Screening and preservation of DNA libraries, DNA

Sequencing and cloning strategies.

GEN 334
Lecture 9
 cDNA clones are copies of mRNAs

• Much of the genomic DNA is not expressed as mRNA

• Many issues about gene function are best addressed by examining
the product that they encode.
• The cDNA copies of mRNA contain primarily sequences that encode
f!nal prote!n

protein.
coded by CDNA

• Therefore, cDNA clones are useful for many studies of gene function.
Construction of cDNA clones
• Use the enzyme reverse transcriptase to copy mRNA into
complementary DNA, called cDNA. This is equivalent to the
template strand of the duplex DNA.
• Use a DNA polymerase to copy that cDNA into the nontemplate
(message synonymous) strand.
• Insert the duplex cDNA product into a cloning vector and
propagate in a host, e.g. E. coli.
cDNA: first strand synthesis
mRNA 5’ AAAAAAA 3’
Anneal oligo-dT primer TTTTT

AAAAAAA 3’
TTTTT 5’
Reverse transcriptase:
RNA-directed DNA polymerase dNTPs
RNase H
AAAAAAA 3’
TTTTT 5’
Hydrolyze remaining RNA with base

TTTTT 5’
Product is complementary DNA, called cDNA. It is equivalent to the template strand of the
duplex DNA.
 cDNA: second strand synthesis
Problem: How to get a primer for 2nd strand synthesis?

cDNA TTTTT 5’
Terminal deoxynucleotidyl transferase dCTPs
add/ transfer term nal dNTPs

CCCC TTTTT 5’

Ligate an adaptor to the 3’ end 5’ GGGG

3’
5’ GGGG
3’ CCCC TTTTT 5’
DNA polymerase dNTPs

5’ GGGG AAAAA 3’
3’ CCCC TTTTT 5’
Duplex cDNA
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 not always go!ng perfect

Ligate duplex cDNA into a plasmid

Duplex cDNA
5’ GGGG AAAAA 3’
3’ CCCC TTTTT 5’

Restriction endonuclease Cut the adaptor

GGGG AAAAA
CCCC TTTTT

Ligate duplex cDNA into a plasmid

Transform the population of cDNA plasmids into bacteria. Result is a cDNA library.
Limitations of cDNA synthesis
• First strand synthesis often does not go to completion.
• Individual cDNA clones will frequently have the reverse
complement of only part of the mRNA.
• Multiple cDNA clones from a single mRNA will be present in
the library
• Priming second strand synthesis is inefficient
• Some methods necessarily result in the loss of sequences at
the 5’ end of the nontemplate strand
How do you find a cDNA clone from the desired gene?

• A cDNA library has >100,000 individual clones.

• It contains copies of as many as 50,000 different
mRNAs .
• The frequency of occurrence of a cDNA from a
given gene reflects the abundance of the mRNA
for that gene.
• Try to find correct 1 clone in about 100,000.
 Strategies for screening cDNA clones

• Brute force screen for abundant cDNAs.

• Hybridization with a gene-specific probe.
• Express the cDNA in the host cell (i.e. make a functional protein
product)
• Specific antisera
• Labeled ligand to a receptor
• Assay for a function (complementation)
• Differential analysis
 Screening by hybridization
Spec f c f lter

Each bacterial Filter replica of

colonies contains a DNA in colonies
single type of cDNA Hybridize with a
plasmid labeled DNA from
m
gene of interest

Detect by
autoradiography t
w ll
sh ne
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Screening for an expressed product

Filter replica of
protein in colonies

Bind an antibody
specific for the
protein of interest
Detect the bound
antibody with an
enzymatic assay
(generating color
or light).
Expression screening in eukaryotic cells
“transfect”

introduce cDNA
+ plasmids into
cells
Epo
Cell line that needs a
cytokine (e.g. IL-3) Expression library:
A transformed cell line that
to grow. Has no Epo cDNA inserts in a
expresses the Epo receptor will
receptor, will not vector
now grow in Epo without IL-3.
grow in Epo. that will drive
The plasmid with the Epo
expression
receptor cDNA can be isolated
in eukaryotic cells
from this cell line.
Differential analysis
CAMA'sofallgens Angle CDNA

• Instead of looking for one particular cDNA, look for

cDNAs from all genes whose expression differs in the
process under study
• Differentiation from mesoderm to muscle
• Response to different nutrients
• Progression through S phase of the cell cycle
• Methods:
seh
• Subtractive hybridization
to

• Differential display
ferent
d!tes!ner
• Hybridization to massively parallel arrays of cDNAs.
Differential analysis applied to muscle
differentiation
d!fferent!ate
convert

myocytes
(muscle)
adipocytes (fat)
mouse 10T1/2
cells chondrocytes
multipotential 5-azacytidine (cartilage)
 Subtractive hybridization
Technique used to isolate a DNA segment that is missing
from one particular sample of DNA.

