Manipulating genomes Flashcards
what is DNA sequencing
allows for the nucleotide base sequence of an organism’s genetic material to be identified and recorded
methods of DNA sequencing
sanger method
high-throughput sequencing
what is the sanger method also known as
chain termination method
overall what is the sanger method
- Dideoxynucleotides pair with nucleotides on template strand in DNA replication
- When DNA polymerase encounters dideoxynucleotide on developing strand – stops replicating
- Termination method
describe the sanger method
4 test tubes – each with diff type of dideoxynucleotides ( A* / T* / C* / G*)
Test tubes incubated – temp allows enzyme function
96 degrees - break H bonds between DNA
50 - primer anneals
60 - DNA polymerase
Primer anneals to start of single stranded template – short section of double stranded
DNA polymerase attached to double stranded section + begins DNA replication using free nucleotides in test tube
At any time – DNA polymerase inserts on dideoxynucleotide by chance – results in termination of replication
Complementary DNA chains varying lengths made
New complementary DNA separated from template DNA
Resulting single stranded DNA separated according to length via gel electrophoresis
what is a primer
short single-stranded sequence with set of bases complementary to those at the start of the DNA fragment
what needs to be in each test tube for the sanger method
DNA to be sequenced as single-stranded template
DNA polymerase
DNA primers
free nucleotides
one of 4 types of dideoxynucleotides ( A* / T* / C* / G*)
when separated how do you actually know the sequence from sanger method
Each test tube only has 1 type of dideoxynucleotide – so you can know terminal nucleotide of each fragment
unique position on electropheresis gel because of unique mass
how would electropheresis separate these fragments
gel will have four wells, one each for A, C, T, and G
fragment that consists of only one nucleotide will travel all the way to the bottom of the gel, and every band above this on the gel represents the addition of one more base
allows the base sequence to be built up one base at a time
overall high-throughput sequencing
New methods of sequencing DNA that are automated, very rapid and cheaper than orig. methods
uses capillary electropheresis
Capillary gel electrophoresis
Separates macromolecules such as nucleic acids through capillary action in a capillary tube
high resolution
capable of separating chains of DNA that vary by only one nucleotide in length
capillary gel electropheresis method
each type of dideoxynucleotide labelled using fluorescent dye
adenine – green
thymine – red
cytosine – blue
guanine – yellow
laser beam used to illuminate all dideoxynucleotides
detector reads colour + position
feeds into computer
bioinformatics
storage, retrieval, and analysis of data from biological studies
computational biology
using computers to study biology – create simulations / models
important of genome sequencing
allows to make comparisons with genomes of other organisms – human genome project
- find degree of similarity = how closely related they are
- useful for looking at organisms that can be used as a model for humans
- evolutionary relationships
allows us to understand genotype-phenotype relationships
- target specific base sequences to knock out + observe effect
aid research + disease control
- genomes of pathogens can be sequences + analysed
- highly infections strains are identified
- personalised medicine
proteome
full range of proteins produced by genome
why is the proteome difficult to determine
non-coding DNA + regulatory genes + alternative splicing
proteome or genome larger
proteome is larger than the genome due to:
Alternative splicing
Post-translational modification of proteins (often takes place in the Golgi apparatus)
Synthetic biology
aims to create new biological parts, devices, and systems, or to redesign systems that already exist in nature
what does synthetic biology involve
involves large alterations to an organism’s genome
operate in novel way more than genetic engineering
Computational bio
Uses data from bioinformatics to build theoretical models of biological systems which can be used to predict what happens in diff circumstances
How can bioinformatics help determine whether a newly sequenced allele causes genetic disease
Base sequence of normal allele and known alternatives held in database as well as AA sequence
Computational analysis allows rapid comparison of sequences w/ newly sequenced alleles
Can create model of new protein structure
Uses of computational bio
Analysing base pair in DNA
Working out 3D structures of proteins
Understanding molecular pathways e.g. gen reg
Identify genes linked to spp diseases
Benefits of using DNA sequencing in studying epidemiology of infectious disease
Allows you to identify pathogen
Sequence DNA and compare to sim microorganisms
Faster than trad methods e.g.culturing bacteria
Can follow routes of infection
Cn identify carriers
Can help find drugs
Variable number tandem repeats
Short nucleotide sequence that is repeated throughout the genome
the number of this varies at any given locus in the genome
how is the number of VNTR repeats determined
inherited
what would a high similarity of VNTRs indicate
closely related
describe the process of genetic profiling / fingerprinting
Obtain the DNA = extracted from the root of a hair / spot of blood / semen / saliva
Increase the quantity of DNA by using PCR
Use restriction endonucleases to cut the amplified DNA molecules into fragments
Separate the fragments using gel electrophoresis
gel immersed in alkali - separate DNA double strands into single strands
transferred onto a membrane by southern blotting
Add radioactive or fluorescent probes in excess
complementary + bind to specific VNTR regions - hybridisation
X-ray images are produced or UV light is used to produce images of the fluorescent labels glowing.
