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Manipulating genomes 6.1.3 - Coggle Diagram
Manipulating genomes 6.1.3
Genetic engineering - inserting DNA fragments (containing gene) into a vector
How to genetically engineer a microbe:
DNA fragment with desired gene is obtained using restriction enzymes
The DNA fragment is inserted into a vector e.g. bacterial plasmid.
The use of DNA LIGASE is needed to seal the DNA.
Procedure:-
1) A bacterial plasmid is removed. This is our vector DNA.
2) Vector DNA cut open using the same restriction enzyme that isolated the DNA fragment containing our useful gene. This makes sure that the sticky ends will be complementary between the vector DNA and the fragment we are trying to insert.
3) Vector DNA and fragment DNA mixed together with DNA ligase. This enzyme joins up the sugar phosphate backbone (this is called ligation)
4) We now have vector DNA joined with our DNA fragment from another organism. This gives us RECOMBINANT DNA
How do we get the vector (plasmid) into the recipient bacterial cell:
Heat Shock
Most common method
alternate the temperature from 0°C to 42°C (in calcium chloride)
bacterial wall and membrane become more porous
1) Ice-cold calcium chloride solution is used
2) temperature repeatedly raised to 42’C to heat shock bacterium wall,
3) this encourages take up of the plasmid
4) Cells that take up the plasmid vector containing the desired gene (e.g insulin production gene) are now ‘genetically engineered’ or transformed bacteria
5) Only 25% success rate when attempting to insert modified plasmid - therefore we need to identify the transformed bacteria
6) Done using marker genes, they are inserted into plasmid vector at same time as desired gene (e.g. insulin)
7) Marker gene codes for antibiotic resistance, bacteria grow on agar containing the antibiotic
8) Only cells containing the marker gene, & therefore also the desired gene, will survive & grow
Electroporation
High voltage electric current disrupts membranes
Electrofusion
Electrical field helps introduce DNA into cells
Transfection
DNA can be packaged into a bacteriophage which can then infect the host cell
Ti plasmids - These bacterial plasmids are genetically modified and inserted back into Agrobacterium which naturally infect certain plants. The bacterium transfers genes into the plant genome.
Gene gun - Not all plants can be infected with Agrobacterium. For cereal crops we need to fire DNA containing the genes into the host cell nucleus. Small pieces of gold are coated with DNA and shot into one of the plant cells. We can then use micropropagation to produce many genetically modified plants.
https://docs.google.com/document/d/1nlji1INUuN7MwC2VJEEcTvTllEd-ObDMoU5K1lRIexE/edit
obtaining the required gene
https://docs.google.com/document/d/1MVLeYt4FuAPMz8ZWwXKwzBYVQrDbIMbFhpgwKzj1hys/edit
obtainIng a human insulin gene:
1) extract mRNA from the cells where the gene is expressed (e.g. pancreas)
2) Use the enzyme Reverse Transcriptase to form a single strand of complementary DNA (cDNA) from the mRNA template
3) The addition of primers and DNA polymerase can make this cDNA into a double stranded length of DNA, whose base sequence codes for the original protein
DNA profiling
Some of the organism's genome consists of repetitive, non-coding sequences.
The number of times these sequences are repeated at specific places in the genome differs from person to person.
The length of these sequences in terms of nucleotides therefore differs too.
DNA is taken e.g. from saliva, blood or hair.
Restriction enzymes are used to cut out these repeating sequences.
Gel electrophoresis is used to separate out the fragments based on their lengths. Larger fragments will travel the shortest distance. This will produce banding patterns on the gel. Different people will show different banding patterns
The banding patterns from different individuals can be compared
Fragments of different lengths will be obtained from these people if the same restriction enzyme is used to cut out the repeating sequences
Applications of DNA profiling:
Not only has it brought about convictions and established the innocence of many suspects and of people previously wrongly convicted, it has been used to:
Identify Nazi war criminals hiding in South Africa
Identify the remains of the Romanov family and to refute a person's claim to be the survivor, Anastasia
Identify remains found in Leicester as those of Richard Ill
Identify victims' body parts after air crashes, terrorist attacks or other disasters
Match profiles from descendants of those lost during WW1 with the unidentified remains of the soldiers who fell on the battlefields in Northern France
https://docs.google.com/document/d/10NhSkwLDyAL6W_lVdVUGacsh5w-ZE_47E3j0h8zSlKs/edit
cutting DNA using restriction enzymes
Scientists can use restriction enzymes to cut DNA.
