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What is a genome?
All of the genetic material an organism contains - all genes possessed by an individual or organism - whole base sequence
What are VNTRs?
Sequence of 20-50 base pairs repeated 50-several hundred times
Number of repeats varies between individuals
What are STRs?
Region of 2-4 bases repeated 5-15 times
Number of repeats varies between individuals
How do you produce a DNA profile?
1. Extracting DNA
- Using PCR, amplify DNA from small sample - artificial replication - produces larger quantities
2. Digesting sample
- Cut DNA strands into fragments using restriction endonucleases (different ones cut at different points; each makes 2 cuts) at restriction sites - (cut at introns)
3. Separating DNA fragments
- Fragments separated using gel electrophoresis. Fragments transferred from gel to nylon membrane using southern blotting -uses gel agarose - or use probes
4. Hybridisation
- Radioactive/fluorescent probes added in excess (short DNA/RNA sequences complementary to known DNA sequence - bind under particular pH/temp); wash off excess probes
- Probes identify short tandem repeats (No. repeats varies between individuals)
5. Seeing the evidence
- Radioactive probe: X-ray
- Fluorescent probe: UV light
- Fragments give a pattern of bars
Describe the process of PCR
Denaturing
1. Temperature increased to 90-95*C
- Denatures DNA by breaking H bonds
- Strands separate/separate double helix
-occurs in thermal cycler - higher temp so more kinetic energy to break H bonds between complementary bases
Annealing
2. Temperature decreased to 55-60*C
- Primers anneal to ends of DNA strands by H bonding to each end of single-stranded DNA in 5' to 3' direction
-there will be a small section of double-stranded DNA at each end of single-stranded molecule
-thermal cycler
Synthesis/ Elongation
3. Temperature increased to 70-72*C
- Optimum for DNA polymerase
- Adds bases to primer, building up the complementary DNA strand
-free nucleotides pair to the exposed bases
-The enzyme taq polymerase is used (obtained from thermophilic bacteria) - catalyses the addition of the nucleotides
Describe the process of Sanger Sequencing
1. DNA mixed with primers, free nucleotides, DNA polymerase and terminator bases - ddnucleotide
2. Place in thermal cycler that rapidly changes temperature at set intervals (96*C to break H bonds and form single stranded DNA; 55*C so primers anneal)
3. At 60*C DNA polymerase builds up new DNA strands by adding complementary nucleotides - synthesis of DNA nucleotides - H bonds form complimentary base pairs
4. Terminator base binds; no more bases added resulting in fragments of differing length. Fragments are separated by length according to capillary sequencing (like GE). Fluorescent markers on the terminator base used to identify final base in each fragment - can't form phosphodiester bond - sequence can no longer elongate - variety of lengths formed.
5. Order of bases in capillary tubes shows sequence of complementary DNA which is used to build up the original DNA sequence - forms developing strands
6. can read by electrophoresis using fluorescence and UV light on nylon membrane - OR use laser box - beam goes through gel as bases pass - smallest fragment goes through first and generates a pattern on the nucleotide - there will be smaller background signals which indicate free nucleotides or any errors.
What is bioinformatics?
Development of the software and computing tools needed to organise and analyse raw biological data
Development of software and computing tools to analyse biological data.
These studies may generate data on DNA sequences, RNA sequences, and protein sequences, as well as on the relationship between genotype and phenotypeHigh-power computers are required to create databases
The large databases contain information about an organism's gene sequences and amino acid/protein sequences
Once a genome is sequenced, bioinformatics allows scientists to make comparisons with the genomes of other organisms using the many databases available.
What is computational biology?
Uses the data analysed using bioinformatics to build theoretical models of biological systems which can be sued to predict what will happen in different circumstances
What is synthetic biology?
design and construction of new and biological parts; redesign of things for useful purposes - - building useful biological devices and systems
-biotech-biophysics-systems bio-molecular bio
how has gene sequencing lead to synthetic biology?
By sequencing a gene, the sequence of amino acids that a gene codes for and so the primary structure of a polypeptide can be predicted.