These images contain patterns of bars (the DNA profile) which are then analysed
how do the restriction endonucleases work in DNA profiling
different restriction endonucleases - cut at different recognition / restriction sites
make two cutes - once through each strand of DNA double helix
hybridisation
radioactive / fluorescent DNA probes added in excess to single stranded DNA fragments on the membrane
bind to complementary strands of DNA
southern blotting
DNA (-ve) from gel electrophoresis is transfereed to a +vely charged membrane e.g. nylon
Fragments are irreversibly bound to the blot, whilst maintaining their relative positions on the gel
DNA probes
Single stranded short piece of DNA with a known complementary sequence to the VNTR
Synthesised chemically and is radio-labelled
how would you see the result of DNA profile if you added radioactive labels to the DNA probes
X ray images of the membrane
how would you see the result of DNA profile if you added flourescent labels to the DNA probes
membrane placed under UV light so fluorescent tags glow
uses of DNA profiling
Tissue typing
= Donor + recipient tissues matches to reduce risk of rejection
Detection of oncogenes
= Can inform medication
Detect mutations
= E.g. – embryo selection
Identify type of viral infection + monitor spread of infectious disease
= PCR covid testing
Identify suspects of crimes // forensic science
= DNA profile of sample compared to samples from suspect / criminal database / victim
= Identify bodies
determine familial relationships for paternity cases
species conservation to help scientists with captive breeding programmes to reduce chances of inbreeding
PCR
polymerase chain reaction
what is PCR used for
in vitro method of DNA amplification
used to produce large quantities of specific fragments of DNA/RNA from very small samples
even just 1 molecule of DNA / RNA
PCR ingredients
small sample of target DNA
2 primers
Taq polymerase
Free nucleotides
Buffer
Thermal cycler
what are the primers complementary to in PCR
Complementary to the 3’ end
why do you need 2 primers for PCR
One for each single strand of the now broken double helix
what is Taq polymerase + where is it found
DNA polymerase
from thermophilic bacteria in hot springs
why do we use taq polymerase
does not denature at high temp
why do we need a buffer in PCR
optimum pH for reactions beaker
why do we need a thermal cycler
automated
provided correct temp for correct time
3 stages of PCR
denaturation
annealing
elongation / extension
describe PCR - denaturation
DNA fragments / primers / DNA polymerase / nucleotides added to thermocycler
heated to 95°C
breaks the hydrogen bonds
forms 2 separate strands with exposed nucleotides
denaturation – separating strands
describe PCR - annealing
temperature is decreased to between 50 - 60°C
primers (forward and reverse ones) can anneal to the ends of the single strands of DNA
describe PCR - elongation
temperature is increased to 72°C for at least a minute
optimum temperature for Taq polymerase to build the complementary strands of DNA
produce the new identical double-stranded DNA molecules
formula for total number of strands formed from PCR
Number of original DNA strands X 2^number of PCR cycles = total number of strands
advantages of PCR
very rapid – millions of copies made in hours
does not require living cells – only base sequence
PCR – needed for the sanger method + DNA sequencing
electropheresis
molecules are separated according to their size/mass and their net charge
why does separation occur in electropheresis
electrical charge
mass / size
type of gel
how does electrical charge aid separation
negatively charged DNA - negatively charged due to the phosphate groups
when placed in an electric field the molecules move towards the anode
how does size aid separation
Different sized molecules move through the gel at different rates
tiny pores in the gel result in smaller molecules moving quickly + larger molecules slowly
how does type of gel aid separation
Different gels have different sized pores
affect the speed at which the molecules can move through them
process of electropheresis for DNA
Create an agarose gel plate in a tank
Wells are cut into the gel at one end
Submerge the gel in buffer solution
Load the fragments into the wells using a micropipette
Apply an electrical current to the tank.
