2.10: molecular techniques Flashcards
what is PCR?
polymerase chain rxn
rapidly amplify a segment of DNA in vitro
for cloning and analysis
PCR reagents
- target DNA sequence
- forward and reverse primers
provides free 3’OH end for DNA pol to replicate from 5’ -> 3’ direction - Taq polymerase (thermally stable, optimal temp: arnd 75)
- free dNTPs in excess
PCR process: denaturation
target DNA and reagents heated to 95C for 1 min
heat -> increase kinetic energy -> break H bonds btwn cbp
dsDNA -> ssDNA
PCR process: annealing
mixture cooled to 55C for 2 min
forward and reverse primers bind to comp seq via H bonds
*excess primers=primer annealing > DNA target seq renaturing
PCR process: elongation
72C for 3 min
Taq pol synthesises comp DNA strand in 5’ -> 3’ direction
by adding free dNTPs to free 3’OH end
using target DNA seq as template
calculation of DNA molecules after each PCR cycle
no of dsDNA molecules = 2^(no of cycles)
no of ssDNA = 2^(no of cycles) x 2
advantages of PCR
- rapid and efficient (each cycle only 3-5min)
- relatively easy (automated)
- sensitive and robust (even from badly degraded material, eg fingerprints)
- specific
- high fidelity (relatively accurate)
limitations of PCR
- primer design
short = may bind to multiple along DNA
long = more specific but costly - limited length of target seq
- error in replication
Taq pol lacks proofreading activity
process of agarose gel electrophoresis
formation of gel
- agarose powder dissolved in TBE buffer
- poured into casting tray
- comb inserted to form wells
- left to cool -> agarose gel
- placed into gel chamber
- submerged in TBE buffer
- buffer maintains pH and ions to conduct direct electric current
- comb removed
- DNA mixed w loading & tracking dye
- loaded into CATHODE
- DNA ladder w known sized fragments can be loaded in 1st well
- voltage btwn 90V to 150V
- DNA fragments migrate accordingly
- electrophoresed gel submerged in stains
what does loading dye contain
glycerol
weighs DNA fragments into wells
what does the tracking dye contain
low and high molecular weight coloured compound
acts as front and back markers of migration
how does agarose gel act as a molecular sieve to separate DNA fragments
DNA fragments negatively charged (phosphate grps)
gel separates DNA fragments by size and shape
smaller fragments migrate faster than larger
compact fragments migrate faster than relaxed
what stains can be used in electrophoresed gel
- methylene blue
- low sensitivity
- safe - EtBr
- higher sensitivity
- carcinogenic
- under UV light
polyacrylamide gel electrophoresis [PAGE]
- higher resolution
- separates DNA by 1 base pair
- more difficult to use
- more expensive
- neurotoxin
what is southern blotting?
after electrophoresis : transferring DNA from gel into a membrane
