6.1.3 manipulating genomes Flashcards
(26 cards)
describe sanger sequencing
- DNA split into 2 single strands
- primers and DNA polymerase added
- fragments separated by gel electrophoresis
- sequence determined by reading fragment length
limitations of sanger sequencing
- slow
- can sequence only one DNA fragment
early methods of DNA sequencing
- DNA sequencing determines the order of nucleotides
- used radioactive labels
- expensive
describe high throughput sequencing
- faster, cheaper
- can sequence entire genomes simultaneously
- used personalised medicine and research
- e.g Human Genome Project
genome comparisons
- compare genomes of species
- reveals evolutionary relationships
- e.g humans and chimpanzees share 98.7% of their DNA
predicting amino acid sequences
- predict the protein that codes for an amino acid
- understand disease causing mutations
- e.g CFTR gene mutation -> cystic fibrosis
DNA profiling
- genetic fingerprinting for:
- forensics - use DNA from blood, hair, saliva to identify criminals
- paternity testing
- risk of genetic disorders
why use Taq polymerase
- found in hot springs
- it denatures at high temperatures
what is the PCR used for
- cloning genes
- detecting viruses - HIV or Covid-19
- forensics
method of PCR
- denaturation, heating(95’) - break the H-bonds between complementary bases
- cooling (55’) - primers anneal to the DNA 3’ to 5’ end
- synthesis (72’) - free nucleotides pair up complementary
- Taq polymerase forms the backbone and form phosphate bonds
method of electrophoresis
- prepare gel box - place agarose gel in it
- position wells to the -ve electrode
- add buffer solution
- use micropipette to add DNA and dye into wells
- run electrophoresis - turn power on
- DNA fragments move to +ve electrode
- smaller fragments travel faster
- after 30 mins view DNA bands to observe fragment sizes - Southern blotting or UV
resitriction enzymes
- cut DNA at specific recognition sites
- break the phosphodiester bonds between adjacent nucleotides
- creates sticky ends
genetic engineering
- reverse transription to form cDNA and isolate desired gene
- same restriction endonucleases to cut plasmid and desired gene to produce same sticky ends
- forms recombinant DNA , insert desired gene into plasmid, using DNA ligase which forms phosphodiester bonds between desired gene and plasmid
- transformation
ethical issues for genetic modification
+
- insect resistant crops = less pesticide use
- animals producing life saving drugs
- GM bacteria in disease research
-
- in humans - not ethical
- scientists using GM bacteria can become infected and lead to a disease outbreak
- monoculture
gene therapy
somatic cell therapy
- fixes gene in body cells - not passed to offspring - short-term
- treat cystic fibrosis, sickle cell
germ line therapy
- fixes gene in gametes - passed onto offspring - long-term
- ethical concerns
process of DNA sequencing (Sanger sequencing)
- DNA is fragmented
- denatured –> single strand
- free nucleotides and fluorescently-labelled terminator bases are added
- DNA polymerase builds new strands by adding free nucleotides
- terminator base stops the strand from increasing
- fragments seperated by size - gel electrophoresis
- observe under UV light - read colours in order of fragment size to find the DNA sequence
why use terminator bases
have H instead of OH on 3’ so cant form phosphodiester bonds with nucleotide
methods of transforming the bacteria membrane to make it more permeable to take in plasmid
- heat shock in a Ca2+ solution
- inc. temp. = more permeable because the bi layer vibrates more - electroporation
- electricity to membrane , creates pores between it - electrofusion
- plasmid in a vesicle
- electric current fuses the bacteria and plasmid
- moves into bacteria
how is plasmid described as a vector
carries the desired gene into host cell
how can you tell if the bacteria has taken up the desired gene
- the use of ampicillin resistant bacteria means when grown on agar plate with ampicillin the bacteria that taken up plasmid survive
- to identify the bacteria that taken up desired gene, the coloured marker gene e.g blue will produce blue colonies if the desired gene no taken up
- if taken up, produce white colonies because blue marker gene is disrupted by desired gene
what are satellite DNA
- short sequences of DNA that can be repeated many times which contain introns, telomeres and centromeres
- the more closely related a person is the more similar the satellite pattern is
- micro/minisatellite
what are microsatellites
- short tandem repeats (everyone has a different repeat of this)
- detected by PCR
- forensics
- fast analysis, small sample needed
what are minisatellites
- variable number of tandem repeats
- gel electrophoresis
- slow, large high quality sample needed
what is PCR used for
to amplify DNA of different lengths
