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Lecture 1 Problem: From an E. coli cell extract, you assay enzyme activity for beta-galactosidase. You divide the extract into two samples, one of which you treat with SDS (sodum dodecyl sulfate). Both samples are further divided into 2 samples each which are alternatively assayed for enzyme activity and subjected to Western Analysis (immunological testing with beta-galactosidase antibody). These are the results:Enzyme Activity Antigenic ResponseExtract only YESYESExtract +SDS NOYES
Give a molecular/biochemical explanation of these results.
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Lecture 2DNA Structure and ReplicationTopics: StructureSynthesisDNA Sequencing & PCR
Reading: Chapter 4: 101-6; 131-7Chapter 9: 372-5Molecular Biology syllabus web site
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All nucleotides have a common structure
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There are five principal bases in nucleic acidsA, G, T, C are present in DNAA, G, U, C are present in RNA
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Nucleotide subunits are linked together by phosphodiester bonds
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Native DNA is a double helix of complementary antiparallel chains held together by: Hydrogen bonding between complementary base pairs (A-T or G-C) Hydrophobic interactions between planar bases
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Forces that maintain DNA as a double strand.
are destroyed by formamide, high pH (NaOH), high temperature
H-bondingHydrophobic interactions (cooperative base stacking)
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DNA can undergo reversible strand separation
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Analysis of DNA denaturationTm= temperature at which half the bases in a double stranded DNA sample have denatured
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Many DNA molecules are circular and local unwinding of circular DNA can produce supercoilingsupercoiledrelaxed
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RequirementsEnzyme: DNA PolymeraseDNA Template3 OH (primer of DNA or RNA)Deoxynucleoside triphosphates: dATP, dGTP, dCTP, dTTPSynthesis is 5 to 3DNA Synthesis
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DNA Synthesis
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Features of DNA Polymerasesactivity functionpolymerase synthesis
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The growing replication fork shows that both strands are synthesized simultaneously
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-Problem- Q: If DNA can only be synthesized in a 5 to 3 direction, and both strands are simultaneously replicated, how can this occur? A: Discontinuous DNA Replication Discontinuous DNA Replication Discontinuous DNA Replication 3553growing fork3553?
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The Solution
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Synthesis of the lagging strand
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DNA Replication Animation
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DNA Sequencing with dye terminators
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3 OH can be used for phosphodiester bondNo 3 OH: DNA synthesis terminatesIn both cases, DNA polymerase will incorporate nucleoside monophosphates, but..
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DNA sequencing: the Sanger (dideoxy) method
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Automated DNA sequencing involves use of four different fluorescent primers allowing the simultaneous detection of all four reactions in one sample.
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DNA Sequencing Animation
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Polymerase Chain Reaction (PCR)
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PCR Animation
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