Download topic B - Institute of Life Sciences

MOLECULAR BIOLOGY 2003-4
Topic B
Recombinant DNA -principles and tools
Construct a library - what for, how
Major techniques +principles
Bioinformatics - in brief
Chapter 7 (MCB)
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Motivation
From Protein to Gene
Isolate protein (in diseased blood)
Determine few aa in the sequence
Synthesize oligonucleotide
……HOW??…….
Isolate the gene
Sequence the gene
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Motivation
From Gene to Protein
Isolate genomic clone (some defect?)
Isolate relevant RNA
Sequence it
Define the aa
Comparative , search in DB
Cloning in expression vector
Protein production , biochemical function
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Additional information
Bioinformatics tools and methods
DB search
Comparative proteomics & genomics
Not in this course…
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7.1 DNA cloning with plasmid vectors
T Recombinant DNA technology depends on the ability to
produce large numbers of identical DNA molecules
(clones)
T DNA fragment of interest is ‘inserted’ into a vector DNA
molecule.
T When a DNA in vector is introduced into a host cell, large
numbers of the fragment are reproduced along with the
vector
T Two common vectors are E. coli plasmid vectors and
bacteriophage λ vectors
7.1 Plasmids are extrachromosomal selfreplicating DNA molecules
Figure 7-1
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7.1 Plasmid cloning permits isolation of DNA
fragments from complex mixtures
Figure 7-4
7.1 Restriction enzymes cut DNA molecules
at specific sequences
Figure 7-5a
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7.1 Selected restriction enzymes
Site for cutting
Length of recognition site
Blunt/sticky
Modification sensitivity
Practically - price, stability, false cut…
7.1 Restriction enzymes cut DNA molecules
at specific sequences
Figure 7-5b
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7.1 Polylinkers facilitate insertion of restriction
fragments into plasmid vectors
Figure 7-8
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7.2 Constructing DNA libraries with λ
phage and other cloning vectors
T Cloning all of the genomic DNA of higher organisms into
plasmid vectors is not practical. Instead vectors derived
from bacteriophage are used.
T A collection of clones that includes all the DNA
sequences of a given species is called a genomic library
T A genomic library can be screened for clones containing
a sequence of interest
6.3 Virus/phage particles can be counted in plaque
assays
Figure 6-14
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6.3 Bacterial viruses commonly used in biochemical
and genetic research
T T phages of E. coli
T Temperate phages (bacteriophage λ)
T Small DNA phages
T RNA phages
7.2 The bacteriophage genome
Figure 7-10
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6.3 Bacteriophage λ undergoes either lytic replication or
lysogeny following infection of E. coli
•~100/cell
•1000 more efficient
than transformation
•Length up to 50 kb
Figure 6-19
Lambda phage -An alternative method
for genomic representation
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7.2 Nearly complete genomic libraries of higher
organisms can be prepared by λ cloning
Figure 7-12
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A library in lambda bacteriophage
A larger genomic piece
More effective host bacterial transfection
Higher yield
A genomic library (25 -50 kb)
A cDNA library representing a tissue/condition
1. How to get all mRNA in a cell?
2. How to apply cloning methods ?
3. How to make mRNA isolable ?
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A cDNA library
1. Isolate ALL RNA (tRNA, mRNA, rRNA, sRNA…)
2. Purifying only mRNA on polyT beads
3. Elute all mRNA
TTTTTTTT
AAAAA
TTTTTTTT
AAAAA
TTTTTTTT
AAAAA
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Recombinant DNA technologies
DETECTION METHODS
Sequencing reaction (tirgul)
PCR and RT-PCR (tirgul)
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7.3 Identifying, analyzing, and sequencing
cloned DNA
T The most common approach to identifying a
specific clone involves screening a library by
hybridization with labeled (radioactively) DNA or
RNA probes.
T Library - A global collection of genomic region,
transcribed sequences, proteins, peptides etc
7.3 The membrane-hybridization assay
Double stranded DNA
Melt
Single-stranded DNA
DNA binds to filter
Filter
Incubate with labeled DNA
Hybridized complemetary
DNAs
Wash away labeled DNA
that did not hybridize to
DAN bound to filter
Figure 7-17
Perform autoradiography
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7.1 Small DNA molecules can be chemically
synthesized
Synthetic DNA is useful for:
generating polylinker sequences
sequencing DNA isolating clones of interest
creating site-specific mutations
Oligonucleotide for PCR
DNA chip technology
…….
