lecture 1 problem: from an e. coli cell extract, you assay enzyme activity for beta-galactosidase....
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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 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.
Polymerase Chain Reaction (PCR)
