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’ ?

The Solution

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.

DNA Sequencing Animation

Polymerase Chain Reaction (PCR)

PCR Animation