MGD - molecular techniques (DNA) Flashcards
what is gene cloning?
- gene from donor DNA is isolated using restriction enzymes (endonucleases - cut at same place)
- the same restriction enzymes are used to cleave the plasmid vector leaving ‘sticky-ends’
- the donor DNA and vector form recombinant DNA via sticky ends
- the vectors are introduced into bacterium (transformation)
- the vectors have antibiotic resistance so those that haven’t taken up the vector lysis
- the gene has been isolated
PCR (polymerase chain reaction) what is it used for
uses heat cycles replicate small amount of DNA for further analysis
can be used to detect viral DNA in diagnostic medicine
how does PCR work?
heat to 92 degrees - break hydrogen bonds between 2 DNA strands
cool to 55 degrees - to allow primers to anneal to DNA template strands
heat to 72 degrees - Taq polymerase (withstand high temp) anneals the 2 DNA strands together (1/2 old 1/2 new - semi-conservative replication)
what is the purpose of DNA electrophoresis
separate DNA according to size using an electric current running through a gel
larger fragments remain at the negative end whilst smaller travel to the positive end
how do you run a DNA electrophoresis
stain samples with ethidium bromide (so they show up in dark room)
- prepare an agarose gel and submerge in buffer (maintains pH)
- pipette DNA samples at the -ve end
- attach to electric current
- DNA moves towards +ve end at rate determined by its size
how do you amplify DNA? name the methods
DNA gene cloning and PCR
how do you separate DNA?
gel electrophoresis
how do you detect small changes in DNA?
restriction analysis
DNA sequencing
how does restriction analysis work? what can affect this process?
uses restriction enzymes (endonucleases) which cut at restriction sites (can be palindromic)
restriction sites can be altered by mutations
analysis via electrophoresis
what happens if a piece of DNA has been subject to a mutation in restriction analysis leading to removal of restriction site??
if mutation removes the restriction site, then the 2 fragments which corresponded to the cut DNA will be absent
loss of DNA fragment
what happens if a piece of DNA has been subject to a mutation creating a restriction site in restriction analysis ?
then the banding pattern will show extra fragment
create extra DNA fragment
what is a palindromic sequence?
a nucleic acid polymer which has the same sequence of bases when read in 5-3 direction on 1 strand and 5-3 on the opposite strand
(sticky ends - left by restriction endonucleases)
what is restriction analysis used for?
digest DNA at a specific sequences (may be palindromic)
when are changes in restriction analysis apparent?
through subsequent DNA electrophoresis (separate through size by running current)
what is required to carry out DNA sequencing via Sangar method?
4 separate DNA samples containing:
- DNA to be sequenced (template)
- DNA primers (attach to template - start process)
- DNA polymerase (anneal new bases to template)
- DNA bases (form new strands
- 1 of 4 labelled terminator bases (per test tube - 4 in total)
