Topic 7 Modern Genetics Flashcards
What is the definition of genome?
All of the genetic information in an organism
What is in vitro PCR used for?
Replicate copies of DNA outside body
Eg DNA profiling, COVID19 testing,
What are the steps of PCR?
- Separate DNA strand - heat to 95C, breaking H bonds exposing bases
- Annealing - cool to 55C, primers added
- Elongation - heat to 72C, DNA polymerase replicates DNA using complementary base pairing
- REPEAT to get more samples
What are the 3 raw materials used in PCR?
- DNA primer
- free DNA nucleotide
- DNA strand
- Taq polymerase
What needs to happen before PCR?
Purify dna (break up cells, filter and suspend)
Why is DNA polymerase from human sources not suitable for PCR use in machine? (2)
- human enzymes can’t work at high temps
- will denature
Why can’t xylem be used in PCR and DNA profiling?(2)
- dead, made of dead material
- so no dna present
What nucleotide do we use for gene sequencing?
Terminator bases or dideoxyribose molecules
They lack -OH on carbon 3
So it stops(terminates) the extension during PCR
How to identify the terminator bases?
Add radioactive markers
What does PCR stand for?
polymerase chain reaction
Describe how a DNA profile can be produced from a small sample of DNA. (6 marks)
- use PCR
- to multiple copies of DNA
- use restriction endonucleases to produce DNA fragments
- gel electrophoresis
- load DNA onto gel (agarose)
- electric current applied
- use florescent tag
Describe how small samples of DNA can be amplified. (4 marks)
- use PCR
- add Taq polymerase
- and primer and free nucleotides
- heat to 95C, cool to 55C, heat to 72C
- repeat several cycles to make several copies
How to DNA sequence?
- PCR steps
- but with terminator nucleotides labelled with fluorescent dye
- DNA polymerase incorporates labelled dideoxynucleotides randomly during replication
- gel electrophoresis
- detect fluorescent dye
What is the human genome?
all the genes of the human species
How are DNA profiles compared? (3 marks)
compare
- total number of bands
- position of bands
- size/width of bands
What are exons and introns?
exons are the gene, coded region/expressed
introns are the non-coded region
What is the role of restriction endonuclease? (1 mark)
cut DNA to produce short sections of DNA
Describe the process of PCR. (3 marks)
- use primers / nucleotides
- heat to 95C to separate strands
- cool to 55C to bind primers
- heat to 72C with Taq polymerase
- repeat to obtain multiple copies
What is proteomics used for?
To study proteins (including enzymes)
How to distinguish between species?
Gel electrophoresis
What are stem cells?
undifferentiated cells with ability to differentiate into many types of cells
What are 3 types of stem cells?
multipotent
totipotent
pluripotent
What does pluripotent mean in terms of stem cells?
can differentiate into many types of specialised cells except placenta
(think p-p)
What does totipotent mean in terms of stem cells?
Can give differentiate into all types of cells
What are the 2 sources of stem cells?
- embryonic
- adult (from bone marrows)
What are some risks/disad of stem cells?
- unethical as embryo killed in the process
- risk of infection during transfer
- may be cancerous
How are iPS cells produced?
- from fibroblasts
- genes for transcription factors are inserted (eg Oct4, Sox2)
- cells regain ability to differentiate
(- encodes transcription factors)
Explain why some cells are not able to become other cell types. (2 marks)
- because they are specilised / differentiated cells
- so genes for other cell types are permanently turned off
- so proteins needed by other cell types not needed
Compare and contrast uses of embryonic stem cells and iPS cells. (5 marks)
Both
- can differentiate indefinitely
- potential to specialise into a number of cell types
ESC / iPSC
- from morula / adult cells
- doesn’t / has gene
- less ethical implications for iPSC
- may be rejected after injection / won’t if own stem cells
What is multipotent in terms of stem cells?
cells that can only become some cell types
Why must stem cells ideally be taken and used by the same person? (2 marks)
- genetically identical
- prevent rejection
- reduce risk of infection
How can stem cells reverse a paralysis in a spinal injury? (2 marks)
- they are totipotent / pluripotent
- so can specialise and differentiate
- to replace damaged cells
How do stem cells differentiate and develop into heart muscle cells? (4 marks)
- stimulus of hormone / chemical
- activation of genes
- only activated genes are transcribed
- mRNA translated
- protein made
- cell permanently modified
How are fertilised eggs used as a source of human pluripotent stem cells? (3 marks)
- fertilised egg allowed to grow for a few days
- in blastocyst
- inner cell mass harvested
- from spare embryos from IVF
What are some uses of stem cells?
