ENGR213_Lecture6_BRAE213

ENGR213_Lecture6_BRAE213 - Lecture 6 PCR A deeper Look PCR...

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Lecture 6 PCR A deeper Look
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PCR a quick Review DNA Melting and Hybridization • Typical Temperatures: – Melting ~90 - 95 C  – Hybridization ~40 - 60 C  
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DNA The Genetic Code for Life Some new Information • The hydrogen bonds can be modified with heat, i.e, – DNA Melting Temperature – DNA Hybridization  t m 64.9 + 0.41% GC ( 29 - 500 LengthofDNA
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PCR a quick Review • Replication of a unique region of the genome to  – Identify an organism – Forensics – Gene replication – Development of a cDNA library Temperature Cycle: ~95 C melting ~50 C Annealing Ramp ~70 C Extension
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PCR a quick Review First Cycle Subsequent Cycles: •Replication is a 2 process where n  is the number of  cycles
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What else are we lacking? • Polymerase w and w/o Exonuclease • Primers (what’s that?) • Probes with “Reporters” • Reagents • Inhibitors
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New Definitions Amplicon  -- ssDNA that is amplified via DNA  Polyermase Exonuclease  -- Enzyme that clips off any  unpaired bases “proof reading” Primer  -- short, single strand of RNA that locates  the start and end of a replication region for PCR Probe  -- piece of RNA or DNA labeled with  Reporter that locates a specific region of DNA by  hybridization
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Some New Definitions Reporter  -- A Radioactive or Fluorescent signal  that is detectable  Exogenous DNA  -- DNA outside an organism Cycle  -- One cycle of PCR replication PCR -- Polymerase Chain Reaction QPCR  -- Quantitative PCR RT-PCR  -- Reverse Transcriptase PCR real time-PCR  -- Real Time PCR ICC-PCR  -- Integrated Cell Culture PCR
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Polymerases Polymerase Exo- nuclease General Info (Prokaryotic) Taq No Thermphilus Aquaticus; bacteria  grows in Hot Springs Pfu Yes Pyrococcus furiosus,  an Archea  which thrives at 100 C, first  discovered in Italy near a volcanic  vent Vent Yes Thermus litoris; isolated from a deep  sea hydrothermal vent Why such polymerase?
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