Presentation on theme: "PCR POLYMERASE CHAIN REACTION Dr. Venkateswaran A."— Presentation transcript:
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 The foundation for this invention was built on the concept put forward by Dr. HarGobind Khurana in 1971
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
PCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 5’3’ 5’ 3’5’ 3’ 5’ 3’5’ 3’ 5’ 3’5’ 3’ 5’3’ 5’
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
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 strandphosphate grouphydroxyl group 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
DNA Between The Primers Doubles With Each Thermal Cycle 0 Cycles Number 1 3 8 2 4 1 2 4 16 5 32 6 64
Theoretical Yield Of PCR Theoretical yield = 2 n-1 x y Where y = the starting number of copies and n = the number of thermal cycles = 107,374,182,400 If you start with 100 copies, how many copies are made in 30 cycles? 2 n-1 x y = 2 29 x 100 = 1,073,741,824 x 100
PCR in Simple Terms An invisible quantity of few molecules of a DNA fragment yields millions of copies of the same DNA fragment in visible quantity in a short time
More Cycles = More DNA Yield is confirmed by Gel Electrophoresis Number of cycles 0 10 15 20 25 30 Size Marker
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
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
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
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.
Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated THANK YOU PCR Animation