This has allowed us to create biological molecules from scratch and so has led to the development of synthetic biology.
biosensors
-Modified bioluminescent bacteria -placed on microchip - glows if air is polluted with petroleum pollutannts
living microbes, enzymes or organelles are linked to electrodes to detect specific substances
detection is done by converting biological reaction products into electrical currents
Novel proteins
Designed proteins have been produced. E.g. one that is similar to haemoglobin and binds to oxygen but not to carbon monoxide
nanotechnology
The science and technology of building devices, such as electronic circuits, from single atoms and molecules.
synthetic biology - genetic code
predicts the aa sequence in protein - helps scientist predict how new protein will fold into tertiary structure
specific example of synthetic biology?
A drug called artemisinin is an antimalarial drug that has been extracted from plants. Using synthetic biology, scientists have created all the genes responsible for producing a precursor to artemisinin. They have successfully inserted these genes into yeast cells, which can then produce artemisinin.
Describe the process of genetic engineering
1. Isolating required gene
- mRNA for specific gene obtained using restriction endonucleases to form cDNA - produces sticky ends and cuts at marker genes
OR
- Treated with reverse transcriptase to form cDNA
2. Placing inside vector
- Cut plasmid (from bacteria) using same restriction enzymes - this ensures that the sticky ends of the DNA fragment are complementary to those of the plasmid
- DNA ligase catalyses insertion of gene into plasmid - forms recombinant DNA - forms phosphodiester bonds
3. Vector carries (recombinant plasmid) gene to target cell
- Method 1: electroporation (Small electric current applied to make membrane of bacteria more porous) - can also be used in eukaryotic cells
- Method 2: Culture in calcium-rich solution and increase the temperature so bacterial membrane more permeable - heat shock - pushes phospholipid bilayer apart - membrane more permeable
-Method 3: Electrofusion - stimulates the plasmid in vesicle and bacteria together.
How does electrofusion work?
Tiny electric current applied to membranes of 2 different cells
This fuses cell and nuclear membranes of 2 different cells forming a hybrid or polyploid cell containing DNA from both
How can plants be genetically modified?
- Using the vector agrobacterium tumefaciens
- Plasmids from the vector have the required gene inserted into them
- Carried directly into plant cell DNA
- Transgenic plant cells form callus (a mass of GM plant cells each of which can be grown into a new transgenic plant)
Describe process of genetic engineering in plants
1. Cut leaf
2. Expose to bacteria carrying weedkiller-resistance gene and antibiotic-resistance gene; allow bacteria to deliver genes into leaf cells
3. Expose leaf to antibiotic to kill off cells that lack new genes; wait for surviving cells to form a callus
4. Allow callus to sprout roots and shoots
5. Transfer plants to soil where they can develop into fully differentiated plants
Pros of GM crops
(pest resistance) Pesticide-resistant crop varieties reduce amount of pesticide spraying/Increased yield
(disease resistance) Crop varieties resistant to common plant diseases can be produced
(extended shelf-life) Less food waste
(growing conditions) Crops can grow in a wider range of conditions
Cons of GM crops
(pest resistance) Non-pest insect-eating predators might be damaged by toxins in GM plant/insect may become resistant to pesticides in GM crops
(disease resistance) Transferred genes might spread to wild populations and cause problems e.g. superweeds
(extended shelf-life) May reduce commercial value and demand for the crop
What is pharming?
positives?
Use of genetic engineering in animals to produce human medicine. There are 2 aspects to it:
1. Creating animal models - addition/removal of genes so animals develop certain diseases
2. Creating human proteins - Introduction of a human gene coding for a medically required protein
-drug can be made in large quantitiesmore available to more people
What are the ethical issues surrounding pharming?
Should animals be genetically modified to act as models of disease?
Is it right to put human genes in animals?
Is welfare compromised during the production of genetically modified animals?
-can have harmful side effects on animals
What is gene therapy?
altering a genotype - using a mechanism to alter a persons gentic material to cure/treat disease by inserting a functional allele
How is gene therapy done if the disorder is caused by two mutated recessive alleles?
A working dominant allele can be added to make up for them, you supplement the faulty ones.
