6.1.3 manipulating genomes Flashcards
DNA sequencing is
working out the sequence of nucleotides
purpose of PCR
amplify (increase the length of ) DNA sample hwen there is not enough to be analysed
differences between natural DNA replication and PCR
- PCR requires addition of primer molecules to make it start
- only short sequences can be replicated
- a cycle of heating and cooling is neded
what do u need for PCR?
- DNA sample
- free nucletodies
- primers
- taq DNA polymerase
PCR 3 steps (and temperatures)
- Denaturation 95 degrees
- Annealing 68 degrees
- elongation 72 degrees
describe steps of PCR
- DNA smaple is mixed with free nucleotides, primers, TAQ DNA POLYMERASE
- DENATURATION: heat to 95 degrees to break the hydrogen bonds between CBP. U now have 2 separate strands
- ANNEALING: cool to 68 degrees so that primers can ANNEAL (bind by hydrogen bonding) to one end of each DNA strand. so now u have a small sectino of double stranded DNA at the end of each strand
- EXTENDING: taq DNA polymerase binds to the double stranded end, temperature is raised to 72 degrees , and the DNAP catalyses addition of free nucleotides to the single stranded. moving in the 5 to 3 direction
- repeat !
what is special about taq DNAP
- taken from thermophilic bacteria
- optimum temperature is 72 degrees
- means that cycles can be repeated as wont denature in the denaturation section
why do u need a primer for PCR?
- taq dna polymerase cannot bind to single stranded DNA
USES OF PCR
- forensic science. amplifying small sections of DNA for further DNA profiling. criminal, parentage
- tissue typing, reduce risk of rejection
- identifying viral infections
purpose of electrophoresis
used to separate different sized fragments of DNA based on length
describe breifly how electrophoresis works and its cmponent
- agarose gel plate covered by a buffer solution. electrodes at each end of tank so a current can flow
- DNA samples pre-digested with restriction endonuclease enzymes
- add the DNA
- DNA is negatively charged due to the phosphate groups, so is attracted to positive electrode
- saller fracgments travel faster
- at the end, remove buffer and add dye to stain the fragments
electrophoresis can alaso be used for
proteins. eg hameoglobin
describe sanger sequencing [for short strands]
- PCR: 4 dishes containing the free nucleotides, DNA polymerase, primers, and a DDNTP fluroescent chain terminator
- thousands of varying lengths are generated due to the adding of the fluroescent base at random positions
- electrophoresis, ethidium bromide and uv to see
- the samller ones travel further
- use it to sequence a genome; what u read from the smallest to the largest is the complementary
improved merthod to sanger
-high throughput
-pyrosequencing
- whole genome sequencing
applications of gene sequencing
- HGP
- genome wide comparisons between individuals and species
- !!!!evolutionary relationships
- !!!!genotype phenotype relationships
- !!!!epidemiology (genome of pathogens, allele for a disease)
- predict the amino acid seequence of proteins
somatic cell therapy
- insert gene into affected body cells
- only affects some cells
- short term (cells die ), must be repeated
germ line therapy
- insert gene into gamete (egg cell)
- long term sokution, all cells and offspring have it
- designer babies
what can u use for somatic and germline to insert the gene
- viral vectors
- liposomes
what do u use as markers in genetic engineering
antibiotic resistant
FARMER probelm with genetic engineering
have to buy new seeds eery year
how does dna sequencing allow for predictino of amino acid
- sequence DNA nucleotides of a gnee
- 3 base pairs = 1 amino acid
bioinformatics
- access to large amounts of data online
- data on DNA sequences AND PROTEIN structures!!!!
- with a universal format
how can DNA sequencing help with a viral outbreak?
- sequence of DNA base pairs = codes for aa sequence
- base pairs of antigens on sufrace
- can create vaccine with specific antigen
Suggest how the interdisciplinary field of bioinformatics may be useful in determining whether a
newly-sequenced allele causes a genetic disease.
- large volume of data hled in computers about DNA sequences and protein structures in a universal format
- holds info about allele and its variations
- rapid computational analysis to compare the allele to existing info
- modelling of protein structure from sequence
Explain why only selected sections of non-coding DNA are used when profiling a human.
- alot of the genome is very similar
- so using CODING sequences would not allow for unique identification
- non coding dna contains diff numbers of SHORT TANDEM REPEATS
importance of using taq DNA polymerase?
- DOES NOT DENATURE AT 95 DEGREES
- allows for the PCR to be cycled many times without reloading the enzymes
4 ways to visualise dna after electrophoresis
- visible stain
- fluorescent tag
- radioactive tag
what must you do to proteins before protein electrohporesis?
- heat
- denature
- expose charged region
what must you add to proteins before protein electrophoresis?
- something negatively charged
- as all proteins are diff charges. this ensures thyere all negative
- so all travel in same dirction, all attracted to anode, can be separated by mass
describe how DNA can be visualised after electrophoresis has been completed? (2)
- add ethidium bromide
- UV light
restriction enzymes
- specific enzymes that cut DNA at specific seqeucnes (phy hydrolysiing sugar phosphate backbone phosphodiester bond)
- can cut to blund ends, or sticky ends (overhanging single strands)
- sceintists will analyse the DNA and then look at the recognition sites on either side to choose the right restriction enzyes
how to do dna profiling
- collect dna sample
- amplify using PCR
- ## separate using gel electrophoresis, use ethidium bromide and UV light to visialose
describe GE for BACTERIA
- GENE probe complementary to the gene u desire, identifies your gene
- use restriction enzymes to remove gene u desire, creates sticky ends
- same restriction enzymes to cut open a plasmid
- CBP of sticky ends
- DNA LIGASE phosphodiester bonds to join sticky ends
- to form the recombinant dna
- (can also insert gene for antibiotic resistance with it so u can add antibiotics and then see which have succesfuly taken up the gene)
- recombinant plasmid mixed with bacteria and put into an ELECTROPORATOR: uses electricity to make bacteria csm more permeable
- bacteria are then grown in large fermenters to make lots
GM plants
- all the same just after, tje bacteria acts as a vector
- so can infect a plant cell and insert its dNA into the genome of a plant cell
advantage of GMO
- eg insulin, no allergic reaction
- increase crop yield, make food more nutritious
- ## pest resistance, save money on pesticide and porbs eg eutrophication less deep
disadvantage gmo
- patented and expensive, farers
- can cross pollinate and contaminate wild species
- ethical
- long term
problems of gene therapy
- ethical
- body may identify viral vector carrying gene as foreign and trigger immune response
- gene could be inserted into wrong place in genome
examples of GOOD things from gm
-insect resistance in soya => higher yield, less use of pesticides so cheaper
- genetically modified pathogens for research
- ‘pharming’ => genetically modified animals to produce pharmaceuticals
(3) why if they have the same adaptation does it not nec mean thyer eclosely related?
- convergent evolution
- adaptatoin
- as subject to the same selection pressure
3 ways to get your gene before genetic engineering
- gene probe with dna sequence comp to that of hte gene u want, restriction enzymes to cut out desired gene
- obtain mRNA for the gene you want, use reverse transcriptase to make the DNA sequence
- sequence the gene, work out base sequence, make that DNA sequence
