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Biology A level - Manipulating genomes Cheat Sheet by

This is an A level OCR A Gateway biology cheat sheet for Chapter 21 of Module 6. Specification reference - 6.1.3

Human genome - introns

Minisa­tel­lites
20-50 base pairs (bp) repeated 50-100 times. Also called VNTRs (Variable Number Tandem Repeats).
Micros­ate­llites
2-4 bp repeated 5-15 times. Also called STRs (Short Tandem Repeats) or stutters
Can repeat a varying amount of times on each homologous chromo­some. The number of repeats is inherited from parents.
Satellite DNA is a repeating sequence of DNA found in introns (non-c­oding DNA).

Producing a DNA profile

1. Extract DNA
Tissue sample is mixed with phenol solution. PCR can be carried out to multiply sample (more detail later).
2. DNA fragme­ntation
Restri­ction endonu­cleases - cut DNA at specific base sequence (restr­iction site).
3. Gel electr­oph­oresis
Used to separate DNA fragments using charge and Mass of DNA fragments (more detail later).
4. Denatu­ration
Hello soaked with alkali to separate strands.
5. Southern blotting
Membrane that single strands of DNA are transf­erred on to.
6. Hybrid­isation
Comple­mentary radioa­ctive or fluore­scent probes are added. Their base sequence is known and so they tag comple­mentary regions so specific genes can be pinpoi­nted.
7. Disclosure
X-rays or UV lights are used depending on what type of tag was used. The result will show lines where specific sequences were tagged and a DNA profile is created.

Gel electr­oph­oresis

Gel electr­oph­oresis

DNA cut up by restri­ction enzymes and mixed with loading dye is placed in wells in agarose gel. Buffer solution is added to maintain a constant pH and a current is passed through with the anode at the opposite end to the DNA.
Because DNA is negatively charged, the fragments will be pulled towards the anode. Smaller fragments travel faster and further. The loading dye allows us to make sure the DNA doesn't fall off the other end.
An alkaline solution is then added to denature the DNA and the other steps of DNA profiling take place (Southern blotting and disclo­sure).

PCR

Polymerase chain reaction (PCR)

PCRis used when only a small sample of DNA is available, for example at a crime scene. The DNA sample along with excess bases, primers and DNA polymerase are mixed and placed in the PCR machine
1. Separating the strands (90-95C)
30 seconds, this denatures the DNA - breaks hydrogen bonds between bases.
2. Annealing the strands (55-60C)
Primers bind to the ends of the DNA strands.
3. DNA synthesis (72-75C)
At least 1 minute, DNA polymerase (Taq polyme­rase) adds bases to the primer, comple­mentary strand is made.
The cycle is repeated as many times as necessary to produce enough DNA for the necessary usage.
 

DNA sequencing (Sanger)

DNA mixed with primers, DNA polyme­rase, excess nucleo­tides and terminator bases (4 separate contai­ners, one for each base).
PCR performed to synthesise DNA.
Terminator nucleo­tides cause transc­ription to stop. This therefore produces different lengths of DNA fragments.
Process similar to gel electr­oph­oresis started. Terminator nucleo­tides contain fluore­scent markers, so base which ends sequence can be identi­fied.
Shortest fragment travels furthest so order of bases can be determ­ined.
Next-g­ene­ration sequencing or massively parallel sequencing is more commonly used than the Sanger method. This is where DNA fragments are put through a plastic slide (flow cell) instead of gel electr­oph­oresis. PCR is then carried out in situ.

Bioinf­orm­atics and comput­ational biology

Bioinf­orm­atics
Stores and organises data.
Comput­ational biology
Uses data to form theore­tical models.

Uses of genome­-wide compar­isons

Human genome
10,000 Genomes Project UK10K.
Genomes of pathogens
Find source of infection.
 
Identify antibi­oti­c-r­esi­stant bacteria.
 
Track progress of an outbreak.
 
Identify target areas of pathogen genome.

