Transcript 02 091904 cpj

Chapter 2
Technical Foundations of
Genomics
Recombinant-DNA techniques used in
genomics
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Contents
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Introduction
Genomic and cDNA libraries
DNA Hybridization and Northern blots
Subcloning in vectors
Restriction-enzyme mapping
DNA sequencing
PCR amplification
Protein expression
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What is the main goal of genomics?
 Sequence the entire genome by cutting
it into small, manageable pieces
(fragments)
 Assemble the entire genome from the
pieces (fragments)
 Make sense of the genome
 Understand how gene expression takes
place?
 How life processes are networked?
 Understand life??
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Introduction
 Genomics built on recombinant-DNA technology
(developed since early 1970s)
 Thorough understanding of recombinant-DNA
techniques
 Prerequisite for understanding genomics
technologies
 Differences between genomics and recombinantDNA technology
 Genomics is high throughput
 Genomics is dependent on computational analysis
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Genomic and cDNA libraries
 Libraries are fragments of DNA cloned into a
vector (microbial) but these are not organized
according their natural arrangement on the
chromosomes
 Libraries are usually constructed before
sequencing (prerequisite)
 Genomic libraries are used for genomewide
sequencing
 cDNA libraries are needed for EST sequencing
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Central Dogma
DNA
Vector
Genomic library
Transcription
Vector
RNA
mRNA
cDNA
cDNA library
Translation
Proteins
Expressed Sequence Tags
ESTs
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Genomic library
 Made from fragments of genomic DNA
 Genomic DNA cut up with restriction enzymes or
randomly broken by mechanical shearing
 Fragments ligated into cloning vectors
 Small insert
 Lambda phage: 20-50 Kbp
 Plasmid: ~10 Kbp
 Large insert
 BACs (Bactetial Artificial Chromosomes) 100-300 kbp
 YACs (Yeast Artificial Chromosomes) ~ 1 MBP
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How to make a genomic library
ori
total genomic DNA
ampR
genomic
DNA
restriction
enzyme
anneal
and ligate
ampR
ori
ori
plasmid (black)
ampR
ori
ampR
same
restriction
enzyme
ori
ampR
transform E. coli;
select for
Amp resistance
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Making a cDNA library
 Step 1: Isolate RNA
 RNA is purified from
tissue or cell line
 The mRNA is then
isolated away from
ribosomal and tRNAs
 Column with oligo dT is
used to bind poly A
tissue or cell
mRNA
polyA
stationary support
polyT
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Step 2: Obtain cDNA
from RNA
 mRNA is treated with
the enzyme reverse
transcriptase (RT)
 The enzyme copies
sequence of mRNA into
first strand of DNA
 Digest RNA with
RnaseH
 Another enzyme (RT) is
used to make second
strand of cDNA
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Step 3: Transformation
 Double-stranded cDNA
is inserted into cloning
vector
 cDNA is ligated into
cloning vector (plasmid
or phage)
 Vector is transformed or
infected into bacteria
plasmid
E. Coli
bacteria
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A=T
C=G
Step 4: Library screening
(both genomic and cDNA)
 Colony DNA is attached
to membrane
 DNA is screened with
labeled probes
 DNA is labeled with
radioactivity
 Labeled DNA is allowed
to hybridize with DNA
on membrane
 After washing, positive
hybridization spots are
identified
selected
colonies
membrane
Radioactive
probe
hybridization
X-ray film
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cDNA to EST
cDNA
library
 For use in EST
sequencing
 Need to array
individual clones
 Library is spread on
bacterial plates
 Individual colonies are
picked
 Colonies are placed in
test tubes or microtiter
plates
Clone 1
2
3
4
5
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Colony picking
 Automatic colony
pickers play key role in
genomics
 Instead of manually
picking one colony at a
time, they identify and
pick multiple colonies
from plates
 Pickers then deposit
each colony into a
microtiter well
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Nucleic Acid Hybridization
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Basis of microarrays for determining gene expression
Process by which complementary strands find each other
A–T and C–G base pairing
Dependent on temperature, salt, sequence, and
concentration (High temp and low salt)
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Northern blot
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Gene expression analyzed by Northern blots
RNA samples undergo electrophoresis
RNA separated by molecular weight
Transferred to membrane
Probe labeled
 Radioactivity or antibody ligand
 Hybridized to RNA on membrane
 Hybridization dependent on time, temperature, salt
concentration, and nucleic acid sequence and
concentration
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Steps in Northern blotting
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Northern blot example
 Example of time course of
gene induction
 Upper panel: RNA after
electrophoresis (18S and 28S rRNA)
 Bands correspond to
ribosomal RNA (EtBR)
 Probe detects two bands
 Lower panel: Lower band
shows rapid induction and
then decline
 Upper band shows slower
induction, but stays induced
for longer
Time after elicitation
0
2
4
6
8
10
12
24
M
0
2
4
6
8
10
12
24
– 4.2
– 2.1
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Northern blot and microarray
0 2 5 6 7 hrs
0 2 5 6 7 9 11 hrs
DMC1 –
DMC1 –
SPS1 –
DIT1 –
SPS1 –
SPS100 –
0 2 5 6 7 9 11 hrs
DIT1 –
SPS100 –
fold
repressed
fold
induced
>20 10x 3x | 3x 10x >20
1:1
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Cross-hybridization
 Hybridization to a related, but not identical,
sequence = cross-hybridization
 Example: A probe from one member of a gene
family is likely to hybridize to all other
members
 Problem in microarrays, particularly cDNA
arrays
 Oligonucleotide arrays prescreened to
eliminate sequences likely to cross-hybridize
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Subcloning
 Propagating fragments
of cloned DNA
 Used for sequencing and
protein production
 Plasmid vectors
 Replicate in bacteria
 Resistant to antibiotics
 Cloning sites
ORI
Region
into which
DNA can
be inserted
Plasmid
cloning
vector
ampr
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Subcloning: vector and fragment
 Vector and fragment to
be inserted must have
compatible ends
 Sticky ends anneal
 Enzyme ligase makes
covalent bond between
vector and fragment
 Use of recombination
instead of restriction
sites
DNA
restriction
enzymes
fragment
cloning
vector
recombinant
plasmid
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Recombination cloning
 Uses site-specific
recombination for
subcloning
 DNA fragment flanked
by recombination sites
 Add recombinase
“Clonase®”
 Moves fragment from
one vector to another
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Transformation into bacteria
 Bacteria prepared for
transformation by
making outer membrane
permeable to DNA
 Become competent
E. coli
host cell
recombinant
plasmid
 DNA added to bacteria
 Heat shock
 Plate on selective media
transformed cell
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Restriction-enzyme mapping
 Used for physical
mapping of DNA
 Restriction enzymes cut
at defined sites
 Palindromic sequences
 Sites are landmarks on
DNA
 Then fragments are
separated by gel
electrophoresis
CGATCG
GCTAGC
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Gel electrophoresis
 DNA fragments are separated by size in electric field
 DNA negatively charged: proportional to size of
fragment
 Separated through gel matrix
 Agarose or acrylamide
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 Separate DNA
fragments are cut with
restriction enzyme
 DNA is visualized with
ethidium bromide
Log MW
Cutting a BAC with restriction enzymes
..
