Genomes Flashcards
satellite DNA
- short sequences of repeating DNA codes within introns, telomeres (ends of chromosomes) and centromeres
- non-coding
- closer related you are, the more similar your satellite DNA will be
mini-satellites
sequences of 20-50 base pairs repeating 50-hundreds of times
micro-satellites
sequences of 2-4 base pairs repeated 5-15 times
comparative genome mapping uses
- allows examining of evolutionary relationships
- identify essential genes for life
- compare pathogenic and non-pathogenic individuals
- identifying genetic polymorphisms - different alleles for a gene
- DNA profiling
DNA profiling steps
- extract DNA - small amount from sample then replicate using PCR
- DNA strands cut into fragments by restriction enzymes at specific sites
- electrophoresis - separates DNA fragments
- hybridisation - fluorescent DNA probes added in excess to DNA fragment that bind to a known complementary sequence
- if radioactive labels added - x-ray images taken, if fluorescent labels added - placed under UV light
uses of DNA profiling
- forensic science - crime
- paternity tests
- identifying who is at risk of developing a disease
polymerase chain reaction (PCR)
- makes millions of copies of a fragment of DNA a few hundred bases long
1. DNA is extracted and heated to 95 degrees to break H bonds between strands - denatured
2. mixture cooled to 50-65 degrees and a primer is added which anneals (binds) to the DNA ends - needed for replication
3. mixture heated to 72 degrees and DNA polymerase added to line up free nucleotides along each template strand
4. 2 copies of DNA are formed
what is different about the DNA polymerase used in PCR?
it’s derived from a thermophilic bacteria (Taq) so it doesn’t denature during extreme heating
restriction enzymes
- cut DNA to get a DNA fragment from a genome
- they recognize specific palindromic sequences (same forwards and backwards eg. GGATCC)
- DNA sample incubated with specific restriction enzyme which cuts out a specific fragment by a hydrolysis reaction
- unpaired bases at the end of the fragment are called sticky ends - can anneal (bind) the DNA fragment to another piece of DNA with complementary bases
electrophoresis
- DNA is digested by some restriction enzymes
- a fluorescent tag if added to the DNA fragments so they can be viewed under UV light
- DNA fragments are put into wells in agarose gel strips containing a buffer to maintain the pH
- DNA fragments of a known length are used as a reference for the fragment size - electric current is passed through the electrophoresis plate and DNA fragments in the wells at the cathode end move through the gel to the anode side because of the negatively charged phosphate groups in DNA fragments
- smaller fragments move faster as they can fit through the gel mesh
- gel is immersed in alkali to denature the DNA - separate the 2 strands and expose bases
- single stranded DNA fragments transferred to nylon membrane which is placed over the gel
- membrane covered in absorbent paper to draw the alkaline solution containing DNA through the membrane
- DNA probes are then used to allow DNA bands to be visible
DNA probes
- short sequence of single stranded DNA with a known sequence
- bind to their complementary sequence to identify specific genes
- can be attached to radioactive/fluorescent labels to allow DNA bands to be visible
human genome project
- started in 1990 to map the entire human genome and make the data accessible to all scientists
- started with sequencing bacteria before applying the technique to humans
Sanger genetic sequencing
- similar process to PCR is performed on the DNA - mixed with DNA polymerase, a primer, normal nucleotides and terminator bases
- Each time a terminator base is included instead of a normal nucleotide, the synthesis of DNA is terminated
- eg. an A terminator will stop DNA synthesis where an A base would be added - this results in many DNA fragments of different lengths
- After many cycles, all possible DNA chains are produced and they are separated according to length by capillary sequencing (similar to electrophoresis)
- the fluorescent markers the terminator bases can show the final base of each fragment - lasers detect the colours and the order of the sequence
- The sequence detected by the lasers is the complementary sequence to the original DNA - fed into computer that reassembles genome by comparing fragements
differences between original and next generation Sanger sequencing
- original - used radioactive or fluorescent labelling of ddNTP’s , modern - uses fluorescent labelling
- original - used gel electrophoresis to separate DNA fragments, modern - uses capillary electrophoresis
- original - involved manual reading of the DNA bands, modern - lasers detect colours and order of DNA bands
- original - slower, less cost-effective, modern - faster, more efficient
next generation sequencing
- millions of fragments of DNA put on a flow cell (plastic slide) and replicated using PCR to form clusters of identical DNA fragments
- uses same principle of adding coloured terminator base so an image can be taken
- all clusters are being sequenced at the same time
- uses very high-tech computers - human genome could be sequenced in days - much cheaper
bioinformatics
- development of software to analyse enormous quantities of data being generated
computational biology
- uses data being analysed by bioinformatics to build models of biological systems to predict circumstances
- uses computational techniques to analyse huge amounts of biodata
- eg. helps use info from DNA sequencing to identify genes linked to diseases
what does analysing genomes of pathogens enable?
