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Manipulating genomes notes
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DNA profilinggenome: all genetic material
introns - non coding regions removed
exons remain
satellite DNA: short sequences of DNA repeated many times
minisatellites + microsatellites
- same positions on chromosomes
- # of repeats varies
- identical twins -> identical pattern
1. extract DNA
- large sample required
- PCR
2. digesting sample
- cut into small fragments w/ restriction endonucleases
- splices at defined points in introns
3. separate DNA fragment
- electrophoresis
- gel + electric current
4. hybridisation
- DNA probes added (attached to radioactive/fluorescent label)
5. seeing the evidence
- X rays if radioactive labels in DNA probes
- UV light if fluorescent labels in DNA probes
PCR
- sample, free nucleotides, primer DNA, DNA Taq polymerase mixed
- primer: anneal complementary strand
step 1 95C DNA denaturation
- separation of DNA strands
- hydrogen bonds between bases break
step 2 55C DNA primer anneal to ends
step 3 72C DNA Taq polymerase
- optimum temp
- adds bases to primer
adv:
quick few hours
less equipment e.g thermocycler
less space no growth medium rq
, less labor intensive completed in machine
safe does not use whole cells
can use lower quality DNA e.g forensics
electrophoresis
- DNA fragments in agarose gel strip wells
- electric current -> DNA moves to anode
- rate of movement depends on mass/length of DNA
- gel resists movement
- smaller = faster
- electric current switched off when fragments reach end
- gel put in alkaline buffer -> denatures + expose bases
- southern blotting
- nitrocellulose paper/nylon membrane put over gel
- covered w/ dry absorbent paper
- fixed in place w/ UV light/80C
DNA sequencing + analysis
terminator bases:
- modified versions of 4 nucleotide bases
- dideoxynucleotides
- e.g A terminator -> stops DNA synthesis at where A would be added
Sanger sequencing
1. DNA mixed w/ primer, DNA polymerase, normal nucleotides + terminator bases
2. mixture in thermal cycler
- equipment used for PCR
- rapidly changes temp at intervals
3. primer anneals -> short DNA double strand
4. DNA polymerase -> free nucleotides added
5. terminator base -> DNA synthesis stopped
- many DNA fragments w/ diff lengths
- all DNA chains produced
6. DNA fragments put into capillary tube
- has fluorescent markers on terminator bases
- lasers detect colors + order
7. computer finds areas of overlap -> identify regions linked w/ specific diseases
massively parallel sequencing
- millions of DNA fragments replicated on slide
- flow cell
- colored terminator bases
- clusters imaged
the human genome project
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