To do subtractive hybridization, both the mutant

and wild-type DNA samples are cut into
fragments of convenient size using a RE. Then
the two sets of fragments are hybridized
cut
the w!thame together. This will give hybrid molecules for all
res
enzy
me
regions of the DNA except the region of the
deletion, which is present only in the wild-type
chromosome.
to prol ferate them
p!ck uptheTumernesent
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 Differential display of RT-PCR products

• Make cDNA from all mRNA in the two different cellular

states (RT = reverse transcriptase).
• Use several sets of PCR primers to amplify a
representative sample of all the cDNAs.
• Resolve those RT- PCR products on a gel.
• Find the products that are present in only one of the
two cellular states being compared.
• Try to isolate the corresponding gene.
Sequence everything, find function later
• Determine the sequence of hundreds of thousands of
cDNA clones from libraries constructed from many
different tissues and stages of development of organism
of interest.
• Initially, the sequences are partials, and are referred to
as expressed sequence tags (ESTs).
• Use these cDNAs in high-throughput screening and
testing, e.g. expression microarrays (next presentation).
 Genomic DNA clones
• Clones of genomic DNA contain fragments of
chromosomal DNA. They are used to:
• obtain detailed structures of genes
• identify regulatory regions
not just exons

ntrons , promoters too

• map and analyze alterations to the genome,

e.g. isolate genes that when mutated cause a
hereditary disease
• direct alterations in the genome
• sequence the genome.
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Construction of libraries of genomic DNA
Partially
digest with
restriction
endonuclease
,
select ca.
20,000 bp RE RE
fragments
Total nuclear DNA from an >
per

organism with, e.g., 3 billion bp

in a haploid genome
RE RE

Mix genomic DNA fragments

with a DNA from a cloning
vector (e.g. lambda) and ligate

cos
... ...
cos cos cos

Package the
concatameric DNA
into phage particles
in vitro
 BAC vectors for large DNA inserts
promoter
S E SacB+: SacBII encodes levansucrase,
Cm(R) E
which converts sucrose to levan,
SacBII a compound toxic to the bacteria.
pBACe3.6
oriF 11.5 kb

Cut with restriction enzyme E, remove “stuffer”

Ligate to very large fragments of genomic DNA

promoter Large insert, 300kb

SacBII
S

Cm(R) SacB-: No toxic levan produced on sucrose

Not to scale.
oriF
media: positive selection for recombinants.
 How many clones make a representative library?

• P = probability that a gene is in a library

• f = fraction of the genome in a single recombinant
• f = insert size/genome size
• For N recombinants, 1-P = (1-f)expN
• ln(1-P) = N ln(1-f)
• N = ln(1-P) / ln(1-f)
• For a lambda library with an average insert size of 17 kb and a genome size of
3 billion bp, then one needs a library of 800,000 clones to have a probability of
0.99 of having all genes in the library.
usually h gher

• For a BAC library, with an average insert size of 300 kb and a genome size of 3
billion bp, then the library size required for P=0.99 is reduced to about 46,000
clones.
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 Screening libraries of genomic clones

Genomic library (ca. 800,000 recombinant phage) plus host E. coli

This s creen re quires 40 plate s w ith 2 0,00 0 phage on e ach plate .

Pour onto
plate and
incubate
Transfer DNA
overnight
from the plaques
onto a membrane
(e.g. nylon)
Each plaque has
DNA from a
different
segment of the
genome * *
* *

()
them
onto
st ck

*
Expose to X-ray film
to generate an
autoradiogram
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Sequence everything: genomics
• Instead of screening for one gene at a time, an entire
genome can be sequenced, and one can use experimental
and bioinformatic approaches to find many (all?) genes of
interest.
• Made possible by
• Substantial increases in speed of sequencing
• Larger insert libraries for larger genomes
• Combination of hierarchical sequencing (based on maps) and
whole genome shotgun sequencing
Genelibraries and screening

Screening procedures
Screening
ampl!fy

Colony and plaque hybridization

colony

Expression screening

Hybrid arrest and release

Chromosome walking (repeat screening)

 Overview of strategies for cloning genes

take fragment
tranaer t
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 Screening
The process of identifying one particular clone
containing the gene of interest from among the very
large number of others in the gene library .
b nd
Spec f callyof nterest
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-

Using nucleic acid probe to screen the library

to gene

1.
based on hybridization with nucleic acids.
2. Analyze the protein product.
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 Screening libraries
Searching the genes of interest in a DNA library

