PCR by aqeel hadithe
- 1. PCR Polymerase Chain Reaction Thermocycler. Typies & Applications BY: AQEEL HADITHE osmania university BIOCHEMISTRY sem. III 1007-13-514-005 akeelgmil@gmail.com 20-9-2014
- 2. ( 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. ----------------------------------------------------------------- ----------------
- 4. 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).
- 5. Materials needed for PCR (RReeaaccttiioonn CCoommppoonneennttss)) -------------------------------------------------------------------------------------- 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)
- 6. 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)
- 7. 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.
- 9. Primer length • 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 )
- 10. 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.
- 11. 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)
- 12. 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 .
- 14. PCR Melting for all the DNA 94 oC long time Melting for only the fragment DNA 94 oC 30x Annealing Short time Primers 50 oC Extension 72 oC Temperature 100 50 0 Time 5’
- 16. 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
- 17. Some of PCR application 1 - 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