. .
Distance
 Binds to DNA and
fluoresces orange
 The sizes of the
fragments are
determined based on a
standard
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DNA sequencing
 Most current sequencing projects use the chain
termination method
 Also known as Sanger sequencing, after its
inventor
 Based on action of DNA polymerase
 Adds nucleotides to complementary strand
 Requires template DNA and primer
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Chain-termination sequencing
 Dideoxynucleotides stop
synthesis
 Chain terminators
 Included in amounts so
as to terminate every
time the base appears in
the template
 Use four reactions
Template
3’ ATCGGTGCATAGCTTGT 5’
Sequence reaction products
5’ TAGCCACGTATCGAACA* 3’
5’ TAGCCACGTATCGAA* 3’
5’ TAGCCACGTATCGA* 3’
5’ TAGCCACGTA* 3’
5’ TAGCCA* 3’
5’ TA* 3’
 One for each base:
A,C,G, and T
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Sequence separation
–
 Terminated chains need
to be separated
 Requires one-base-pair
resolution
 See difference between
chains of X and X+1
base pairs
 Gel electrophoresis
 Very thin gel
 High voltage
 Works with radioactive
or fluorescent labels
CAGTCAGT
+
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Sequence reading of radioactively
labeled reactions
 Radioactive labeled
reactions
A
T
C
G
–
 Gel dried
 Placed on X-ray film
 Sequence read from
bottom up
 Each lane is a different
base
+
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Sequence detection
 To detect products of
sequencing reaction
 Include labeled
nucleotides
 Formerly, radioactive
labels were used
 Now fluorescent labels
 Use different fluorescent
tag for each nucleotide
 Can run all four
reactions in same lane
TAGCCACGTATCGAA*
TAGCCACGTATC*
TAGCCACG*
TAGCCACGT*
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Sequence reading of fluorescently
labeled reactions
 Fluorescently labeled
reactions scanned by
laser as particular point
is passed
 Color picked up by
detector
 Output sent directly to
computer
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Summary of chain termination
sequencing
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Polymerase chain reaction
 Used in sequencing, diagnostics, comparative
genomics, etc.
 Uses thermostable DNA polymerase
 Able to function near boiling temperature
 Two primers complementary to sequences at 5’
and 3’ of region to be amplified
 Double-stranded DNA template
 Performed in thermal cyclers programmed to
raise and lower temperature
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PCR machines
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PCR reaction: annealing primers
 Template melted into
two strands by high heat
 > 90 degrees C
 Primers anneal to both
strands
 Polymerase makes a
copy of both strands
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PCR reaction: amplification
 Temperature raised to
melt newly made DNA
 Primers allowed to
anneal as temperature
drops
 Polymerase elongates
new second strand of
DNA
 Process repeated
 Exponential increase in
DNA
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Protein expression
 Important for proteomics
 Need large amounts of recombinant protein for
the following:
 Structure determination
 Antibody production
 Protein arrays
 Proteins made in bacteria, yeast, and insect
cells
 Then must purify the recombinant protein
away from other proteins
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Protein expression vectors
 Protein expression
vectors have the
following:
 Inducible promoters
 Tags for purification
 Histidines
 Epitopes
 Proteins
 Coding sequence
inserted in frame
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Making recombinant protein
 Expression vector transformed into bacteria
 Bacteria grown to saturation
 Compound added for induction
 e.g., IPTG
 Protein accumulates in bacteria
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Protein purification
 Contents of bacteria run
over column
 Tagged proteins bind to
column
 Examples
 Nickel column for Histagged proteins
 Anti-myc antibody
column for Myctagged proteins
 Elution yields purified
protein
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Recombinant protein
 Gel electrophoresis of
recombinant protein
shows the following:
 Soluble proteins
 Column flow-through
 Purified protein
 Four fractions from
column
1
2
SHR::MBP
SDS-PAGE
4
5
6
7
kDa
124 –
83 –
42 –
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Summary
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Libraries
Hybridization and Northern blots
Subcloning
Restriction-enzyme mapping
Sequencing
PCR
Protein expression
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458