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 betagalactosidase antibody).
These are the results:
Enzyme Activity
Extract only
Extract +SDS

Antigenic Response

YES

YES

NO

YES

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; 131-7

Chapter 9: 372-5

Molecular Biology syllabus web site

All nucleotides have a common structure

Copyright (c) by W.

There are five principal bases in nucleic acids

A, G, T, C are present in DNA

A, G, U, C are present in RNA

Copyright (c) by W.

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.

H-bonding
Hydrophobic interactions
(cooperative base stacking)
are destroyed by formamide, high pH
(NaOH), high temperature

DNA can undergo reversible strand separation

Copyright (c) by W.

Analysis of DNA denaturation

Tm= 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

supercoiled

relaxed

DNA Synthesis
Requirements
1. Enzyme: DNA Polymerase
2. DNA Template
3. 3 OH (primer of DNA or
RNA)
4. Deoxynucleoside
triphosphates: dATP, dGTP,
dCTP, dTTP

DNA

DNA Synthesis
H
H

Incoming dNTP

animation

PPi
2P

Features of DNA Polymerases

activity

function

polymerase

synthesis

exonuclease

editing

(to remove non H-bonded base)

proof-reading

exonuclease

primer

removes only H-bonded base)

removal

The growing replication fork shows that both strands are

synthesized simultaneously

replicated, how can this occur?

5
3
3
5

5
3

growing

A: Discontinuous DNA Replication

Discontinuous DNA Replication

3
Discontinuous DNA Replication

fork

The Solution

Synthesis of the lagging strand

DNA Replication Anim

ation

DNA Sequencing with dye

terminators

In both cases, DNA polymerase will incorporate

nucleoside monophosphates, but..

3 OH can be used for

phosphodiester bond

No 3 OH: DNA synthesis

terminates

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