How is gene therapy done if the disorder is caused by a mutated dominant allele?
The dominant allele is silenced, e.g., by sticking a bit of DNA in the middle of the allele so it doesn't work anymore.
How is the new allele inserted into cells in gene therapy?
Using vectors, such as altered viruses, plasmids or liposomes.
Somatic cell gene therapy
-Involved replacing mutant allele in somatic cells
-Altering the alleles in body cells, particularly the cells that are most affected by the disorder.
Viral vectors often used to introduce alleles into goblet cells/target cells
Only temporary - somatic cells have limited life and stem cells
will have same faulty gene
Not passed onto offspring - Somatic therapy does not affect the individuals sex cells, so any offspring could still inherit the disease.
-Somatic gene therapy - ex vivo
via virus/viral vector outside the body -blood or bone marrow is extracted and exposed to virus - cells grown in lab then transgenic cells return to person by injection in vein
uses adult stem cells
Somatic gene therapy - viruses
-uses retroviruses and adenoviruses
-viruses unable to cause disease
-edit genetic material with desired genetic material - virus targeted cell and gets dna by binding to receptors and then transfer dna into cell
problems with using viruses as gene delivery
-even tho modified to be non-virulent - can still cause immune or inflammatory response in patient
-patient may become immune to virus making further deliveries impossible
-may insert allele into patients genome that may disrupt gene involved in regulating cell division which increases risk of cancer or disrupt the regulation of the expression of other genes.
however adenoviruses or retroviruses can be modified to not divide to produce cancer
pros of gene therapy
Gene therapy could prolong the lives of people with life threatening genetic disorders.
Gene therapy could give people with genetic disorders a better quality of life.
Germ line therapy would allow carriers of genetic disorders to protect their offspring from having the disorder.
Germ line therapy could decrease the number of people that suffer from genetic disorders and cancer.
cons of gene therapy
-ethical concerns...try to create perfect person
-unintended side effects
-unknown consequences
-religious concerns
-not always successful
-might not be long term
-expensive
-unreliable delivery method
-invasion of privacy
The body could identify vectors as foreign bodies and start an immune response.
An allele could be inserted into the wrong place in the DNA.
An inserted allele could get over expressed producing too much of the missing protein.
The effects of the treatment in somatic therapy may be short lived.
The patient may have to undergo multiple treatments.Difficult to get the allele into specific body cells.
Somatic cell therapy - liposomes
(non-viral)
-small spheres packaged within small spheres of lipid bilayer to make liposomes
-less likely to cause immune response
-fuse with membrane of the cell
aerosol inhaler
liposomes packed into inhaler - sprayed through the nose - pass through the PM of cells lining the respiratory tract and nuclear envelope to host genome cell - host cell will express desired protein e.g. CFTR protein for CF - forms noraml chloride ion channel
but epithelial cells lining respiratory tract replaced every 10-14 days - treatment has to be replace in short regular intervals
Mitochondrial DNA (mtDNA)
genetic material in the mitochondria of the cytoplasm of a cell; only inherited from the mother
example of somatic gene therapy:
Cystic fibrosis, a genetic disorder that is very damaging to the respiratory system, so somatic therapy targets the epithelial cells lining the lungs.
-lack functioning CTFR gene.
CF has two reccessive faulty alleles - add dominant gene or replace defective gene.
artificial chromosomes
cloning vectors that can accommodate very large pieces of dna, producing recombinant dna molecules resembling small chromosomes
Germ line gene therapy
Healthy allele inserted into germ cell (eggs)
Individual born healthy
Passed onto own offspring
Illegal with humans in most countries due to ethical and medical concerns
Altering the alleles in the sex cells, this means that every cell of offspring produced from these cells will be affected by the gene therapy and they won't inherit the disease.
What are some of the ethical concerns with GLT?
