Recombinant DNA Technology Flashcards
restriction enzymes
they cut DNA at specific recognition sequences
where do restriction enzymes cut
phosphodiester backbone
when restrcition enzymes break the double strdand what do they produce
two 3-oh and 2 5-p base pair overhanging termini
how can recombinant dna be made
any remaning single strand breaks are fixed by dna ligase by catalysing the ormatino of a new phosphodiester bond
RFLP analysis
taking a dna fragment with a restriction enzyme that can cleave dna at a specific sequence
what can rflp analysis be used
for diagnosing diseases
how dna fragments can be analysed
agarose gel electrophoresis and southern blotting
limitations for rflp analysis
only works if the restriction enzyme site is affected by the disease-causing mutation or if the disease involves a length polymorphism
to fragment a gene it has to be inserted into a cloning vector
limitiation for southern blotting
slow process and requires a lot of starting material which can be limited
southern blotting process
dna fragments separated by electrophoresis
denatured and transfeered to nitrocellulose paper
radiolabelled probe added for hybridization
labelled probe hybridized visualised by autoradiobiography
what technique is used to amplify and isolate dna sequences for research purposes
polymerase chain reaction (pcr)
how pcr works
region of interest located from dna smaple
dna melted into 2 single strands through denaturation
2 primers made to match specific part of single strand and annealed
elongated by dna polymerase
dna polymerase used for pcr
taq polymerase
they can withstand denaturation step at high temperatures and elongates dna at 72°C
what can pcr be used
forensics
medicine
evolutionary studies
where do the primers extend
at the 3’ end
