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 it is too short 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
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
- 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
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
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 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
DNA probes, electrophoresis
- short single stranded length of DNA comp to the DNA being investigated
- can be labelled with a fluorescent or radioactive marker
- so that u can locate specific DNA sequences
describe sanger sequencing
- 4 dishes containing the bases, DNA polymerase, and a DDNTP chain terminator
- thousands of varying lengths are generated, then oassed through a gel by electrophoresis, the samller ones travel further
- use it to sequence a genome
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)
- predict the amino acid seequence of proteins
somatic cell therapy
- insert gene into affected body cell
- short term (cells die )
germ line therapy
- insert gene into egg cell
- long term, offspring have it
- designer babies
what can u use for somatic and germline
- 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