The smaller mass / shorter pieces of DNA fragments will move faster + further from the wells
smallest fragments reach end - turn off electric current
gel placed in alkaline buffer solution
The fragments are not visible
must be transferred onto absorbent paper or membrane via southern blotting
fixed in place by UV / heat
Probes are then added
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
why do we put the final gel in a alkaline buffer solution
denature DNA fragments
two strands separate
why are probes added in electropheresis
allow VNTRs / sequences to be identified
A radioactive label 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 which fluoresces (shines) when exposed to ultraviolet (UV) light, creating a pattern of coloured bands
how are proteins prepared for electropheresis
Denaturing (to break the disulfide bonds)
manipulating the proteins into rod shapes (negatively charged) to allow separation by size
membrane for southern blotting
nylon membrane
sanger method final product
DNA sequence of COMPLEMENTARY STRAND
overall steps of genetic enginnering
isolate the gene
multiply DNA fragment - PCR
formation of recombinant DNA
transferring the vector
what are the two ways to isolate a gene
cut the DNA or isolate mRNA
describe isolating gene - cutting DNA
restriction endonucleases cut 2 DNA fragments unevenly
sticky ends – regions with unpaired / exposed bases
make it easier to insert the desired gene into DNA of diff organism
difference in cuts in DNA profiling + genetic enginnering
blunt cuts vs sticky ends
describe isolating gene - mRNA
isolating mRNA for gene – reverse endonucleases
using reverse transcriptase
produce single strand of complementary DNA
- cDNA
advantages of isolating mRNA
easier to identify desired gene – particular cell will make very specific types of mRNA +
mRNA + cDNA – no introns
e.g. – beta cells in pancreas make insulin – a lot of insulin mRNA
what do you do after you isolate the gene
multiply DNA fragment - PCR
what do you do after you multiply DNA fragment - PCR
form recombinant DNA
how do you form recombinant DNA
Same restriction endonuclease cuts plasmid
Complementary sticky ends to DNA fragment
Line up sticky ends
DNA ligase forms phosphodiester bonds between sugar-phosphate groups
what are usually the vectors
Usually bacterial DNA / plasmids
advantages of plasmids as vectors
Replicate independently
plasmid gets int host cell + combines with host DNA – form recombinant DNA
Have marker gene e.g antibiotic resistance – can tell if bacteria have taken up gene by growing in media containing antibiotic
how can we tell the vector has taken up the plasmid
Plasmid – has second marker gene – show that plasmid contains recombinant DNA
Restriction enzyme inserts desired gene in this marker gene
If inserted right – marker gene wont function
put the first 3 steps into a diagram
what do you do after forming recombinant plasmid
transfer the vector
what is transformation
Plasmid with recombinant DNA – inserted into host cell (bacteria)
two methods to transfer the vector
calcium solution or electroporation
transferring the vector - calcium
Culture bacterial cells + plasmids in calcium rich solution
Increase temp
Causes bacterial membrane to become permeable + plasmids enter
transferring the vector - electroporation
Small electrical current applied to bacteria
Membranes – become very porous + plasmids move in
Also be used to get DNA fragments directly into eukaryotic cells
DNA – pass through cell + nuclear membrane
Control power or damage membrane
how else can you make GM cells
electrofusion
describe electrofusion
Tiny electric currents applied to membranes of 2 diff cells
Fuses cells + nuclear membranes
Forms hybrid / polyploid cell
Contains DNA from both
Produce GM plants
Animal cells – do not fuse easily – membrane have diff properties
Used in monoclonal antibodies
how to form GM plants - M1
Agrobacterium tumefaciens – causes tumours in healthy plants
Desired gene placed in the Ti plasmid + marker gene (antibiotic resistance / fluorescence)
Carried into plant cell DNA
Transgenic plant forms callus
what is a callus
mass of GM plant cells
how do you test which bacteria have taken up the plasmid
expose host to an antibiotic that kills cells that lack the new genes
wait for surviving cells to form a callus
how to form GM plants - M2
Electrofusion
Remove plant cell wall by cellulases
Electrofusion to form new polyploid cell
Plant hormones to stimulate growth of new cell wall
Callus formation
Transgenic plant formarion
transgenic organism
organism contains nucleotide sequences from a different species
why can we genetically modify
universal, meaning that almost every organism uses the same four nitrogenous bases
same codons code for the same amino acids in all living things (meaning that genetic information is transferable between species)
mechanisms of transcription and translation are also universal which means that the transferred DNA can be translated within cells of the genetically modified organism
vectors list
Plasmids - transfer DNA into bacteria or yeast
Viruses - transfer DNA into human cells or bacteria
Liposomes - fuse with cell membranes to transfer DNA into cells
what are restriction endonucleases
class of enzymes found in bacteria
used as a defence mechanism bagainst bacteriophages (viruses that infect bacteria)
e enzymes restrict a viral infection by cutting the viral genetic material into smaller pieces at specific nucleotide sequences within the molecule.
