100
50
0
Series1
Series2
1 2 3 4 5 6 7 8
Series1 0 25 50 60 65 73 80 83
Series2 0 0 0 0 0.1 0.1 0.2 0.4
Series3
Series3
Series4
IP (no net charge): ampholytes, set up pH gradients
Anion column: DEAE-sephadex
Cation column: phosphocellulose
Columns filled with porous resins.
Void volume: volume of buffer surrounding the beads
Source: Chapter 20:
Important feature of pol I can be cleaved by mild proteolytic treatment
into two polypeptides: a large fragment (the Klenow fragment),
which has polymerase and proofreading (3’→5’ exonuclease)
activities; a small fragment with 5’→3’ exonuclease activity.
Klenow fragment used in molecular biology when DNA synthesis
required and destruction of one of the parental DNA strands, or primer
Klenow fragment used to perform DNA end-filling and can used
to sequence DNA
Crystal structure of the Klenow fragment in 1987,------ first
look at fine structure of a DNA-synthesizing machine.
Revise: DNA replication major stages and role od DNA Pol I
and DNA pol III
Self Reading the major DNA poIII Subunits (Chapter
21,Weaver)
Source Chapter 21, Weaver
The Pol III core does not function processively by itself, can
replicate only a short stretch of DNA before falling template.
By contrast,core plus Beta-subunit can replicate DNA
processively at a rate approaching 1000 nt/sec.
Beta-subunit forms a dimer :ring-shaped. This ring fits around a
DNA template and interacts with the Alpha-subunit of core
Experimet
What is Clamp? What is Clamp Loader??
Chapter 21: Weaver
High-throughput sequencing allows
very rapid sequencing of genomes----- if genome of one member of
species already sequenced.
In pyrosequencing, nucleotides added one by one, and incorporation of
a nucleotide detected by release of pyrophosphate---- chain of
reactions to a fl ash of light.
Another method ,developed by Illumina company, uses short pieces
of DNA amplified in tiny, closely spaced patches on a support surface.
These DNA pieces sequenced by adding fluorescent, chain-terminating
nucleotides,color of whose fluorescence reveals identity.
colors visualized with a microscope fitted with CCD camera.
After each round of DNA elongation, fluorescent and chainterminating groups removed and process repeated to obtain whole
fragment’s sequence.
Protein Engineering with Cloned Genes: Site-Directed
Mutagenesis
Using cloned genes, introduce changes , thus altering amino acid
sequences of protein products.
Mutagenized DNA can be made with double-stranded DNA, two
complementary mutagenic primers, and PCR. Simply
Digesting PCR product with DpnI removes almost all of the wildtype DNA, so cells can be transformed primarily with mutagenized
DNA.
Protein Engineering with Cloned Genes: Site-Directed
Mutagenesis
Measure the conc. Of transcript, same labeled primer uesd for
Sequencing-( used as marker for the extended labeled primer.
The 5’ end-first base of transcript can be predicted
Run-off transcription: checking the efficiency and accuracy of
in vitro transcription.
A gene truncated in the middle and transcribed in vitro in
presence of labeled nucleotides.
RNA polymerase runs off end and releases an incomplete
transcript. Size of this run-off transcript locates transcription
start site, and amount of this transcript reflects the efficiency of
transcription.
In G-less cassette transcription, a promoter fused to dsDNA
cassette lacking G’s in nontemplate strand, then construct
transcribed in vitro in absence of GTP.
Transcription aborts at end of cassette, yielding a predictable
size band on gel electrophoresis.
Cut the cloned gene-whose transcription is to be measured
Summary:
Nuclear run-on transcription : ascertaining which genes
active in a given cell by allowing transcription of these genes
to continue in isolated nuclei. Specific transcripts can be
identified by their hybridization to known DNAs on dot
blots.
The run-on assay can also be used to determine
the effects of assay conditions on nuclear transcription.
Measuring Transcription Rates in Vivo
Summary:
To measure activity of a promoter, one can link it to a
reporter gene, such as genes encoding b-galactosidase, CAT,
or luciferase
Reporter gene products indicate activity of promoter.
One can also use reporter genes to detect changes in
translational efficiency after altering regions of a gene that
affect translation.
Gene expression can be quantified by measuring accumulation of
protein products of genes.
Immunoblotting
Immunoprecipitation
Chapter 2:
Objectives:
-The nature of Genetic Material
-transformation in bacteria
-chemical nature of polynucleotides
-DNA structure
-experimental background
-double helix
-a variety of DNA structure
-Physical chemistry of Nucleic acids
-
Chapter 2:
Objectives:
-The nature of Genetic Material
-transformation in bacteria
-chemical nature of polynucleotides
-DNA structure
-experimental background
-double helix
-a variety of DNA structure
-Physical chemistry of Nucleic acids
-
Molecular Biology and A brief History
-What is Molecular Biology?
Study gene function and structure @molecular level
-Genes exist in form of
alleles(dominant/recessive/heterozygot)
- mitosis and Meiosis ----self reading
-Chromosomes arranged in linear fashion
-farther apart genes----more likely recombination
Nature of Genetic material
DNA as genetic material ----Griffith experiment
Transforming unit =DNA
•Tools used: ultracentrifugation,electrophoresis,
Spectrophotmeter, Elementary chemical
-Mistaken notion:DNA include repeat of 4
nucleatides
-watson&Crick:Nucleotids not in equal proportion
Hershay experiment Findings:
-Phage protein out, DNA got in confirmed?
Chemical nature of polynucleotide:
-DNA/RNA composed of nucleotides,base in 1’
&P joins 3’, 5’ of sugar
DNA 3D structure improved by:
-study helix in protein, double helix DNA
-X-ray diffraction
-Chargaff findings
-Franklin (regular repeating
structure/Paradox?
-Length of one turn=34A,10bp /turn
--spacing between bp of helix=3.4A
-Watson&Crick:DNA d helix,sugar-p outside,bp
inside
-AT&CG,antiparallel stands
--replication
-Physical Chemistry of NA.
-Figs. B,A(right handed)&Z (left)forms
-cells exist in B form,small fraction Z
-RNA-DNA ---A form
-Forms differ in residue/turn,distance required
/turn
Separating 2 DNA strands:
-G C contents: ratio G/C&A/T fixed but
%G+C(content) vary
Refer table 2.3 ,22-73% reflect physical differences
in DNA
Tm: definition, hyperchromic shift
GC effect:
1- +ve correlation with Tm increase? 2- +ve
correlation with Density(CsCl
-
-complexity of DNA
-Repetitive DNA
-Hybridization?
DNA sizes expressed 3 ways?
1-length/bp/length
10bp/turn=34A----2-Mwt (one pair nucleotide)x660
3-No of bp
Self solving problems –Chapter 2
-complexity of DNA
-Repetitive DNA
-Hybridization?
DNA sizes expressed 3 ways?
1-length/bp/length
10bp/turn=34A----2-Mwt (one pair nucleotide)x660
3-No of bp
How do we measure DNA sizes
Electron microscope
Problems?p.35
Relationship between DNA size
&genetic capacity?
Problem p.35