DNA Replication_PCR
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DNA Replication and the Polymerase Chain Reaction Timothy G. Standish, Ph. D. ©2000 Timothy G. Standish History The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention A special DNA polymerase (Taq) is used to make many copies of a short length of DNA (100-10,000 bp) defined by primers Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry ©2000 Timothy G. Standish What PCR Can Do PCR can be used to make many copies of any DNA that is supplied as a template Starting with one original copy an almost infinite number of copies can be made using PCR “Amplified” fragments of DNA can be sequenced, cloned, probed or sized using electrophoresis Defective genes can be amplified to diagnose any number of illnesses Genes from pathogens can be amplified to identify them (i.e., HIV) Amplified fragments can act as genetic fingerprints ©2000 Timothy G. Standish How PCR Works PCR is an artificial way of doing DNA replication Instead of replicating all the DNA present, only a small segment is replicated, but this small segment is replicated many times As in replication, PCR involves: – Melting DNA – Priming – Polymerization ©2000 Timothy G. Standish Initiation - Forming the Replication Eye Origin of Replication 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ ©2000 Timothy G. Standish Extension - The Replication Fork 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ Primase Lagging Strand Okazaki fragment 5’ RNA Primers 3’ Single-strand binding proteins 5’ DNA Polymerase 5’ 3’ Helicase Leading Strand 5’ 3’ ©2000 Timothy G. Standish Functions And Their Associated Enzymes Function Melting DNA Enzyme Helicase è SSB Proteins è Topisomerase è Polymerizing DNA è DNA Polymerase Providing primer Joining nicks Primase è Ligase è ©2000 Timothy G. Standish Components of a PCR Reaction Buffer (containing Mg++) Template DNA 2 Primers that flank the fragment of DNA to be amplified dNTPs Taq DNA Polymerase (or another thermally stable DNA polymerase) ©2000 Timothy G. Standish PCR Temperature 100 Melting 94 oC Extension Annealing Primers 50 oC 50 0 94 oC 72 oC T i m e 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ 3’ 5’ 5’ 5’ 3’ 5’ 5’ 5’ 3’ 5’ 5’ 5’ 30x Melting 5’ 3’ 3’ 5’ ©2000 Timothy G. Standish 3’ Temperature 100 Melting 94 oC PCR 50 0 T i m e 3’ 5’ 5’ 3’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC PCR 50 0 T i m e 3’ 5’ Heat 5’ 3’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC 50 0 PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC T i m e 3’ 5’ 5’ 5’ 5’ 3’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC 50 0 PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC 30x T i m e 3’ 5’ Heat 5’ 5’ Heat 5’ 5’ 3’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC 50 0 PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC 30x T i m e 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ ©2000 Timothy G. Standish Temperature 100 50 0 3’ 5’ 5’ Melting 94 oC PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC 30x T i m e 5’ 5’ 5’ 3’ Heat 5’ 5’ Heat 5’ ©2000 Timothy G. Standish Temperature 100 50 0 3’ 5’ 5’ PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC 30x T i m e 5’ 5’ 5’ Melting 94 oC 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC 50 0 3’ 5’ 5’ Melting 94 oC Extension Annealing 72 oC Primers 50 oC 30x T i m e 5’ 5’ 5’ PCR 5’ 3’ Fragments of defined length 5’ 5’ 5’ 5’ 5’ 5’ ©2000 Timothy G. Standish DNA Between The Primers Doubles With Each Thermal Cycle Number 1 2 0 1 Cycles 4 8 16 32 64 2 3 4 5 6 ©2000 Timothy G. Standish More Cycles = More DNA Size Number of cycles Marker 0 10 15 20 25 30 ©2000 Timothy G. Standish Theoretical Yield Of PCR Theoretical yield = 2n x 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 x y = 230 x 100 = 1,073,741,824 x 100 = 107,374,182,400 ©2000 Timothy G. Standish How The Functions Of Replication Are Achieved During PCR Function PCR Melting DNA è Heat Polymerizing DNA è Taq DNA Polymerase Providing primer è Primers are added to the reaction mix Joining nicks è N/A as fragments are short ©2000 Timothy G. Standish ©2000 Timothy G. Standish
