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PCRPolymerase Chain Reaction
Thermocycler.Typies & Applications
BY: AQEEL HADITHE osmania university BIOCHEMISTRY sem. III 1007-13-514-005
20-9-2014
( Very good mind behind
this techniques )
Kary Banks Mullis - 1983
a scientist working for the ( Cetus Corporation )
A Biotech Company of USA - northern California when he came up with the idea for the polymerase chain reaction. --------------------------------------------------------------------------------- Developed PCR in 1983
won the Nobel Prize in 1993.
Polymerase Chain Reaction• Polymerase chain reaction (PCR) :- Is nucleic acid amplification technology , that allows small amounts of genetic material to be amplified into billions of copies in just a few hours .
---( enzymatically replicating of DNA without using a living organism, such as E. coli or yeast )
--- The techniques was developed based on the discovery of the (biological activity at high temperatures of DNA polymerases ) Which found in thermo.philes (bacteria that live in hot springs).
Materials needed for PCR (Reaction Components)Reaction Components)
--------------------------------------------------------------------------------------
1- Target DNA (the DNA you want to copy)
( DNA region to be amplified )
The DNA can be from - animals - plants - viruses - bacteria.
Range concentration - 1-2 µl ( for a total reaction mixture of 10 µl)
2 - Free Nucleotides (A, T, C, G) :- PCR Nucleotide Mix is a premixed solution containing the sodium salts of (De.oxy nucleotide triphosphate ) dATP, dCTP, dGTP and dTTP, -- each at a concentration of 10mM in water. -- They are the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Range concentration - 0.5 µl (for 10µl reaction mixture)
3- DNA Primers ( not RNA Primes ) -- Two primers ( forward & reverse ) -- short.long -20 nucleotides sd –DNA ( oligonucleotides ) that are synthesized to correspond to the ( beginning and ending ) of the DNA stretch to be copied .-- They are complementary to the 5' or 3' ends of the DNA region
-- Optimal length of PCR primers is ( 18-22 bp ) Range conc. - 1 µl ( for a total reaction mixture of 10 µl)------------------------------------------------------------------------------------------------------------------------------------
* We can also use RNA primer in PCR But There are two main reason why we are not using it :-
-- first point :- is ( tability ) DNA primer is more stable than RNA
-- second point :- is ( hybridi.zation ) DNA primer bind to template more efficient than RNA primer.
• Primers: Short artificial DNA sequences which define the DNA sequence to be amplified as they bind (anneal) to the DNA template and act as starting points for the DNA polymerase.
• - specificity and the temperature of annealing are ----- ( partly dependent )on primer length
• The primers should be ~20 bases ( long enough for adequate thespecificity ), and short enough to bind easily to the template at the annealing temperature.
• the primers should not be too short ----- ( as specificity decreases )
Primer length
Primer.. Secondary Structures : IN PCR we must avoid Cross homology --- Primers designed for a sequence must not amplify other genes in the mixture.
Primer Secondary Structures ---- produced by intermolecular interactions
• Lead to poor or no yield of the product.
Such as :-
– Hairpins intermolecular interaction within the
primer ( in the primer itself )
- Self Dimer intermolecular interactions
( between the two primers ),
- where the primer is homologous to itself.
They reduce the product yield.
4 - Taq Polymerase (heat stable DNA Polymerase III)
An enzyme that ( able to work in high Temo. 95 C )moves along the segment of DNA, reading its code and assembling a copy Range 0.2ul of (in 10µl of reaction mix)-------------------------------------------------------------------------------------------------------------------
5- Buffer solution•Contains Divalent cations like Mg+2 -- Mg2+ (cofactor that DNA Polymerase III needs to work) -- Provides suitable chemical environment for optimum activity and stability the DNA polymerase and other components of the reaction. Range - 1µl ( for a total reaction mixture of 10 µl)---------------------------------------------------------------------------------------------------------------------------------------------------------------------
6 - Sterile deionized water It’s quantity is variable--------------------------------------------------------------------------------------------------------------------------------------------------------------------------
7 - Thermocycle PCR machine (machine that changes temperatures)
PCR is repeated cycling of three steps:
1. Denature DNA
The DNA is heated to 95° C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single stranded DNA.
2. Primer Annealing
The mixture is cooled to 50° C. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.
3. Extension
The reaction is then heated to 72° C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template.
4. Go to Step 1 ( 20 - 35 X )
--------------------------------------------------------------------------------------
- These steps are repeated 20-35 times.
- In PCR, amplification is exponential because for each cycle, the DNA made in the previous cycles can also serve as template .
PCR
Melting for all the DNA
94 oClong time
Melting for only the fragment DNA
94 oCShort timeAnnealing
Primers
50 oC
Extension
72 oC
Tem
pera
ture
100
0
50
Time
30x
5’
Types of the PCR
• Conventional (basic) PCR • Restriction fragment length polymorphism (RFLP)PCR • Multiplex tandam PCR (MT-PCR)• Nested PCR• Random amplification of polymorphic DNA PCR (RAPD)• Amplified Fragment Length Polymorphisms (AFLPs ) PCR• Reverse Transcriptase PCR (RT-PCR)• Real time PCR (Rtime-PCR)• Colony PCR• Hot Start PCR• Asymmetric PCR• Long PCR• Allele specific PCR
Some of PCR application1 - DNA fingerprinting
2 - Production of DNA for sequencing
3 - Mapping the human genome
4 - The isolation of a particular gene
5 - Generation of probes
6 - Cloning a Gene encoding a known protein
7 - Amplification of ( old DNA - cloned DNA from Vectors )
8 - Detecting Bacterial or Viral Infection
a- AIDS infection
b- Tuberculosis (Mycobacterium tuberculosis) 9 - Genetics Diagnosis a - Diagnosing inherited disorders - Cystic fibrosis - Muscular dystrophy - Haemophilia A and B - Sickle cell anaemia b- Diagnosing cancer c- Blood group typing
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