Screening libraries for specific genes

(is finding needle in the haystack)

Outline
I. Isolating individual clones
II. Screening by sequence
A. Hybridization
B. PCR
III. Screening by protein
structure/biological function
 Screening the library

1. Southern hybridisation (DNA-DNA hybridisation)

Using a labeled DNA probe to detect an identical
or similar sequence in the genomic or cDNA
library.

DNA can be labeled:

- radioactively using 32P
- Non-radioactively - fluorescent tags
- hapten (protein tags)
- biotin tags
- cross-link enzymes
(chromogenic assays)
 Plaque-lift hybridization-
using a lambda library

Can do this
multiple times
(replicate
experiments)
Colony screening
Colony or plaque hybridization
1. Colonies / plaques are attached to nylon membranes.
2. Colony hybridisation more difficult – should grow colonies on
membrane
3. Denature DNA with an Alkaline solution (1.5 M NaCl + 0.5 M NaOH)
4. Attach ssDNA onto membrane
- baking at 80oC in a vacuum oven
- UV cross linking
4. Prehybridisation = prevent non-specific binding of probe to membrane
Hybridization buffer
5X SSC - promote hybridisation
Dextran sulphate - increase hybridisation rate
Denhardt’s solution - blocks DNA binding sites on nylon
SDS - acts as a detergent, reduces background
Denatured salmon sperm DNA - blocks non-specific sites
Nowadays premade hybridization buffers are commercially available
and they are inexpensive and very convenient.
Plaque Colony or plaque hybridization Contd..

5. Hybridisation
Add probe DNA to pre-hybridisation
solution. Incubate at / over 55oC.
6. Stringency washing.
- low stringency - increase salt concentration
(1X SSC); reduce hybridisation temperature (55oC)
- high stringency - low salt concentration (0.1x
SSC) high temperature (65oC)
7. Autoradiography
Place membrane and X-ray film in a
cassette with an intensifying screen
8. Identify colony of interest from master
plate.
9. Confirmation by secondary/tertiary
screening

Colony
• A probe is a piece of DNA or RNA used to detect specific nucleic
acid sequences by hybridization (binding of two nucleic acid chains by
base pairing) .

• What can be used as a probe?

1. heterologous gene
- borrowed gene available from another species to identify a
similar gene in a new species.
- can reduce the stringency of hybridisation and stringency of
washing to identify similar but not identical gene.

2. cDNA clone
- use the cDNA version to identify the full-length clone from a
genomic library.

• They are radioactively /non-radioactively labeled so that the

hybridized nucleic acid can be identified by exposure to X-ray film.

• The size of probes ranges from a few nucleotides to few kilobases.

• Originally they may be double-stranded, but the working probes
must be single-stranded.

• Short probes (oligonucleotide probes) can be made by chemical

synthesis and they are single-stranded.

• Suppose we have cloned a specific gene in yeast and want to find

its homologous gene in human, then we may use the specific
yeast gene or a synthesized sequences from the conserved
sequence as probe to detect its homologous gene from the human
genomic library.

• If the gene sequence is unknown, but protein is known or partial

sequence of it is known, then oligonucleotide probes (degenerate
probe) /”guessmers”can be made and used for hybridization
under low stringency conditions.
 Degenerate probes
The protein is sequenced to determine the amino acid
sequence
- identify a segment of the protein (4-7 amino acid
sequence) which consists of a series of amino acids
of limited degeneracy

Probe for a gene from a sequenced protein: eg.

his-phe-pro-phe-met
 Radioactive labeling
Isotopes: 32P, 33P, 35S or 3H

Labeling of new strands during in vitro DNA or RNA synthesis.

In this type of procedure, DNA or RNA polymerase is used to

make labeled DNA or RNA copies of a starting DNA.

The in vitro DNA or RNA synthesis reaction requires that at

least one of the four nucleotide precursors carries a labeled
group.

Labeling of DNA by in vitro DNA synthesis is normally

accomplished using one of following methods:
End labelling
Nick-translation
Random primed labeling
PCR-mediated labeling
 End labeling
This type of procedure involves addition of
labeled group to one or a few terminal
nucleotides.