- investigate cancer
- test new drugs
- therapy eg gene therapy, treating diseases and accidents
How to therapeutic cloning of stem cells?
- take nucleus of somatic cell (so has genetic info)
- take egg cell and remove nucleus
- combine
- harvest and clone
What are the 4 genes used in iPS cells to reprogram cells?
Oct4
Sox2
kIF4 - cancer causing
cMyc - cancer causing
How to genetically engineer?
- Isolate the gene to transfer
- Insert the gene into the vector
- Use vector to transform the host genome by inserting DNA into it - forming recombinant DNA
What are 2 methods to isolate and cut genes?
- restriction endonucleases
- use mRNA and reverse transcriptase
Why are there specific types of restriction endonuclease?
binds to specific base sequences
where each site is
What do restriction endonucleases do?
- cut DNA at different restriction sites
- leave sticky ends
- complementary
- so is easy to insert
How to use reverse transcriptase to produce DNA fragments?
find mature mRNA
use reverse transcriptase (from retrovirus) to make double stranded complementary DNA
ADs for using reverse transcriptase and mRNA > other methods of gene isolation?
- easier to get complete gene than from DNA
- available in greater quantities
- introns already removed in mRNA, bacteria can’t remove introns so is simpler
What are properties of a good vector?
- targets correct cells
- ensures gene is inserted, so expressed by host
- no significant side effects
plasmids
double stranded
replicates independently
found separately from main DNA
How to get recombinant plasmids into bacteria first (then implant to animal or plant or whatnot)? (1 mark)
by heat shock
What is the bacterial pathogen that infects plants (used to genetically modify plants)? Causing Crown Gall disease
Agrobacterium tumefaciens
need to know
What is the effect of Agrobacterium tumefaciens?
infects root cells by plasmid via pilli
aka Ti plasmid
plant will grow tumour in roots
infects plant cells with transgene
What are the 3 methods to transform animal cells?
- viruses (engineered and injected)
- liposomes
- microinjection (into nucleus of cells)
gene transformation and cystic fibrosis
How to identify a gene is transformed?
florescent marker / transgene / marker gene
or
Replica plating: insert gene to bacteria with antibacterial resistance
grow on agar and compare growth
Describe how soya bean plants can be mass genetically engineered to produce large number of GM soya bean plants. (4 marks)
- use restriction enzyme to cut DNA
- use ligase to insert and join DNA
- use vector
- use Agrobacterium to infect
- cloning to produce large numbers
Describe how a fungus can be modified to make a spider toxin. (3 marks)
- isolate code for spider toxin
- use restriction endonuclease
- gene inserted into fungus using a vector eg gene gun
- clone the genetically modified fungi
What do transgenic plants mean?
genetically modified cells
In DNA profiling, what type of DNA fragments travel furthest?
Smallest fragment
Travels fastest and furthest
Describe evidence, other than gel electrophoresis, a scientist can use to establish evolutionary relationships between species. (4 marks)
- if they can interbreed and produce fertile offspring
- compare anatomy, behavioural and physical characteristics
- molecular phylogeny
- ecological niche, and where they can be found
- DNA sequencing
What are knockout mice and how are they useful?
- mice with one or more genes silenced
- to investigate gene function
-create animal models of disease to allow research - test for treatments
What is the process of producing transgenic plants?
- extract plasmid
-insert gene - infection plan w bacteria
-grow
How to genetically modify soya beans?
What is the benefit of it?
- linoleic acid (polyunsaturated) to replace oleic acid (mono saturated)
- oxidised less easily so prolongs shelf life and healthier
What are advantages of widespread use of genetic modification?
- improve nutritional value
- greater crop yields
- less need for pesticides
What are disads of genetic modification?
- reduced biodiversity
- unknown effects on health
- can result in herbicide-resistant weeds
What is replica plating
- technique to produce multiple near-identical arrangements of organisms on agar plates from a single plate for comparisons
- test for antibiotic resistance marker genes by including antibiotics in new plates
- usually test recombinant DNA - check if incorporated or self-ligated (not taken up)
How to replica plating?
- one master plate
- stamp covered in sterile velvet to imprint
- have agar plats w diff antibiotics
- transfer colonies
- incubate and compare growth
How can plasmids be taken up by bacteria?
Heat shock