Hybridization to identify the interested DNA or

its RNA product noteveryate

Radiolabeled probes which is complementary to a

>
-
a

1.
region of the interested gene
Probes:
• An oligonucleotide derived from the sequence
of a protein product of the gene
• A DNA fragment/oligo from a related gene of
another species
2. Blotting the DNA or RNA on a membrane
3. Hybridize the labeled probe with DNA membrane
(Southern) or RNA (Northern) membrane
 Transfer the DNA in the plaque or colony to a
Nylon or nitrocellulose membrane

Phage DNA bind to

the membrane directly Bacterial colonies must be lysed to release
DNA on the membrane surface.

Hybridization (in a solution

(Alkali treatment)
Containing Nucleic acid probe)

antibody or enzyme
X-ray film(radio- Wash to remove unhybri- (modified
actively labeled ) dization probe and visualize nucleotide labeled

Line up the hybridizated region or

repeated hybridization
 Expression screening

Identify the protein product of an

interested gene

1. Protein activity
2. Western blotting using a specific
antibody
 Expression screening
If the inserts are cloned into an expression
sites, it may be expressed. Therefore, we can
screen for the expressed proteins. However,
this screening may miss the right clone
 Expression screening
Antibodies can be used to screen the
expression library.
The procedure

‘Plaque lift’ (taken by placing a membrane

on the dish of plaque)

Immersed in a solution of the antibody

Detected by other antibodies

Repeat cycles of screening to

isolate pure plaques
 Hybrid arrest and screen
Individual cDNA clones or pools of clones can be
used to hybridize to mRNA preparation

Hybrid arrest :translate the mRNA population

directly, and the inhibition of translation of some
products detected.

Hybrid release translation : purify the hybrids and

the hybridized mRNAs released from them and
translated, it identifies the protein encoded by the
cDNA clone
Definition: To clone the desired gene by repeated isolating
adjacent genomic clones from the library.

to obtain overlapping genomic clones that represent

progressively longer parts ofa particular chromosome .
 Process:
1. Prepare a probe from the end insert .

2. The probe are used to re-screen the library by

colony or plaque hybridization

3. Analyzed the new isolate clones and posited them

relative to the starting clone.
some will be overlapping.

4. Repeated the whole process using a probe from

the distal end of the second clone.
 Vector arm Genomic clone insert Vector arm

Prepare probe from

}
Restriction
ends of insert
Re-screen genomic
library

Restriction map new

genomic clones
}

}
Prepare new probes from distal ends of least overlapping insert.
Re-screen genomic library . Restriction map new genomic clones

Chromosome walking
 Screening of genomic library

• Once the genomic library has been created, it is screened to

identify the genes of interest. One of the most common library
screening technique is called colony hybridization.
• In the process of library construction, phage vectors are used
then the process of identification of genes of interest involved
is the plaques hybridization
 Colony hybridization

• Colony Hybridization is the screening of ibrary with a

labeled probe (radioactive, etc.) to identify a specific
sequence of DNA, RNA, enzyme, protein, or antibody.
 get r d of the th ngs
not DNA
wh ch are .

L
konThere
you don't
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 Plaque hybridization :
probe >
- sea of DNA( ?)

• The plaques are screened by a technique ,based on the

hybridization of oligonucleotide probe to target DNA.
• DNA is transferred directly from the Petri dish to the
filter, which is then incubated with labeled probes
 l brary
thousands
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 Samanlıkta gne aramak ?!

Screening libraries for specific genes (finding the needle

in the haystack)

I. Isolating individual clones

II. Screening by sequence
A. Hybridization
B. PCR
III. Screening by protein
structure/biological function
IV. Gene identification--diseases
check performance/funct on of prote n

reference
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 You want to clone Improved
a gene from the library
human genome… construction
Partial digest: Sau3A
(BamHI compatible
ends)

Phosphatase
So you follow the protocol for
* mak ng the l brary s not an easy th ng Ligate to lambda

Package

Or…buy a kit/premade library…

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 Basic “lytic” phage life cycle
100’s to 1000’s of
plaques (individual
phage infections)
capdeseaps d
encod ng for

nfect onsat on

Lawn of E. coli

But…which lambda clone

(plaque) has the gene of
interest????
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 about calculat on n the f nal.
there w ll be no quest on

How many recombinant DNA molecules are required in a

library to get complete coverage of a genome?

p = probability of getting a specific

piece of DNA
ln(1-p)
N=
ln(1-f) f = fractional size of clone DNA relative
to genome