This enables a gene to be cut out and inserted into DNA from another organism to produce recombinant DNA
Recombinant DNA is used to produce genetically engineered organisms which new characteristics.
When a gene is cut out, short chains of unpaired bases are often left at either end. These chains are called sticky ends
Sticky ends allow the gene to be inserted into new DNA which also has sticky ends which match.
To seal DNA together ,another enzyme is needed called ligase.
We can use restriction enzymes to cut out a useful gene.
Restriction enzymes cut in specific places
They recognise PALINDROMIC SEQUENCES also called recognition sequences and cut in these places.
The 'shape' of the sequence is complementary to the enzyme active site.
If the same sequence is found at both ends of your chosen fragment then this piece of DNA will be cut out. Useful for example if you want to cut out a gene.
Restriction enzymes use hydrolysis reactions to break bonds.
The cut often leaves 'sticky ends'. These are short lengths of unpaired bases used to bind the DNA to another piece of DNA which has complementary sticky ends e.g. when inserting human DNA into a bacterial plasmid.
https://docs.google.com/document/d/1UPX8OCjfV_ZAS3uQpD9h3w2M6K_lrT-LCrcATjr1Ijo/edit
Separating different sized stands of DNA by electrophoresis
How it works
Electrophoresis is used to separate DNA fragments based on size
A gel plate is used and is usually made of agarose (a type of sugar)
DNA is cut into fragments of different sizes using restriction enzymes
Buffer solution is placed on top of the gel to conduct electricity
DNA is negatively charged and moves towards the positive terminal
Smaller strands find it easier to move through the gel
Takes 2 hours to complete
Dyes and stains are often used to see the DNA fragments
Fragments can be lifted from the gel for further analysis
Electrophoresis is similar in many ways to chromatography
e.g. mixture, size, resistance to travel etc.
A procedure uses an extricate current to separate out DNA fragments, RNA fragments or proteins depending on their size
Steps
1) Agarose help poured into tray & left to solidify
2) Crete a row of well at 1 end of the gel
3) Position the gel tray with wells near negative electrodes
4) Add buffer solution so the gel is covered with it
5) Add your fragmented DNA sales together with loading dye. This helps samples sink to the bottom of wells & makes them easier to see
6) Keep volumes the same in each case.
7) Record which DNA sample you have added to each well
8) Connect gel box to power supply
9) Electric current passes through gel
10) DNA is negative charged so the fragments will move towards the positive terminal
11) Small fragments move faster.
12) After 30 mins, turn off power and stain the surface of the gel
13) The strains will now become visible
https://docs.google.com/document/d/1S0GwmHuYigp4BQvCBMO7VuBmD24SaZY0IVDJrr3-uMA/edit
Copying DNA -
Polymerase chain reaction (PCR)
How to produce multiple copies of a DNA fragment using polymerase chain reaction (PCR)
1) Materials:-
DNA sample (to be copied)
Free nucleotides (building blocks for your new DNA copies)
Primers (short pieces of DNA, complementary to the ends of the sample
DNA polymerase (the enzyme that builds new DNA)
2) Heat mixture to 95°C. this breaks the H bonds between the double stranded DNA
3) Cool to 55 °C so primers can bind (anneal) to the strands.
4) Heat to 72 °C so DNA polymerase can work.
5) DNA polymerase lines up the free nucleotides alongside each template strand. Two new complementary strands are formed.
6) We now have two copies of the fragment and one cycle of PC is complete.
7) Repeat the whole cycle again. Each cycle will double the number of DNA molecules.