1) Kinase end labelling:

The 5′-terminal phosphate of the

oligonucleotide is replaced in an
exchange reaction by the 32P-
labeled γ-phosphate of [γ-32P]ATP
using Polynucleotide kinase
2) Fill in end labelling
a) creating recessed 3’ ends
- using restriction enzymes at low
concentrations
b) end fill with radioactively labelled
dNTPs
- using Klenow fragment of DNA
polymerase-1 and incubation at 37oC,
60 min.
c) excess labelled dNTPs are removed
from the probe
- by Column purification –(sephadex
G50 spun column)

Disadvantages of end labelling-

- Results in low specific activity
probes
- Need a lot of start DNA for probe
conversion
- Long incubation time up to an hour
- Excess label has to be removed.
Nick translation
a) Random nicks along the dsDNA
- is created by using pancreatic
DNase I at low concentration.
b) use DNA polymerase-1 and
labelled dNTPs.
- Incubate at 15°C for 60 min
-DNA polymerase-1 has 5’-3’
polymerase activity and 5’-3’ and
3’-5’ exonuclease activity
- adds nucleotides to the 3’ end
while degrading the 5’end
- the end result is that a significant
portion of the DNA molecule is
radioactively labelled.
- results in high specific activity
probe ( 109 dpm μg-1)
c) Excess labeled dNTP removed
- Column purification - done by
passing the reaction mixture
through a sephadex G50 spun
column
Nick translation:
Advantages -
- high specific activity probes (109 dpm μg-1)
- requires only 50 ng of DNA for labelling
- high labelling efficiency
- shorter reaction times (30-60 min) for probe
preparation

Disadvantages -
- higher concentration of DNase1 can result in the
reduction in probe length.
- Excess label has to be removed.
- Obsolete method when compared to Random
labelling
Random primer labeling
a) Denature dsDNA to ssDNA
- Boil for 2 min; can use as little as 25 ng of start DNA
b) Add a cocktail of random hexanucleotide primers
- primers bind to complementary sequences along the
ssDNA strand
- dNTP mix of which one nucleotide is labelled
c) Use klenow DNA fragment or a modified T7 DNA
polymerases to add radioactively labelled nucleotides
- Adds dNTPs by its 5’ - 3’ polymerase activity
- Incubate 30 min at 37oC for klenow fragment of DNA
- polymerase and 2-3 min at 37°C for T7 DNA polymerase
Advantages
highest specific activity
probes (5 x 109 dpm μg-1)

requires only 25 ng of DNA

for labelling

high labelling efficiency -

70-80% incorporation of
radioactivity

No need for column

purification

shorter reaction times with

T7 DNA polymerase (5 min)
 Nonradioactive labeling and detection
Non-isotopic labeling systems involve the use of nonradioactive probes.

Direct nonisotopic labeling: A nucleotide which contains the label

that will be detected is incorporated. Often such systems involve
incorporation of modified nucleotides containing a fluorphore
(fuorescein, rhodamine, Texas red), a chemical group which can
fluoresce when exposed to light of a certain wavelength

Indirect nonisotopic labeling: Usually featuring the chemical coupling

of a modified reporter to a nucleotide precursor.
After incorporation into DNA, the reporter groups can be specifically
bound by an affinity molecule, a protein or other ligand which has a
very high affinity for the reporter group.
Conjugated to the latter is a marker molecule or group which can be
detected in a suitable assay .
 Indirect nonisotopic labeling systems
The biotin-streptavidin system utilizes the extremely high affinity of
two ligands: biotin (a naturally occurring vitamin) which acts as the
reporter, and the bacterial protein streptavidin, which is the affinity
molecule.
Biotin and streptavidin bind together extremely tightly with an affinity
constant of 10-14, one of the strongest known in biology.
Biotinylated probes can be made easily by including a suitable
biotinylated nucleotide in the labeling reaction.

Digoxigenin is a plant steroid (obtained from Digitalis plants) to which a

specific antibody has been raised.
The digoxigenin-specific antibody permits detection of nucleic acid
molecules which have incorporated nucleotides containing the
digoxigenin reporter group

A variety of different marker groups or molecules can be conjugated to

affinity molecules such as streptavidin or the digoxigenin-specific
antibody. They include various fluorophores or enzymes such as alkaline
phosphatase and peroxidase which can permit detection via colorimetric
assays or chemical luminescence assays, etc.
western blotting Protein based hybridization/
immunological screening
Involve use of antibodies that specifically recognize antigenic
determinants (epitopes) on the polypeptide
synthesized by the target clone.

Western hybridization is the commonest method

and can be used when,

1. a protein is known but there is no information regarding the gene,

this is the only way of screening for the gene.
2. Requires, Expression libraries and Development of an antibody
against the protein of interest
3. Expression libraries
- cDNA libraries constructed in expression vectors ( λgt11 or λ-Zap)
- fusion protein with a secretary signal
4. Antiserum/ antibody
- the protein is injected
into rabbits and antiserum
isolated.