N = number of clones needed

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 ln(1 - p)
N=
ln(1 - f)

p = probability of getting a specific piece

of DNA = 99%

f = fractional size of clone DNA relative to genome = 17000 base pairs (lambda
capacity) / 3 x 10 9)

N = number of clones needed = 810,000

cDNA cloning: this calculation is harder…

ln(1 - 0.99)
N= = 810,000
ln[1 - (1.7 x 104 / 3 x 109)]
 Screen by hybridization

 Very fast
 Applicable to a large number of clones
 Can identify clones that are not full length

 But you need to know at least some of the

sequence of the gene you are after (more on this
later)
 Design of nucleic acid probes
1) Known sequences: eg. previously cloned cDNA to
locate position in genome (identical match exists in
library--stringent hybridization conditions)
related genes from other spec es

2) Probes for non-identical but related sequences: finding

a related gene in another species (non-identical match-
-reduce stringency of hybridization)
3) Probing for a gene from a sequenced protein: eg.
his-phe-pro-phe-met
4) Screen by PCR

make synthetic “mixed

probe” (typically 16-mers)
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“guessmers”: long, degenerate oligo probes

• 40-60 nts, alternative to short, “mixed probe”

• Codon uncertainty mostly ignored
• Most common codon used
• Increased length improves specificity
• Inosine substitutions at uncertain positions
• Inosine pairs with all 4 bases
• Low stringency hybridizations
 hybr!d!zat!on process
w ll be a quest on

repl ca g!b! düşün !

or!entat!onoftha alan
same

“Colony
hybridization” for
ID of clones
(like Southern blotting
but without DNA
isolation/gel
electrophoresis)
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 Plaque-lift hybridization--
using a lambda library

Can do this
multiple times
(replicate
experiments)
 bu 3
ç!z!m de
adım
olab l s
yeterl!
DNA hybridization

1. The DNA of samples containing the putative target DNA is denatured,

and the single strands are kept apart, usually by binding them to a
solid support, such as a nitrocellulose or nylon membrane
DNA'nın b r yere bağlanması gerek r
>
- sol d membrane

2. The probe, which is often 100 to 1,000 bp in length, is labeled,

denatured, and mixed with the denatured putative target DNA under
hybridization conditions
3. After the hybridization reaction, the membrane is washed to remove
nonhybridized probe DNA and assayed for the presence of any
hybridized labeled tag
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 There are at least two possible sources of probes for screening a
genomic library

we share homology w th other spec es

1. Heterologous probe: Cloned DNA from a closely related organism. In this

case, the conditions of the hybridization reaction can be adjusted to permit
considerable mismatch between the probe and the target DNA to
compensate for the natural differences between the two sequences
2. A probe can be produced by chemical synthesis. The nucleotide sequence
of a synthetic probe is based on the probable nucleotide sequence that is
deduced from the known amino acid sequence of the protein encoded by
the target gene
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 Immunological screening of a gene library (colony
immunoassay)

1. Cells from the transformation reaction are plated onto solid agar medium
under conditions that permit transformed, but not nontransformed, cells to
grow
2. From the discrete colonies formed on this master plate, a sample from each
colony is transferred to a solid matrix, such as a nitrocellulose or nylon
membrane
3. The cells on the matrix are lysed, and their proteins are bound to the matrix
4. The matrix is treated with a primary antibody that binds only to the target
protein get r d of unwanted prote n
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 Immunological screening of a gene library (colony
immunoassay)
second ant body- labell ng

1. Unbound primary antibody is washed away, and the matrix is treated with
a secondary antibody that binds only to the primary antibody
2. Any unbound secondary antibody is washed away, and a colorimetric the best way

reaction is carried out. The reaction can occur only if the secondary
antibody, which is attached to an enzyme (E) that performs the reaction,
is present
3. A colony on the master plate that corresponds to a positive response on
the matrix is identified
4. Cells from the positive colony on the master plate are subcultured
because they may carry the plasmid–insert DNA construct that encodes
the protein that binds the primary antibody.
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 1

color attachment
 Screening a genomic library for enzyme activity

1. Cells of a genomic library are

plated onto solid medium
containing the substrate for the
enzyme of interest
2. If a functional enzyme is
produced by a colony that
carries a cloned gene encoding
the enzyme, the substrate is
converted to a colored product
that can be easily detected
Gene cloning by functional complementation

1. Host cells that are defective in a certain

function, e.g., A−, are transformed with
plasmids from a genomic library
derived from cells that are normal with
respect to function A, i.e., A+
2. Only transformed cells that carry a
cloned gene that confers the A+
function will grow on minimal medium
3. The cells that show complementation
are isolated, and the insert of the vector
is studied to characterize the gene that
corrects the defect in the mutant host
cells