- findings source of infection eg. bird flu
- identify antibiotic-resistant strains of bacteria so antibiotics are only used when needed
- tracking progress of an outbreak
- identify regions in pathogen genome that may be useful in development of drugs
proteomics
- study of amino acid sequence of an organism’s whole protein complement
spliceosomes
- enzymes that join exons to be translated together to give mature mRNA
- may join the same exons in a variety of diff ways - single gene produces diff versions of mRNA - codes for diff amino acids so diff phenotypes
synthetic biology
- includes genetic engineering, using enzymes in drug production, synthesis of new genes to replace faulty ones, synthesis of entirely new genomes
recombinant meaning
- combining DNA from more than one source into a single organism
genetic engineering - 1. isolating the desired gene, 2 methods
technique 1. restriction enzymes cut DNA at a specific restriction site leaving sticky ends to make it easier to bind with the plasmid
technique 2. isolate mRNA and use reverse transcriptase to produce strand of complementary DNA (cDNA) which is then extracted
genetic engineering - 2. placing the gene in a vector
- plasmids are used as they replicate independently
- plasmid will have 2 marker genes eg. blue marker gene which is interrupted by desired gene and antibiotic resistant which will remain in tact
1. plasmid is cut using same restriction enzyme - complementary to sticky ends to DNA fragment at a marker gene (eg. blue marker gene)
2. once the DNA fragment is lined up with the plasmid, DNA ligase forms phosphodiester bonds between the 2 strands on DNA fragment and plasmid
3. this plasmid now has recombinant DNA
genetic engineering 3. transferring the vector
transformation:
- moving isolated plasmid back into bacteria
- method 1 - culture bacterial cells and plasmids in solution with Ca2+ and increase temp - bacterial membrane becomes permeable and plasmids enter
- method 2 - electroporation - small electrical current applied to bacteria - makes membrane porous and plasmids move into cells
- method 3 - electrofusion - plasmid is in vesicle, electric current applied to vesicle and bacteria. This causes them to fuse together and the plasmid moves into the bacterial cell
genetic engineering - 4. mass production of transgenic bacteria (bacteria on agar plate)
3 situations could’ve occured:
- bacteria are placed on an agar gel plate with nutrients and ampicillin
- bacteria which haven’t taken up the plasmid will not appear on the plate - no antibiotic resistant gene so killed by antibiotic
- bacteria which has taken up a plasmid which has not taken up the DNA fragment (desired gene) will survive and appear blue - has antibiotic resistant gene and blue marker gene in tact
- bacteria which have taken up the plasmid containing the desired gene will grow but appear white - blue marker gene interrupted by desired gene and antibiotic resistant gene in tact
electrofusion
- tiny electric currents are applied membranes of 2 cells
- this fuses the cell and nuclear membranes of the 2 cells together forming a hybrid or polypoid cell - contains DNA from both
- used in GM plants, animal membranes have diff properties and don’t fuse as well
when is electrofusion used?