Potential impact unknown
Cannot give consent
Process irrevocable
Designer babies
what is the genome split into (two)
the genome is split into extrons and introns
exons - code for the protein
introns - do not code for protein - gene expression control
Introns - in depth
introns have satellite DNA
- this contains VNTRs and STRS
- variable number tandem repeats - always appear on same area in each chromosome - Minisatellite - variable as they can be repeated an random number of times
- short tandem repeats- smaller repeats - also always appear on same place on each chromosome. - Microsatellite
tandem repeats - repetitive segments of DNA which do not code for proteins
Why agarose gel?
because it is alkaline - helps to break down fragments to single strand DNA - helps it move faster across electrophoresis
sources of DNA
Skin, sweat, blood, mucus, saliva, tissue, semen, urine, hair, ear wax
Uses of DNA profiling
To identify potential suspects
To exonerate individuals
To identify crime and casualty victims
To establish paternity/maternity/family relations
-half of STRs fragments come from mother and then from father
analysis of disease
To match organ donors
STR for dna analysis
highly variable short repeating lengths of DNA
used instead of restriction fragment length polymorphism - was laborious and no longer used
STR sequence separated by electrophoresis
each STR is polymorphic - but the number of alleles in the gene pool for each one is small
13 STRs analysed at the same time
very sensitive - can pick up a trace of DNA and can be stored for years - must be careful to avoid contamination
DNA profiling - disease
DNA profiling (along with Next Generation DNA sequencing) can be used to identify individuals that are at risk of developing particular diseases, as research shows that certain VNTR sequences are associated with an increased incidence of particular diseases eg. cancers and heart disease
protein electrophoresis can detect the type of Hb present and aid diagnosis of sickle cell anaemia
Huntingtons disease can also be detected
Southern blotting
A hybridization technique that enables researchers to determine the presence of certain nucleotide sequences in a sample of DNA.
Involves using a nylon sheet which can soak up the DNA and makes it easier to process after electrophoresis
What is PCR used for?
-replicating DNA sequence many times
-rapid detection of diseases
- doubles amount of strands we have
e.g. 1-2-4-8-16-32-64-128...
Thermal cycler
used to complete pcr reactions - changes and controls temp at programmed timings to trigger different steps
controls length of time in each stage.
Taq polymerase
A heat-stable form of DNA polymerase extracted from bacteria that live in hot environments, such as hot springs, that is used during PCR technique
thermophilic bacteria - thermophilous aquatics
-its optimum temperature is high enough to prevent annealing of the DNA strands that have not been copied yet
In pcr why do we use taq polymerase instead of human polymerase
human would denature at higher temperature - less stable
How does PCR differ from DNA replication?
Only short requested, of up to 10 000 base pairs, of DNA can be replicated, not entire chromosomes. It requires the addition of primer molecules to make the process start, and a cycle of heating and cooling is needed to separate the DNA strands, bind primers to the strands and for the DNA strands to be replicated.
what is the in vitro method of DNA amplification/ replication
PCR
what nucleotides does sangers method use and why
modified nucleotides called dideoxynucleotides - stop replication.
Dideoxynucleotides have a slightly different structure to the nucleotides (which can be referred to as deoxynucleotides) found within the DNA of organisms
Dideoxynucleotides can pair with nucleotides on the template strand during DNA replicationThey will pair with nucleotides that have a complementary base
When DNA polymerase encounters a dideoxynucleotide on the developing strand it stops replicating, hence this method of sequencing is referred to as the chain termination method
- doesnt have oxygen - on ribose it is a H instead of OH on C3 - so we cannot form phosphoidester bond
gene sequencing - building up bases
The resulting single-stranded DNA chains are then separated according to length using gel electrophoresis
The gel will have four wells, one each for A*, C*, T*, and G*A 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. E.g.
If the band on the gel that travels furthest comes from the C* well, scientists can see that the first base in the sequence is C. If the next furthest band comes from the T* well, the second base in the sequence is T, and so on
This allows the base sequence to be built up one base at a time
Cloning DNA in bacteria
another way (other than PCR) to amplify genes; 1) ligation of the DNA sequence of interest with vector DNA fragments 2) once the recombinant molecule is formed it can be inserted into the bacteria strain, through transformation, identical copies of DNA produced
1. isolate gene using restriction enzymes from the bacterium
2.DNA inserted into bacterial plasmid/ vector
3. Then put into an bacteria Coli bacterium host that when cultured, divided many times, enables plasmid DNA insert to be copied many times -
first automated dna sequencing
flourescent dyes used instead of radioactivity used to labe terminal bases - glow when shone with laser ba=eams - processed into autoradiograms
pyroseq
What are cells that take up a desired gene called?