Method: 3’ to 5’ direction systematic exposure of reactive sites
7.3 Oligonucleotide probes design
Probes - Unique (20 mer - 420 = 1012)
For protein - 7 aa
Degenerative probes
Figure 7-19
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7.3 Oligonucleotide probes are designed
based on partial protein sequences
Labeled - 5’
polynucleotide
kinase
Figure 7-19
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Probes may be
Synthetic poly-nucleotide
mRNA (or any genomic/ genetic string)
Antibodies
Peptides
Drug…
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7.3 Specific clones can be identified based
on properties of the encoded proteins
Figure 7-21
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Typical Questions in Molecular
research
1. Homologue gene from yeast to man ?
2. An antibody in serun of diseased condition ?
3. Cancer cell -Any new gene is expressed ?
4. Treated with drug -which transcript is suppressed?
5. Parenthood, criminal evidence - profile?
6. And many, many more….
7.3 Gel electrophoresis resolves DNA/protein
fragments of different size
Figure 7-22
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7.3 Visualization of restriction fragments
separated by gel electrophoresis
Figure 7-23
7.5 Southern blotting detects specific
DNA fragments
Figure 7-32
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7.5 Northern blotting detects specific mRNAs
Figure 7-33
Western Blot - your protein
Separation on gel a protein mixture
Blotting (solid phase matrix)
Specific probe (antibodies)
Detection method (enzyme, Radiolabel, luminescence…)
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7.3 DNA sequencing: the Sanger method
Four separate polymerization
reactions are performed
Figure 7-29a
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7.3 DNA sequencing: the Sanger (dideoxy)
method
Figure 7-29b,c
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7.3 Few words on ESTs
Large set of expressed cDNA
partially sequenced 200-500nt
Over 3 million public
In silico cloning
Very important source for finding new genes,
alternative spliced etc.
Figure 7-26
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Few words on PCR
Alternative technology to ‘classical’ cloning
Method to recover minute amounts of DNA (crime scene)
Method to detect Alternative splicing
Method to introduce mutations
Many more….
Figure 7-26
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7.6 Producing high levels of proteins from
cloned cDNAs
T Many proteins are normally expressed at very low
concentrations within cells, which makes isolation of
sufficient amounts for analysis difficult
T To overcome this problem, DNA expression vectors can
be used to produce large amounts of full length proteins
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7.6 E. coli expression systems can produce
full-length proteins
Figure 7-36
7.6 Even larger amounts of a desired protein
can be expressed with a two-step system
Figure 7-37
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7.4 Bioinformatics
T Bioinformatics is the rapidly developing area of computer
science devoted to collecting, organizing, and analyzing DNA
and protein sequences
T Using searches based on homologous sequences, stored
sequences suggest functions of newly identified genes and proteins
New technologies
New opportunities to understand the molecular level of life
The role of Bioinformatics
in the Human Genome Project
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7.4 The C. elegans genome encodes numerous
proteins specific to multicellular organisms
Analyzing complex mixtures
A cellular snapshot
T Detect the presence and the amounts of
complementary nucleic acids in complex mixtures
including total cellular RNA
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7.8 DNA microarrays: analyzing genome-wide
expression
T DNA microarrays consist of thousands of individual gene
sequences bound to closely spaced regions on the surface of a
glass microscope slide
T DNA microarrays allow the simultaneous analysis of the
expression of thousands of genes
T The combination of DNA microarray technology with genome
sequencing projects enables scientists to analyze the complete
transcriptional program of an organism during specific
physiological response or developmental processes
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7.8 A yeast genome microarray
Figure 7-39
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DNA Chip technology
The yeast genome -a snapshot
Clustering of expression data
“the cell program”
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7.8 Changes in yeast gene expression as
cells deplete glucose from the growth media
Figure 7-40a
DeRisi: Coordinated regulation of
functionally-related genes
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DeRisi: Expression Time Course
DNA Chip technology
Currently, up to 20,000 DNA
samples, or clones, can be
arrayed on each microarray.
DNA samples can include
genes with known functions
DNA samples can also
include gene fragments
(ESTs) whose function is
unknown.
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DNA CHIP analysis
in determine the ‘profile’ of a specific type of cancer
Microarray Applications
• Detect expression of thousands of genes
• Many applications
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Identification of complex genetic diseases
Drug discovery and toxicology
Mutation and polymorphism (SNP) detection
Pathogen analysis
Detect patterns of gene expression between tissues
or disease states
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7.3 Additional material
For your own fun
Technologies
7.3 Pulsed-field gel electrophoresis separates
large DNA molecules
Chromosoe
Separation
Chromosome specific
libraries
Figure 7-26
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7.5 Specific RNAs can be quantitated and
mapped on DNA by nuclease protection
Figure 7-34a,b
7.5 Transcription start sites can be mapped
by S1 protection and primer extension
Figure 7-35
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RNA association
Eukaryotic tissue
10,000-15,000 mRNA
~5 copies
~4,000 copies
~100,000 copies
abundance
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