PCR by AQEEL hadithe
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Polymerase chain reaction (PCR) is a technique used to amplify small segments of DNA across several orders of magnitude. PCR uses DNA polymerase to replicate a target DNA segment using primers that flank the region of interest. The reaction involves cycling between high and low temperatures to denature the DNA, anneal primers, and extend new strands. Kary Mullis developed PCR in 1983, allowing for rapid amplification of DNA without using living cells. PCR has many applications, including DNA fingerprinting, sequencing, genetic testing, and detecting bacterial/viral infections.
PCR by AQEEL hadithe
- 1. PCR Polymerase Chain Reaction Thermocycler. Typies & Applications BY: AQEEL HADITHE osmania university BIOCHEMISTRY sem. III 1007-13-514-005 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 (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)
- 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. • 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
- 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 o C long time Melting for only the fragment DNA 94 o C Short timeAnnealing Primers 50 o C Extension 72 o C Temperature 100 0 50 Time 30x 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