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Brief Review of DNA Replication:. Nearest Neighbor Analysis (Kornberg 1956):. In vitro DNA synthesis •Template DNA •DNA polymerase •dNTPs. *dATP. *dCTP. *dGTP. *dTTP. •Allow DNA replication to take place •Denature DNA •Treat with micrococcal nuclease-
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Nearest Neighbor Analysis (Kornberg 1956): In vitro DNA synthesis •Template DNA •DNA polymerase •dNTPs *dATP *dCTP *dGTP *dTTP •Allow DNA replication to take place •Denature DNA •Treat with micrococcal nuclease- yields mononucleotides with 3’ terminal phosphates
ssDNA treated with micrococcal nuclease: A A C C C * G G T T T T C ssDNA: *
Exercise: List all possible dinucleotides (analysis of nearest 5’ neighbor) Which must be present in equal frequencies if the DNA strands are: Parallel ?Antiparallel?
ApA ApC ApG ApT CpA CpC CpG CpT GpA GpC GpG GpT TpA TpC TpG TpT Exercise: List all possible dinucleotides (analysis of nearest 5’ neighbor) Which must be present in equal frequencies if the DNA strands are: Parallel ?Antiparallel? ApC = TpG CpA = GpT ApG = TpC CpG = GpC ApT = TpA CpT = GpA ApC = GpT CpA = TpG ApG = CpT GpA = TpC
DNA Melting *Temperature at which DNA strands are ½ denatured is the melting temperature or Tm *GC content of DNA has a significant effect on Tm