BB211: Cell and Molecular Biology

Dr Eve Lutz
Department of Bioscience
Recombinant DNA technology:
Lecture 4
Making & Screening Libraries:
Selection of a specific clone from
a pool of recombinants
References for lectures on recombinant DNA technology please
read the following:
Chapter 16
Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

What is a DNA library and why construct one?

A DNA library consists of cloned DNA fragments representing
the entire genome of an organism (genomic DNA library) or of the
protein-encoding genes only (cDNA library).

Genomic library: Contains entire DNA content of an organism

Suitable for determining genomic DNA
sequence
Requires chromosomal DNA isolation

cDNA library: Contains entire protein-encoding DNA content

Messenger RNA used as a starting material

Messenger RNA reverse-transcribed into
cDNA
Requires mRNA isolation

Isolating and replicating sections of the chromosomal DNA of an

organism in order to examine its properties (e.g. DNA sequence,
sequence of protein encoded). DNA is replicated after cloning
into vectors - usually plasmids or viruses that can exist in multiple
copies in cells.

Choosing the appropriate library

1) cDNA or genomic?

Do we want to obtain fragments of the complete genome e.g. to

get DNA sequence, look at promoter regions, see what introns are
in DNA (genomic library). Or do we want to clone protein-encoding
genes and use them for protein expression (cDNA library).

2) What vector system?

What sizes of inserts do we want to clone? Do we want to be able

to generate proteins directly? Do we want to be able to produce
single-stranded DNA and sequence it directly?

3) Which host system to use?

Usually either Escherichia coli or Saccharomyces cerevisiae.

Having created a gene library; how do we identify

a desired recombinant?
If we wish to identify a specific recombinant from a gene library,
and we know some of the sequence of that gene, then a good
method to use is DNA hybridisation. However, in many cases we
may not have this information. Another way of identifying
recombinants is to use expression screening to look for the
production of protein.

Selecting the probe

If the gene or similar sequence has been cloned from another cell
type or species then it is possible to use that cDNA or genomic
DNA fragment or an oligonucleotide derived from the gene
sequence. If the protein of interest has been isolated, then it
may be possible to raise antibodies against it, or to sequence it in
order to derive the gene sequence from the amino acid sequence -
see how to design degenerate probes. Otherwise some sort of
functional assay may have to be used to detect expression of the
desired gene.

Plate out library

Gene libraries can contain millions of different clones and it is
necessary to have a means of isolating positive clones from the
rest of the collection. Plasmid libraries (in E. coli transformants)
are often plated on to agar plates containing the selection
antibiotic to allow colonies to grow. Phage libraries are incubated
with E. coli, then plated into soft agar to allow plaques to form.

Once a library is plated onto agar, nitrocellulose filters or nylon

membranes are laid on the surface of the plates. Plaques or
colonies are 'lifted'when the filter/membrane is carefully peeled
off the plate. The filter is a replica of the original plate.
Bacterial cells within colonies are 'broken' to release their
contents by soaking membrane in detergent (SDS) and protease.
For phage, the cells have already been lysed and DNA is exposed.
In both cases, DNA is denatured with alkali and bonded firmly to
the membrane by baking at high temperature or using UV light to
cross-link it to the filter.

Several rounds of isolation may be required to purify individual

clones
Large libraries are usually plated at high density, so it is difficult
if not impossible to pick individual positive spots or clones.
Mixture of clones from first pick can be replated at lower density
and reprobed to pick out individual positives

DNA hybridisation
This is achieved by labelling a small probe - often by incorporating
radioactivity into it (see also Southern Blotting). For instance, if
we chemically synthesise an oligonucleotide, of the same sequence
as the gene of interest, this can be labelled with a radioactive
phosphate (32P) from radiolabeled ATP. This is done by removing
the 5' phosphate of the oligonucleotide using alkaline
phophatase, and replacing it with a radiolabelled phosphate
using polynucleotide kinase.

Fig. 7-20, Lodish et al. (4th ed.)

An alternative is to incorporate one or more radiolabeled
nucleotides to the 3' end of the probe using terminal
transferase.

Oligonucleotide probes can be as small as 20 nucleotides. It is

important to remember that the DNA probe must be specific for
the gene of interest and should not cross-react with other
recombinants - that is anneal to sequences present in other
clones. The probability of a specific 20 nucleotide sequence
occurring is very low, i.e. 1 in 420 (1 in ~1012) nucleotides.

The membrane is soaked with a solution of the radiolabeled

probe, to allow it to hybridise to the correct gene. The membrane
is washed extensively to remove non-specifically bound probe, and
the colonies/plaques to which the probe remains bound are
visualised by autoradiography.
 Plaque hybridisation

Fig. 7-18, Lodish et al. (4th ed.)

Expression screening
This is frequently done using libraries in phage λ expression
vectors such as λgt11. Target proteins are produced as fusion
proteins wit beta-galactosidase where the insert DNA is cloned
into the lacZ complex which is present in the lambda gt11 vector
DNA. Expression can then be switched on using IPTG. In a similar
method to the DNA hybridisation method, proteins expressed
from the library are bound to membranes or filters. The
filters/membranes are then incubated with a specific antibody to
detect the desired protein.
 Fig. 7-21, Lodish et al. (4th ed.)

There are many different expression screening methods that can

be used to isolate a particular gene, and are designed according to
what is known about the protein function.

Sometimes it is necessary to be able to clone adjacent pieces of

DNA. A technique known as chromosome walking is used to move
systematically along a chromosome from a known location and to
 clone overlapping genomic clones that represent progressively
longer parts of a particular chromosome. Chromosome walking is
used as a means of finding adjacent genes (positional cloning), or
parts of a gene which are missing in the original clone as well as to
analyse long stretchs of eukaryotic DNA

webpage last updated 20/2/03

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