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PCR POLYMERASE CHAIN REACTION

PCR POLYMERASE CHAIN REACTION. Dr. Venkateswaran A. PCR. Not a discovery – It is an Invention It is in one line – Making millions of copies of a DNA fragment Kary Mullis - inventor of pcr – won Nobel prize in 1993

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PCR POLYMERASE CHAIN REACTION

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  1. PCRPOLYMERASE CHAIN REACTION Dr. Venkateswaran A.

  2. PCR • Not a discovery – It is an Invention • It is in one line – Making millions of copies of a DNA fragment • Kary Mullis - inventor of pcr – won Nobel prize in 1993 • The foundation for this invention was built on the concept put forward by Dr. HarGobind Khurana in 1971

  3. Thermal Cyclers

  4. Thermal Cyclers • Thermal Cyclers used • Reactions in Micro titre plates or tubes • Process in short is Cycles of Heating the reaction mixture

  5. Ingredients of PCR 1 D/W 2 10X Buffer • MgCl2, EDTA • dNTPs • Primers • DNA Template • Taq

  6. Ingredients of PCR • 10X Buffer -- BSA, DMSO, Glyserol, Detergents, Gelatine – Allows Macromolecular crowding • Mg ions 1.5 mM soln, K ions, (Mn ions) • dNTPs – 0.2 mM of each of the 4 dNTPs • Primers – 2 – a)complimentary to the 5’ end b) complimentary to the 3’ end of the DNA fragment to be amplified. • DNA fragment to amplified – The Templates 100- 3000 bases upto 10000 bases. Errors more in longer fragments • Taq / Pfu / MuLV-RT

  7. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers 72 oC Temperature 50 50 oC 0 T i m e 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ PCR

  8. STEPS • Initialisation – Heat to 95 degrees C – hold for 1-9 mins– in Hot-Start PCR • Denaturation – Heat to 95 degres C and hold for 20-30 secs– Hydrogen bonds between the 2 strands broken and the dsDNA converted to ssDNA- Templates. • Annealing – Temp brought down to 50 -65 degrees C (3-5 degrees below Tm of the Primer) hold for 20-30 secs. The enzyme binds the Primers to the complimentary sites in presence of Mg ions

  9. STEPS • Extension / elongation-Heat to 72 degrees C (if Taq is used)and hold for 30 secs. DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand • 32- 35 Cycles of Denaturation, Annealing and Elongation • Final elongation – Heat to 72 degrees C and hold for 12-15 mins to allow complete extension of all the amplicons • Final hold – at 4 -15 degrees C for indefinite time for short time storage of the reaction

  10. Number 1 2 4 8 16 32 64 0 Cycles 1 2 3 4 5 6 DNA Between The Primers Doubles With Each Thermal Cycle

  11. Theoretical Yield Of PCR Theoretical yield = 2n-1x y Where y = the starting number of copies and n = the number of thermal cycles If you start with 100 copies, how many copies are made in 30 cycles? 2n-1 x y = 229 x 100 = 1,073,741,824 x 100 = 107,374,182,400

  12. PCR in Simple Terms An invisible quantity of few molecules of a DNA fragmentyields millions of copies of the same DNA fragment in visible quantity in a short time

  13. Number of cycles 0 10 15 20 25 30 Size Marker More Cycles=More DNAYield is confirmed by Gel Electrophoresis

  14. Applications • 1) Gene Sequencing • 2) Molecular Diagnosis – Genetic Disorders, Infectious Diseases eg. a) HIV virus before Ab are detectable, b) TB bacilli 1 per 1,00,000 human cells can be amplified and detected. c) Cancers – early detection certain Cancers. d)Mutations in growth cotrol genes (ras) for cancers. e) Monitoring Chemotherapy in Leukaemia – To stop treatment when abnormal DNA is eliminated – Ideal in Leukemias f) To detect recurrance of Cancers g) DNA in artificial knee / hip joints to detect infection

  15. Applications • 3) Forensic Use–a) Paternity Testing b) Identification at the time of Immigration c) Traces of DNA from sites of crime like assaults, murder, rape etc from dried blood, semen, saliva, single complete hair, DNA material of the accused from the inside of finger nail tips d) An individual’s Genetic profile is highly distinctive because many Genetic Loci are highly variable – DNA is highly stable if protected from water air and light and can remain intact indefinitely

  16. Applications • Historians and other scientists – a) Russian czars, b) 7,000 years old Egyptian Mummies, c) 40,000 years old Mammoth preserved in Ice in the Arctic d) to learn Evolutionary relationships between organisms, e) genes of microorganisms that cannot be cultured g) Mitochondrial DNA from various modern human populations indicating that Homo Sapiens originated in Africa supporting Fossil evidence

  17. Applications • DNA from saliva, hair, skin, excreta of organisms/animals/birds that cannot be caught. To estimate population size of animals / birds from their excreta in a given area. DNA in the GI Tract of Carnivores reveal Food web interactions.DNA in products made from endangered species like powdered Rhinocerus horn sold as aphrodisiac. DNA from food prepared from the meat of endangered species.

  18. THANK YOU PCR Animation Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated

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