerprinting?
* AKA Genetic profiling
* Tool used in forensics, paternity testing, medical diagnosis based on the fact that the DNA of all individuals (except identical twins) is unique.
* Every person has a unique banding profile because half of the bands you have in common with your mum and half with your dad.
67
-What are genetic diseases caused by?
A mutation in specific genes. rDNA technology has enabled doctors to diagnose and treat people with genetic conditions.
68
-How might we LOCATE a mutated form of a gene (allele) within the entire human genome to diagnose if an individual has a genetic condition?
In order to diagnose a patient it is necessary to locate the mutant allele– to do this we use DNA probes and a technique called DNA hybridisation
69
-So how are DNA probes used?
1. Sequence of the mutated gene/allele is determined by DNA sequencing OR genetic libraries are used (HGP) – many bases sequences of most genetic diseases are known
2. DNA probe is made/synthesised that is complementary to the base sequence of the allele you are trying to locate.
3. DNA probe made is then labelled
Radio active tag
OR florescence tag
4. PCR is used to make large amounts of the probe So that we have plenty
5. DNA from a patient is treated to separate the DNA into single strands – probe is added
6. DNA is cooled to allowing joining of probe to ONE DNA strand due to CBP
7. If the DNA from the patient contained mutated allele – probe will hybridise – The DNA is then washed to removed unbound probe
8. The DNA fragment that has probe bound will be identifiable due to the label – using X ray film or special UV light and microscope
70
- What is hybridisation?
* This is when a section of DNA (that is single stranded) binds with another single stranded portion of DNA that has complementary base pairs
71
Before DNA hybridisation what must happen to the DNA? How is this achieved?
The DNA must be separated (denatured) into single strands (breaking the hydrogen bonds) – done by heating
72
- Once the DNA strands separate what is likely to happen next when you add the probes?
The probes will bind (hybridise (anneal)) the complementary portions of the DNA molecules or the separated DNA will rejoin with its partner strand
73
What is genetic screening?
* To identify the presence of a mutant allele - usually if a family history
* Probability of having a child with the disease.
* Potential parents can then receive guidance anbt implications of having this kid
74
75
What can you geneticallys creen? Why?
* **OF ADULTS** - Find out if they carry the faulty allele – *could help them decide whether to have children or not.*
* **OF EMBRYOS (IVF)-** Find out if their embryos (‘pre-implantation genetic diagnosis) are affected – *potentially only have ‘healthy’ embryos implanted so can only have a healthy baby*
* ***OF EMBRYOS In Vivo*** *– during the pregnancy (CVS)*
76
Give examples of genetic screening being used
* People with family history of Huntington's can be screened
* Babies are screened at birth for CF – so treatment can begin as soon as possible
* Can help determine how people will respond to drug treatment
* Identify health risks – certain cancers (next slide)
77
-Why are people worried Genetic screening?
* People are worried as it may lead to discrimination with insurance companies and employers if people at a high risk of developing a condition
78
-How is TSG impacted by genetic screening
* Screening is also valuable in screening for cancer causing genes e.g. TSG
* Mutations in TSG – therefore cell division is not slowed down
* Mutations in BOTH TSG alleles must be present to inactivate the gene and initiate development of a tumour
People who inherit a mutation in ONE allele are at a greater risk of cancer
79
What ways can genetic screening be done?
* Single probes – as discussed previously **_OR_**
* Microarray (uses multiple probes) – screen for many genetic mutations at the same time.
* Microarray is a glass plate has hundreds of probes attached in rows– add the DNA sample – it has the ability to detect multiple genetic disorders!
80
What are personalised medicines?
Medicines tailored to an indivdiual’s DNA based on iodea that doctors can predict your response to drugs and prescribe the most effective based on their genetic info.
81
* What are examples of personalised medcines?
Biomarkers, prescribing painkillers, vitamin E
82
Describe genetic counselling
involves advising ppl abt screening and explaining these results.
Identifying if someone is a carrier, the type of mutated allele and the most effective treatments.
