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Trends in Biotechnology
110516 Constructing and
Screening a DNA Library
Constructing and Screening a
DNA Library.
DNA libraries help to map and
sequence genomes, and are
screened for the target DNA.
Genomic Library.
Contains DNA fragments that
represent an entire genome.
Created by the following steps:
a) Total nuclear DNA is isolated
and cut with a restriction enzyme.
b) A cloning vector is also cut with
the same enzyme.
c) The two DNAs are mixed in a
test tube and placed into host cells.
d) The host cells are selected for
the recombinant DNA by antibiotics.
e) Colonies (if not using a
bacteriophage) or plaques (if using a
bacteriophage) on bacterial plates
represent successful transformation.
f) A collection of colonies or
plaques represents a library.
g) Can calculate how many clones
are needed to represent a genome.
Fig. 3.15 The
steps involved
in the
construction of
a DNA library
(Genomic).
cDNA Library.
Made from mRNA, and shows only
genes expressed by a cell at a
given time.
Reduces the amount of DNA to be
cloned because all the genome
is not being used.
Made this way:
a) Get mRNA from cells, use the
enzyme reverse transcriptase to make
one strand of DNA from the mRNA.
b) Degrade mRNA with a ribonuclease
(an enzyme that breaks down RNA) or
an alkaline (알칼리의) solution.
c) Makes the second DNA strand with
DNA polymerase.
d) Add double-stranded DNA pieces,
called “DNA linkers,” to the new DNA,
and put the recombinant DNA into a
vector.
The RNA that is used has already
been processed and does not
contain regulatory elements
such as promoters.
Fewer clones represent a library,
making screening less laborintensive.
Fig. 3.16
The
synthesis
of a
cDNA.
Fig. 3.16 The
synthesis of a
cDNA.
Screening Libraries.
DNA inserts are found by a
screening process called
“nucleic acid hybridization.”
A known DNA sequence is
used as a probe to find the
clones or plaques with the DNA
sequence we want.
Screening libraries are made this
way (Figure 3.17):
a) Bacterial colonies are
transferred from a bacterial plate
to a nylon or nitrocellulose
membrane.
b) Membranes are treated to
lyse the cells.
c) DNA on the membrane is
denatured, and single-strand
DNA probes that are labeled
attach to the desired DNA.
d) Unattached probe is washed
off, and the label gives off light to
expose photographic film.
Fig. 3.17 Colony hybridization is used to
identify bacterial cells that have a
specific recombinant plasmid.
Fig. 3.18 Probe hybridization (cDNA or DNA)
to signal-stranded DNA in cloned DNA.
Expression Libraries.
1. Made with a cloning vector
that contains the required
regulatory elements for gene
expression, such as the
promoter region (Table 3.2).
2. Can insert into host cells to
produce a protein or create a
library.
3. Useful for finding a clone
with the gene or cDNA of interest.
4. DNA probes or antibodies (a
process called “antibody
binding”) can be used to find a
DNA sequence. Antibodies can
also be used to find proteins by
Western blotting.