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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
Hybridization and Northern blots
Subcloning
Restriction-enzyme mapping
DNA sequencing
PCR amplification
Protein expression
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Introduction
 Genomics built on recombinant-DNA technology
 Thorough understanding of recombinant-DNA
techniques
 Prerequisite for understanding genomics technologies
 Differences between genomics and recombinant-DNA
technology
 Genomics is high throughput
 Genomics is dependent on computational analysis
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Genomic and cDNA libraries
 Libraries are fragments of DNA cloned into a
vector
 Libraries are usually constructed before
sequencing
 Genomic libraries are used for genomewide
sequencing
 cDNA libraries are needed for EST sequencing
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
 Plasmid
 Large insert
 BACs
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Step 2: Obtain cDNA
from RNA
 mRNA is treated with
the enzyme reverse
transcriptase
 The enzyme copies
sequence of mRNA into
first strand of DNA
 Another enzyme is used
to make second strand
of cDNA
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Step 4: Library screening
 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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Steps in Northern blotting
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Northern blot example
 Example of time course of
gene induction
 Upper panel: RNA after
electrophoresis
 Bands correspond to
ribosomal RNA
 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
0
2
2
4
4
6
6
8
8
10
10
12
12
24
24
– 4.2
– 2.1
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Northern blot and microarray
0 2 5 6 7 hrs
DMC1 –
0 2 5 6 7 9 11 hrs
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Recombination cloning
 Uses site-specific
recombination for
subcloning
 DNA fragment flanked
by recombination sites
 Add recombinase
“Clonase®”
 Moves fragment from
one vector to another
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
 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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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*
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
+
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
+
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
PCR machines
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Protein expression vectors
 Protein expression
vectors have the
following:
 Inducible promoters
 Tags for purification
 Histidines
 Epitopes
 Proteins
 Coding sequence
inserted in frame
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Making recombinant protein
 Expression vector transformed into bacteria
 Bacteria grown to saturation
 Compound added for induction
 e.g., IPTG
 Protein accumulates in bacteria
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
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