Presentation on theme: "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."— Presentation transcript:
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 Response Extract only YESYES Extract +SDS NOYES Give a molecular/biochemical explanation of these results.
Lecture 2 DNA Structure and Replication Topics: Structure Synthesis DNA Sequencing & PCR Reading: Chapter 4: 101-6; Chapter 9: Molecular Biology syllabus web siteweb site
Copyright (c) by W. H. Freeman and Company All nucleotides have a common structure
Copyright (c) by W. H. Freeman and Company There are five principal bases in nucleic acids A, G, T, C are present in DNA A, G, U, C are present in RNA
Nucleotide subunits are linked together by phosphodiester bonds
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
Forces that maintain DNA as a double strand…. are destroyed by formamide, high pH (NaOH), high temperature H-bonding Hydrophobic interactions (cooperative base stacking)
Copyright (c) by W. H. Freeman and Company DNA can undergo reversible strand separation
Analysis of DNA denaturation T m = temperature at which half the bases in a double stranded DNA sample have denatured
Many DNA molecules are circular and local unwinding of circular DNA can produce supercoiling supercoiledrelaxed
Requirements 1.Enzyme: DNA Polymerase 2.DNA Template 3.3’ OH (primer of DNA or RNA) 4.Deoxynucleoside triphosphates: dATP, dGTP, dCTP, dTTP 5.Synthesis is 5’ to 3’ DNA Synthesis
H DNA H H H Incoming dNTP PPi 2 P animation
Features of DNA Polymerases 5’ 3’ activity function polymerase synthesis 3’ 5’ exonuclease editing (to remove non H-bonded base) “proof- reading” 5’ 3’ exonuclease primer removal removes only H-bonded base)
The growing replication fork shows that both strands are synthesized simultaneously
-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 3’ 5’ 3’ growing fork 3’3’ 5’5’ 5’5’ 3’3’ ?
Synthesis of the lagging strand
DNA Replication Animation
DNA Sequencing with dye terminators
3’ OH can be used for phosphodiester bond No 3’ OH: DNA synthesis terminates In both cases, DNA polymerase will incorporate nucleoside monophosphates, but…..
DNA sequencing: the Sanger (dideoxy) method
Automated DNA sequencing involves use of four different fluorescent primers allowing the simultaneous detection of all four reactions in one sample.