They are genetically engineered, and are called 'transformed' - transgenic
GM pathogens
this research helps aid the development of vaccines and treatments for diseases.
e.g. scientists found tumour cells have receptors on membrane for poliovirus- poliovirus recognises and attacks themby genetically engineering the poliovirus to inactivate the genes that cause poliomyelitis, scientists can use it to attack and kill cancer cells without causing disease- this leads to a cancer treatment.
pros and negatives of GM pathogens
+previously untreatable diseases can now be treatedResearchers follow strict protocols, so risk of anything bad happening is low
-tampering w. viral genomes could cause existing ones to be more harmful to the hostscientists researching the pathogen could become infected and cause a mass outbreakthe GM version could go back to its original form and cause a mass outbreakin the wrong hands, knowledge of GM pathogens could lead to the development of bioweapons
the owning of GM
scientists often share knowledge and skills, however companies can patent their genetic modification
This means they control who uses the product and how long for the owner will make money- this is a positive as scientists will compete to come up with new beneficial ideas.
However, expensive for some, e.g. poor farmers can't afford GM seeds.
GM plants
can be modified for a) resistance to herbicides and insecticides, b) resistance to pests, c) enrichment with vitamins. This helps to increase price/ yield.e.g. soya bean plants, reduced by insect pests, have been modified w. a gene in bacterial thuringiensis (Bt) that codes for a protein that is toxic to some of the insects.
GM plants pros and cons
organisms with desired characteristics produced quicker
increased nutritional value, crop productivity, yield
Reduced environmental impact, don't have to spray herbicides, insecticides etc.
-biotech companies charge farmers more for GM seeds, major struggle for farmers in developing countries
pollen from GM crops may contaminate nearby non-GM crops so not organic anymore.
herbicide-resistance could create 'superweeds', same with insects.
GM toxins could harm non-target species e.g. butterflies.may encourage monoculture
Decreased biodiversity- the GM plants are genetically identical
GM livestock
using GM livestock to produce pharmaceutical drugs is called 'pharming'
e.g. injecting DNA fragment that codes for antithrombin production into goat embryo
'biopharm' sheep and goats would then produce useful proteins, such as...1) the human blood protein known as AAT in sheep milk 2) antithrombin (stops clotting) in goat milk
What is a bacteriophage?
A virus that infects bacteria. The bacteriophage infects the bacteria by injecting its DNA into it. The desired gene can then integrate into the bacterial DNA.
electroporation
If a plasmid is used as a vector...
1) bacterial cells + vector put in electroporator
2) electrical field increases permeability of bacterial membrane, so plasmids are taken in
why does GM need exposed bases in the first step?
Because they are easier for plasmids to recognise
why use mRNA at the start of GM
it is mature so saves more time and energy converting to cDNA as there are no introns to get rid of
what is electrophoresis?
using an electrical current to separate molecules by size and net charge.
how does electrophoresis work? - step 1
Agarose gel is poured into a gel tray and left to solidify.
A row of wells is created at one end of the gel - using a comb.
The gel tray needs to be put into a gel box with the end of the gel tray with wells closest to the negative electrode.
Then add buffer solution to the reservoirs at the sides of the gel box so the surface is covered.
electrodes at each end connected to a power supply to allow electrical current to pass through.
electrophoresis - step 2
submerge gel into the electrolyte solution
Add a set volume of loading dye to each fragmented DNA sample.
Add a set volume of DNA sample to each well, using a micropipette .
precautions in step 2 of electrophoresis?
Make sure that the tip of the micropipette is in the buffer solution and just above the opening of the well.
Don't stick the tip too far into the well or the bottom could be pierced.
Record which DNA sample has been added to each well.
Use a clean micropipette tip each time.
why is loaded dye added to dna fragments?