- GM of plants (not animal cells as they have diff properties and don’t fuse as well)
- monoclonal antibodies - combining a cell producing a single type of antibody with a tumour cell - divides rapidly. These are used to identify pathogens in animals and plants, disease treatment eg. cancer
electroporation
- encourages uptake of plasmid by giving it an electric shock - makes temporary pores in the cell membrane
- increased permeability so more likely to take up the recombinant plasmid
genetic engineering prokaryotes uses
- bacteria and other microorganisms have been GM to produce hormones, clotting factors for haemophiliacs, antibiotics, vaccines, enzymes
genetic engineering plants - 2 methods
method 1
1. a desired gene (eg. pesticide production, herbicide resistance) is placed in a plasmid of a bacteria, with a marker gene eg. antibiotic resistance, fluorescence
2. this is carried directly into the plant cell DNA
3. the transgenic plant cells form a callus, which grows into a new transgenic plant
method 2
- fusing 2 plant species cells together
1. plant cell wall removed by cellulases
2. electrofusion to form new polyploid cell
3. plant hormones to stimulate growth
4. callus forms and many new cloned transgenic plants grow
reasons that genetic engineering of pathogens is restricted to only military research facilities
- health and safety of researchers and the public
- could be used for purposes of biological warfare
pros of GM crops
pros:
- pest resistance - reduces amount of pesticide spraying, protecting environment and helping farmers, increases yield
- disease resistance - reduces crop loss, increases yield
- herbicide resistance - reduce competing weeds, increase yield
- extended shelf life - reduces food waste
- crops can be grown in a range of conditions
- nutritional value increased
- medicines and vaccines
cons of GM crops
- pest resistance - non-pest insects and insect eaters might be damaged by toxins in GM plants, insect pests may become resistant to pesticides
- disease resistance - transferred genes may spread to wild populations
- herbicide resistance - biodiversity reduced, superweeds
- extended shelf life can reduce commercial value and demand
- ppl could be allergic to diff proteins in GM crops
issues with patenting new technology
- when someone discovers a new technique, if they get a legal patent, no one can use it without paying
- those in less economically developed countries who most need the flood/drought resistance, high yields, nutritional value may not be able to afford the GM seed
- these countries also rely on using the seed from one year to plant the next - patenting makes this impossible
reasons for GM animals
- disease resistance
- modify physiology in farmed animals for higher yield
pharming
- production of human medicines
- creating animal models - addition or removal of genes so animals develop a disease to act as a model of development for new therapies
- creating human proteins - human gene introduced to genetic material of a fertilised cow, sheep or goat egg along with a promoter sequence so the gene is expressed only in the mammary glands. The fertilised egg is returned to the mother, the animal is born and when it matures and gives birth, its milk contains the desired human protein to be harvested
ethical issues with pharming
- using animals as models for human disease
- putting human genes into animals - cannot be certain it will not cause harm
- does GM animals reduce them to commodities?
- is welfare compromised during the production of genetically engineered animals?
somatic cell gene therapy and uses
- replacing a mutant allele with a healthy allele in the affected somatic (body) cells
- can help people with a wide range of diseases eg. retinal disease, immune diseases, leukaemia
somatic cell gene therapy disadvantages
- effects of treatment may be short-lived
- may need to undergo multiple treatments
- body may start an immune response against the replaced allele
- disease could still affect offspring because their germ cells are unaffected
germ line cell gene therapy
- inserting a healthy allele into the germ cells (reproductive cells in an unborn baby) or an embryo immediately after fertilisation
- the individual would be born healthy and pass on the normal allele to their offspring
reasons that germ line cell therapy is illegal in humans
- potential impact on intervening with someone’s germ cells is unknown
- human rights of the unborn child could be violated as it’s done without consent and it’s irreversible
- it could also eventually enable people to choose desirable or cosmetic characteristics of their offspring