Applications
Used to amplify DNA samples for sequencing (determine base sequences)
Sequencing allows us to do:
Tissue typing - DNA from donor & recent can be sequenced to ensure compatibility & reduce risk of rejection
Detection of oncogenes - mutations can be detected & in cancer patients we can determine what type of mutation has occurred & give most appropriate treatment
Detecting mutations - a sample of DNA can be analysed for he presence of a mutation that leads to genetic disease. Patients can be screened for a defective recessive allele. Feral cells (IVF) from 8 day old embryo can be screened to analyse feral DNA before implantation
Forensic science - small quantities of DNA can be amplified for DNA profiling to identify criminals of to a certain parentage
Identifying viral infections - small quantities of viral DNA can be detected in host cell DNA to verify, e.g. HIV or hepatitis C infections
Research - amplifying DNA from an heist extinct sources, e.g. Neanderthals bones to see how their DNA differs from ours
Applications of gene technology - the PCR
The polymerase chain reaction was developed in 1983
It is a way of amplifying (copying many times) a sample of DNA in vitro (outside the body).
It takes only a few hours to produce enough DNA to analyse.
It is useful in forensic science either in detective work were only a minute drop of blood has been found at a crime scene or in archaeology when trying to identify remains.
It is essential that the sample of DNA to be copied is not contaminated with any other DNA.
PCR is said to 'produce the hay stack from the needle'.
Meathod:
The piece of DNA to be amplified e.g. from a white blood cell, is heated to 95°C for 20 seconds. This causes the hydrogen bonds between the base pairs to beak so the two strands separate.
A solution containing DNA polymerase, nucleotides be a buffer and primers is added. (Primers are short pieces of DNA containing 20-30 base pairs, which act as signals to the enzymes indicating 'start copying here'.
The mixture is allowed to cool to 55°C - 60°C for 20 seconds. This allows the primers to anneal (join) to the DNA strand. Excess primers are used to prevent the original DNA strands from the sample just rejoining.
The mixture is heated tom 72°C for 30 seconds. The DNA polymerase binds at each end of the primer allowing free nucleotides to build a complementary strand along the exposed portion of DNA. As a result two identical pieces of DNA are obtained as the original sample of DNA is doubles
The cycle is repeated usually 20-30 times. Each time the number of copies of the target DNA is doubled.
Inserting desired genes in plants to create genetically engineered plants
Agrobacterium tumefacciens
Naturally infects plants to insert desired genes into it
Often used as vector to insert desired genes into plant
This bacterium infects plants 7 transfers DNA from a Ti (tumour inducing) plasmid into chromosomal DNA of a plant
25 genes from the Ti plasmid cause a tumour to grow in the plant providing a habitat for the bacteria
Provides scientists a way to inert genes into plant cells
https://docs.google.com/document/d/1DGXZV79SFrzAIq89M1XL9Yf29nnjs1sjlgrEYvgOknE/edit
Identification of transformed bacteria by replica plating
(Used for human insulin)
3 possible outcomes:
1) Bacteria fail to take up a plasmi
2) Bacteria take up a plasmid but without the desired (insulin) gene
Ligase has worked too quickly
3) Bacteria take up plasmid with desired (insulin) gene
Using replica plating to see which bacteria have taken up the correct plasmid
Special plasmids are taken from bacteria who have natural resistance to 2 antibiotics, Ampicillin & Tetracycline
Any ‘normal’ bacteria which takes up the plasmid wld be resistant to both bacteria
Procedure:
1) Grow all the bacteria on normal agar to produce many colonies
2) Transfer some cells to Ampicillin agar. Only bacteria which have taken up the plasmid can grow
3) Take some surviving cells from this agar & grow on tetracycline agar
https://docs.google.com/document/d/1L-X-jRIJipibj1LgStj9-rbB4WN0X6E3FppoccxNc1U/edit
Gene therapy
= altering genes in body cells, any offspring will have disease
Genetic disorders = inherited disorders
Caused by abnormal genes/chromosomes
Gene therapy involves altering alleles inside a cell
Recessive disease - you need to insert a working dominant allele
Dominant disease - must silence the gene
E.g. insert DNA in the middle of the allele so it no longer works
How do we get alleles inside cells:
Vectors
Virus vectors
Plasmid vectors
Liposomes
Disadvantages of treatment:
Short lied effects of treatment, need multiple treatments
Difficult getting alleles into specific body cells
Immune response to vector
Allele could go into wrong place & cause cancer
Allele could be over expressed & you produce far too much protein which could be toxic in high amounts
2 types of gene therapy - 1 is legal & 1 is illegal
1) Somatic therapy (legal)
Altering alleles in BODY CELLS which are affected, e.g. cystic fibrosis
Any offspring will still have the disease
(See above)
2) Germline therapy (illegal)
Altering alleles in the SEX CELLS
Could cure successive generation but also has other ethical complications
Could harm future generation if mistakes are made
Gene technology - testing for faulty genes, microarrays, sequencing & comparing genomes
Gene probes
Used to test I someone has a faulty gene
Using nuclei acid problems to locate specific genes.