5. Western hybridisation
- Proteins from plaques
/colonies are blotted onto membranes
- Incubate membrane with primary antiserum for the protein
- Remove unbound antiserum
- Incubate with a secondary antibody-enzyme conjugate which binds to the
primary antibody.
- Add the enzyme substrate and incubate.
- If it is a chromogenic sustrate, you will see a dark color corresponding to
the plaque or colony. If it is a chemiluminescent substrate expose to X-ray
film.
- Go back to the master plate and pick the right colony indicated in the
autoradiogram and do secondary and tertiary screening to get a single
plaque/colony. Use the fragment as probe to do Northerns using total RNA
of the host.
 Western blotting
The plaque lift method
(Note colony lift similar to this except it lifts a bacterial colony)
Detect antibody with secondary antibody conjugated to
reporter enzyme for direct/indirect visualization
 Functional analysis of expressed protein
• This method depends on the biological activity of the protein.

• When a protein as well as its function is known, but there is no

information regarding the gene, then functional assays can be used.

• Often referred as functional cloning or functional complementation

• In this strategy.

• A particular DNA sequence (clone in question) compensates for a

missing function in a mutant cell and thus restores the wild-type
phenotype.

• Employed for functional cloning of transcription factors and to isolate

cDNAs of several metabolic enzymes of Humans.

• Complementation can be performed in E. coli, yeast or in transgenic

animals or plants.

• This requires a cDNA library or genomic library/BAC library.

Screening by Functional Complementation
• Requires strain unable
to produce desired
product/function
• Cloned DNAs must be
in expression vector or
include elements
required for expression
• Select for restoration of
lost function
• A typical example is the possibility of cloning the mouse deafness
associated gene Shaker-2, and from there its human homolog,
DFNB3.

• Shaker-2 mutation has been previously mapped in the mouse

genome and BAC clones corresponding to this region in wild-type
mice was prepared.

• The DNAs are microinjected into Shaker-2 eggs and the

transgenic generation was screened for restoration of a normal
hearing phenotype.

• This resulted in the identification of a BAC clone corresponding to

the functional Shaker-2 gene.

• The gene encoded a cytoskeletal myosin protein.

• The identified sequence was used as a probe to screen the human

genomic library resulting in the identification of the equivalent
corresponding human gene.
Functional
complementation
of Shaker gene
Shaker-2 mice have
defects in the inner
ear, poor balance, and
deafness

The shaker 2 gene encodes myosin XV

Mutations in the human homolog can
cause deafness
 Yeast Two-Hybrid screening

• Biological processes require protein-protein interactions that

can be defined by noncovalent molecular interactions.

• This method is good for cloning transcripitional activators.

• Transcriptional activators are proteins that bind to DNA and

stimulate transcription of nearby genes.

• In the two-hybrid assay, two fusion proteins are created:

-the protein of interest (X) (bait), which is constructed
to have a DNA binding domain attached to its N-terminus,
and
-its potential binding partner (Y) (hunter), which is fused
to an activation domain.
• If protein X interacts with protein Y, the binding of
these two will form an intact and functional
transcriptional activator.

• This newly formed transcriptional activator will then

go on to transcribe a reporter gene, whose protein
product can be easily detected and measured.

• In this way, the amount of the reporter produced can

be used as a measure of interaction between our
protein of interest and its potential partner.
 Normal Transcription.
Normal transcription
requires both the DNA-
binding domain (BD) and
the activation domain (AD)
of a transcriptional
activator (TA).

The yeast two-hybrid technique

measures protein-protein
interactions by measuring
transcription of a reporter gene.
If protein X and protein Y interact,
then their DNA-binding domain and
activation domain will combine to
form a functional transcriptional
activator (TA). The TA will then
proceed to transcribe the reporter
gene that is paired with its
promoter.
Bait and Hunter Plasmids. The
yeast two-hybrid assay uses two
plasmid constructs: the bait
plasmid, which is the protein of
interest fused to a GAL4 binding
domain, and the hunter plasmid,
which is the potential binding
partner fused to a GAL4
activation domain.

The ‘bait’ and ‘hunter’ plasmids

are introduced into yeast cells by
transfection. Once transfection
has occurred, cells containing
both plasmids are selected for by
growing cells on minimal media.
Only cells containing both
plasmids have both genes
encoding for missing nutrients,
and consequently, are the only
cells that will survive.
 Review Questions: (Library Screening)
1) What are the methods employed for screening DNA libraries?

2) How do you perform screening of a plant genomic phage-library?

Explain the procedure.

3) What are probes?

4) How do you label a probe? Discuss the various methods involved

and their principles.

5) Comment on non-radioactive labeling.

6) How do you perform Western blot-mediated screening of a cDNA

expression library? Explain the procedure.

7) Explain the principle involved in Yeast Two-Hybrid screening.