lecture 1 problem: from an e. coli cell extract, you assay enzyme activity for beta-galactosidase....

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 YES YES

Extract +SDS NO YES

Give a molecular/biochemical explanation of these results.

Lecture 2

DNA Structure and ReplicationTopics:

Structure

Synthesis

DNA Sequencing & PCR

Reading: Chapter 4: 101-6; 131-7

Chapter 9: 372-5

Molecular Biology syllabus web 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 DNAA, 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

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

Requirements1. Enzyme: DNA Polymerase2. DNA Template3. 3’ OH (primer of DNA or

RNA)4. Deoxynucleoside

triphosphates: dATP, dGTP, dCTP, dTTP

5. Synthesis is 5’ to 3’

DNA Synthesis

DNA SynthesisH

DNA

H

H

H

Incoming dNTPPPi

2 Panimation

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

Discontinuous DNA Replication

3’5’

5’

3’

growing

fork

3’

5’

5’

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