Beside labelling probes with radioactive isotopes, fluorescent dyes to ‘tag’ the probes
With the probe binds to the single-sided DNA they will have the fault
Microarrays
People can be screened for genetic diseases using microarrays
It contains a series of single stranded DNA sequences attached to a solid surface
The sequences code for a variety of different genes found in humans
Method:
1) mRNA Is taken from a normal human cell
2) We use reverse transcriptase to produce complimentary DNA (cDNA) strands
3) The cDNA is fluorescently labelled green
4) We repeat this process from a cancerous cell but this time we label the cDNA red
5) We now chop up all the cDNA into fragments & apply to the microarray
Significant results:
Any spots on the microarray which are green means only the ‘normal’ cDNA has bound & not the ‘cancer’ cDNA. This is significant as it means that normal cells only contain those particular genes
Any spots which are red means only the ‘cancer’ cDNA has bound. This is significant cause only the cancer cells would contain those particular genes
Sequencing DNA - chain termination method
1) Make multiple copies of the template strand using PCR (using DNA polymerase)
2) Add free nucleotides, primers & DNA polymerase
3) Add labelled terminator bases (fluorescent)
4) Many different lengths of DNA are produced
5) DNA fragments separated by electrophoresis
6) A laser detects colours on the gel plate & a computer reads the order of bases
This method is called the chain termination method of DNA sequencing
Invented by Fred Sanger
1981 - he published the sequence of the human mitochondrial genome
37 genes or 16,569 base pairs)
1984 - Epstein-Barr virus genome sequenced
1995 - genome of a flu bacterium sequenced
Sequencing whole genomes
The chain termination method is only used for DNA fragments up to 750 base pairs long
To sequence an entire genome we need to chop it up into smaller fragments
These fragments are sequenced in turn & then put back in order with help of computer
1) Restriction enzymes cut the DNA into 100,000 base pair fragments
2) Fragments inserted into BACs (bacterial chromosomes). Theses are man-made plasmids. Each fragment goes into a different BAC
3) BACs inserted into bacteria
4) Bacteria divide, creating colonies of identical cells all containing the specific fragment. We know have a genomic DNA library - a large supply of all the fragments
5) DNA extracted from each colony, cut up using restriction enzyme into overlapping pieces of DNA
6) Each piece is sequenced (chain termination) & put back in order to give full sequence
7) DNA from all the BACs in order by computers to give complete genome
Comparing genomes
Comparing different species:
Helps with evolution studies
closely related species evolve away from each other more recently will share similar DNA
Humans & chimps share 94% similar DNA
Dog % bears share 92% with each other
Understand ways in which genes interact during development are controlled (homeobox genes)
Medical reach
Genes associated with cancer can be found in other animals e.g. mice
These animals can be used as models for research
Comparing the same species:
Trace early migration
Genetics of human disease
BRCA1 gene linked to breast cancer, comparing sufferers to non-sufferers (screening)
Individualised medical treatment (genomic medicine)
https://docs.google.com/document/d/1QyAbBKCZto7NpN1_ILZ7_JxcMePVoCNXjj06bqVKICE/edit
synthetic biology
The interdisciplinary science concerned with designing and building useful biological systems.
examples & their applications
Information storage
Storage of digital information on a strand of DNA e.g. like a memory stick
Production of medicines
E-Coli and yeast have been engineered to produce the anti-malarial drug Artemisinin. Normally only available from a specific plant
Novel proteins
Similar to Haemoglobin but does not mind to carbon monoxide
Biosensors
Modified bacterium which glow if petroleum pollutants are present in the air
Nanotechnology
Amyloid fibres for making biofilms for adhesion
https://docs.google.com/document/d/1pdYh9SIbynSAn0mzhkNF-m3TKeaTzgkMUs8wsjYbdMg/edit
comparing genomes