Sequencing for classi­fic­ation

Identi­fying species (DNA barcoding)
Identify sections of the genome that remain the same within species. Conserved regions in animals are in mtDNA and in plants chloro­plast DNA.
Evolut­ionary relati­onships
Can calculate rate of mutations --> See how long ago two species had same DNA in common ancestor.

Genomics and proteomics

Genomics is the study of genome.
Proteomics is the study and amino sequencing of organisms' entire protein comple­ment. More proteins exist than genes.
Splice­osomes
Enzyme complexes which cut out introns and some exons out of pre-mRNA. Exons can be rearranged differ­ently, therefore one section of DNA can code for many proteins.
Protein modifi­cation
Proteins modified by other proteins. Can be lengthened or shortened.
Splice­osomes and protein modifi­cation are some of the reasons why existing proteins do not reflect the genome of an organism.

Synthetic biology

Design and constr­uction of novel artificial pathways, organi­sms...
Genetic engine­ering.
Industrial contexts - fixed/­imm­obi­lised enzymes and drug produc­tion.
Synthesis of new genes e.g. treat cystic fibrosis.
Synthesis of new organism. New nucleo­tides developed (other than ACTG) incorp­orated in DNA introduced in bacteria.
 

Isolating desired gene

mRNA is isolated from target cell using restri­ction endonu­cleases and treated with reverse transc­riptase to create comple­mentary DNA (cDNA).
Plasmid geneti­cally engineered to have markers, cut by restri­ction endonu­cleases (same ones used for DNA for comple­mentary sticky ends).S­econd marker is added to show plasmid contains recomb­inant gene. This marker should be corrupted when DNA is added.

DNA ligase fuses cDNA and the plasmid by forming phosph­odi­ester bonds. The recomb­inant plasmid is placed in the host cell. The bacteria multiply in a fermenter.
Could also use electr­ofu­sion: merge two cells + their DNA to form polyploid cells. More used in plants, animal polyploids usually don't survive.

Isolating genes

Genetic engine­ering in different organisms

Prokar­yotes
Easily geneti­cally modified for hormones, antibi­oti­cs...
Plants
Agroba­terium tumefa­ciens which usually forms tumours. Desired gene inserted in plasmid, then in plant DNA. Forms a callus of GM plant cells
 
Can also use electr­ofusion - Tiny electric shocks used to fuse the cells and nuclear membranes of two different cells together. Also used for producing monoclonal antibo­dies.
Animals
Cell membranes are harder to manipulate than plant cell membranes. Engine­ering used for medically important proteins and curing human genetic diseases.

Ethics for GM plants

Pest resistance
+ Less pesticide spraying
 
- Non-pest insects are also affected
Disease resistance
+ Less crop loss
 
- Superweeds (if genes spread).
Herbicide resistance
+ Less compet­ition, higher yield
 
- Lower biodiv­ersity, superweeds
Shelf-life extended
+ Less food waste
 
- Lower commercial value and demand
Growing conditions
+ Can grow in a wider range of conditions e.g. flood resistant
 
- N/A
Nutrit­ional value
+ Higher nut. val.
 
- Allergies can develop due to new proteins
Medical uses
+ Medicines and vaccines
 
- N/A
Patenting
+ N/A
 
- Companies charge for seeds, can't harvest seeds, people who may need it the most cannot afford it

Ethics for GM animals

GM pathogens used for research - modify virus to insert new genes in cells e.g. swine fever-­res­istant pigs, faster­-gr­owing salmon...
Pharming
Using animals to produce human medicines and research subjects.
 
Medicine - From GM fertilised animals, human protein collected from milk...
 
Research - Knock-out mice engineered to develop cancer for research.
Issues
- Human genes in animals
 
- Reduce animals to commod­ities
 
- Welfare compro­mised

Gene therapy in humans

Germ line cell gene therapy
Germ cells - Sex cells / embryo post-f­ert­ili­sation.
 
Insert healthy gene in germ cell.
 
Illegal for human embryos - violation of human rights of unborn child, concerns of long term impacts...
Somatic cell gene therapy
Replace mutant allele with healthy one using viral vector.
 
Higher rates of success, but still issues to fix e.g. mutant alleles passed on rather than healthy ones.
                   
 

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