summarise Genetic engineering into diagram
why do we use the Ti-plasmid for plants
Soil bacterium infects plants by inserting the Ti-plasmid DNA into the plant genome
Liposome
DNA is wrapped in a lipid molecule which can pass the lipid membrane by diffusion
Getting the gene into the recipient cell
Microinjection - injecting the plasmid
Heat shock w/ calcium salts
Electroporation
Electrofusion
describe heat shock w/ calcium salts
Reducing the temp to freezing and rapidly increasing to 40 degrees - increases permeability
Ca^2+ surrounds DNA (-ve), reduces repulsion, increases permeability
Why do bacteria take up plasmds
Reproduce asexually - no genetic variation
Taking up plasmids from surroundings increases genetic variation, allows selection and evolution
why do we like making recombinant proteins from eukaryotic cells rather than prokaryotic
these cells will carry out the post-translational modification - due to the presence of Golgi Apparatus
advantages of GM to produce recombinant human proteins
More cost-effective to produce large volumes
Faster to produce
Reliable supply available
engineered to be identical to human proteins / have modifications that are beneficial
moral or ethical or religious concerns against using cow or pork produced proteins
Less allergic reactions
Insulin
Bacteria plasmids modified to include human insulin gene
Inserted into E coli via transformation
Identified + isolated transgenic bacteria
Express human protein insulin
Extracted + purified
advantages of GM plants / animals
Better than selective breeding
Organisms with the desired characteristics are produced more quickly
All organisms will contain the desired characteristic
The desired characteristic may come from a different species/kingdom
uses of GM crops
Resistant to herbicides – increases productivity / yield
Resistant to pests – increases productivity / yield
Enriched in vitamins – increases the nutritional value
Golden rice
reduce the impact of farming on the environment due to there being less need to spray pesticides
Insect resistance – soya
soya beans – susceptible to insect pests
genetically modified the already herbicide-resistant variety of soybean) by inserting a gene for the Bt toxin
gene is taken from the bacterium Bacillus thuringiensis
produce their own insecticide
insect ingests parts - alkaline conditions in their guts activate the toxin
the toxin is harmless to vertebrates as their stomach is highly acidic
killing the insect
BUT insect populations developed resistance
uses of GM livestock
produce pharmaceutical drugs – pharming
biopharma sheep and goats have been genetically modified to produce a number of useful human proteins in their milk
the human blood protein known as AAT in sheep milk
the human protein antithrombin (stops blood clotting) in goat milk
uses of GM pathogens
modified to shed light on their metabolism, drug resistance as well as how it causes damage to its host
aid research
act as vectors – infect cells – modified so can not replicate when inside host cell
ethical issues of GM
Biotech companies charge farmers more money for GM seeds vs non-GM seeds to try and make back the money they have invested in their product
Seeds can not be kept from GM crops to regrow the crop the following year because GM crops do not “breed true”
Buying seeds year upon year can be a major struggle for farmers in developing countries – only buy from patent holder
lack of long-term research on the effects on human health
Organic farmers have complained that the pollen from GM crops may contaminate nearby non-GM crops that have been certified as organic
Environmentalists are concerned about the reduction in biodiversity for future generations
more vulnerable to extinction
Herbicide-resistance genes could transfer to weed plants resulting in “superweeds”
antibiotic-resistance genes that are commonly used as marker genes in genetic engineering could transfer to pathogenic organisms that would then be untreatable with antibiotics - “superbug”
Patenting – people in less developed countries prevented from using GM crops by patents // unable to afford
pest resistance / disease resistance / herbicide resistance - perceived pros + cons
Gene therapy
involves using various mechanisms to alter a person’s genetic material to treat, or cure, diseases
somatic gene therapy
- replacing mutant allele with healthy allele in affected somatic cell
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
germline gene therapy
insert healthy allele into germ cell – egg / embryo immediately after fertilisation
