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Polymerase Chain Reaction - Coggle Diagram
Polymerase Chain Reaction
what is pcr?
a technique that takes specific sequence of DNA of small amount and amplifies it to be used for further testing
principle of pcr
purpose
to amplify a lot of double-stranded DNA molecules with same size and sequence by enzymatic method and cycling condition
Condition
Denaturation of dsDNA template
Annealing of primers
Extension of dsDNA molecules
Denaturation
Temperature: 92-94C
double stranded DNA melts into single stranded DNA
Annealing
Temperature: ~50-70C
primers bind to their complementary sequences
Extension
Temperature: ~72C
Time: 0.5-3 mins
DNA polymerase binds to the annealed primers and extends DNA at the 3' end of the chain
Components
magnesium chloride: 0.5-2.5mM
buffer: pH8.3-8.8
dNTPs: 20 - 200uM
Primers: 0.1-0.5 uM
DNA polymerase: 1-2.5 units
target DNA: <1 ug
DNA sequence of target region must be known.
Primers- typically 20-30 bases in size.
Thermo-stable DNA poymerase - Eg. Taq polymerase which is not inactivated by heating to 95C
DNA thermal cycler - machine which can be programmed to carry out heating and cooling of samples over a number of cycles
things to try if PCR does not work
If no product produced
:
check DNA quality
reduce annealing temperature
increase magnesium concentration
add dimethylsulphoxide (DMSO) to assay
use different thermostable enzyme
throw out primers (make new stock)
If extra spurious products bands presents
:
increase annealing temperature
reduce magnesium concentration
reduce number of cycles
try different enzyem
Advantages of PCR
small amount of DNA is required per test
result obtained more quickly (usually within 1 day for pcr)
usually
not
necessary to use radioactive material
PCR is much more
precise
in determining the size of alleles (essential for some disorders)
PCR can be used to detect point mutations.
Application of PCR
Molecular identification
molecular ecology
DNA fingerprinting
Classification of organisms
Genotyping
Sequencing
bioinformatics
genome cloning
human genome project
Genetic engineering
site-directed mutagenesis
gene expression studies