Polymerase Chain Reaction (TYPES OF PCR (HOT START PCR (-Hot condition to…
Polymerase Chain Reaction
of primer and dNTP
of joining dNTP
-Two primer that complement to the 3' interest segment are added to the denatured DNA,then the temperature is lowered.
-The primer that hybridised will synthesis the DNA when the adddition of nucleotide supply and DNA polymerase.
-DNA polymerase begin to anneling at 72 degree celcius.
Then,heated to 94 degree celcius to melt and form duplex.
-The cycle is repeated to produce many DNA copies.
Used to amplify specific DNA sequences in compex mixture where the sequence ends is known
-RNA @ DNA template
-Forward and reverse primer
-Reaction buffer (Tris ,ammonium ion @ potassium ion)
TYPES OF PCR
HOT START PCR
-Hot condition to activate enzyme
-Enzyme released from beads until it reach annealing
-Primer, Mg+2,dNTP @ polymerase added during anneling tempereture.
-To remove impurities and deal with old DNA sample.
-Amplify the target DNA with low copy no.
-1 fragment and 2 pairs of primer.
-increase the sensitivity if wrong fragment are amplify.
-Amplified the PCR target simultaneously.
-Used more than 1PCR set.
-Primer ratio 50:1 - 100:1.
-Amplify one strand of DNA more than other.
-used in DNA sequencing.
-the process is not simultaneous. ( forward first then reverse)
-Screened bacteria or yeast clones.
-The bacteria or yeast colony pick up and put in the PCR master mix.
-The amplification of DNA lengths up to 30 kb and beyond
-Useful when the target is accurate.
-Proficient polymerase along with accurate polymerase Pfu required.
-Anneling temperature is 65°c that higher compare to normal PCR.
-Then, the anneling temperature reduce to 1°c every cycle.
-Reduce non-specific DNA amplification and allow enrichment of correct product over non-specific product.
Random Amplified Polymorphic DNA
does not require any specific knowledge of the DNA sequence of the target organism
for all kind of genome
Its run along the entire genome
use 10 mers primer (more specific, easy to design the primer)
primers will or will not amplify a segment of DNA (all by chance)
RAPD is an inexpensive yet powerful typing method for many bacterial species
Variable Number Tandem
• Non-coding regions
• Several to many copies of the same sequence
• Large amount of variation among individuals in
the number of copies
From Relative quantity to absolute
Droplet Digital PCR workflow
Droplet readings converted to a digital signal
Dynamic range of detection
Commercially available machines
-QuantaSoft (Life Technologies)
-Bio Rad QX100
APPLICATION OF PCR
Detection of mutations
• Detection of specific species of organisms
• Prenatal sex determination
• Paternity testing
• Diagnosis of genetic diseases
Reverse transcriptase PCR (rt-PCR
• Use RNA / mRNA as a template
• Usually associated with expression studies
• Can be quantitative
Block – Pelteir effect for heat transfer
Heated air – reduce pelteir effect
Block with thermal conductive field