Loading dye helps the samples to sink to the bottom of the wells and makes them easier to see.
electrophoresis - step 3
Put a lid on the gel box and connect the leads from the gel box to the power supply and set it to the required voltage.
This causes an electrical current to pass through the gel.
Let is run for about 30mins and turn off the power supply.
Stain the DNA fragments by covering the surface of the gel with staining solution then rinsing gel with water.
-not visible - transfer to absorbant paper on nitrocellulose - heat to separate two strands- add probes and use x-ray image or UV light. - produces a pattern
How do DNA fragments move through the gel?
DNA fragments are negatively charged, - due to phosphate - so they move through the gel towards the positive electrode at the far end of the gel.
Small DNA fragments move faster and travel further through the gel, so the fragments separate according to size.
Cathode to Anode - negative to positive
what must be done to proteins before electropheresis
Proteins can be positively charged or negatively charged, so they are mixed with a chemical that denatures the proteins so that they all have the same charge.
prep before electrophoresis
_prepare fragments_
-mplify by PCR]
-Restriction endonuclease - cuts into fragments
Microarrays
Thousands of nucleic acid sequences are arranged in grids on glass or silicon. DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amts of complementary binding. Used to profile gene expression levels or to detect single nucleotide polymorphisms (SNPs).
-quickly compare gene expression in different cells or tissue samples
-can place number of probes on filled surface -dna microarray - reveals presence of mutated alleles that match fixed probes - becuase sample of dna will anneal to complimentary base pairs
process of microarray
-take mRNA from samples - reference it to and test dna sample - change to cDNA using reverse transcriptase - two different coloured flourescent labels - place cDNA on microarray - sticks to probes - when both bind to specific probe it reveals flourescence of both colours indicating presence of sequence in test dna - test with different coloured lasers - use computer analysis and target therapies
probing uses:
-locate specifc gene in GM
-identify specific alleles
-Identify gene in multiple alleles - comparison studies
-locate same gen in variety of different genomes
DNA probe
a molecule labeled with a radioactive isotope, dye, or enzyme that is used to locate a particular sequence or gene on a DNA molecule
- short single stranded legnth of dna being investigated - VNTR regions
Radioactive label - probing
-phosphorous isotope - causes probe to emit radiation that makes x-ray film go dark - creating dark bands
- can be revealed by exposure to photographic film
-fogmark produced - because of radiolabelled nucleotides
-DNA probe anneals to complimenatry sequence
flourescent dye
e.g. ethidium bromide - flouresces - shines when exposed to uv light - coloured bands
seperating proteins
seperate proteins not dna fragments - by size/mass
-uses detergent such as SDS - sodium dodecyl sulfate
-equalises the charge on molecules
-allows protein to seperate by mass as they travel through gel
-prepared for electrophoresis
-denature disulfide bods - manipulate into rod shapes - negative charge - allow sepration by size
uses:
can be used to analyse types of Hb proteins for diagnosis of condition
sickle cell aneamia - HbS not HbA
or
aplastic anaemia;leukemia - higher amount of HbF then HbA
Step one Pyrosequencing
DNA fragmentation (nebulization; double-stranded DNA into fragments by high pressure of nitrogen gas flow)
A section of DNA is cut into fragments, split into single strands and then a strand from each fragment is attached to a small bead.
Step 2 Pyrosequencing
PCR is used to amplify the DNA fragments on each bead.
Step 3 Pyrosequencing
Each bead is put into a separate well.
Step 4 Pyrosequencing
Free nucleotides added to the wells attach to the DNA strands via complementary base pairing. The four different types of nucleotides are added to the wells on after the other, over and over again for 100 cycles.
Step 5 Pyrosequencing
The wells also contain specific enzymes, which cause light to be emitted when a nucleotide is added to the DNA strand. More than one nucleotide can be added at a time if the bases are the same, so the intensity of the light can vary.
Step 6 Pyrosequencing
Computers analyse the occurrence and intensities of the light emitted in the different wells, after each type of nucleotide is added and process this information to interpret the DNA sequence.
