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BSL2016 / 2018 – Lecture 7 – cDNA libraries
cDNA synthesis results in the generation of 1000’s of cDNA molecules.
All these cDNA molecules are derived from individual mRNA’s
mRNA’s are the product of active, transcribed, genes ( including, in
our example, the product of the gene coding for the cysteine rich
protein.
cDNA molecules have to cloned in order to characterise them,
and to find the cDNA molecule you are after.
cDNA molecules are usually (but not always) cloned into plasmids
BSL2016 / 2018 – Lecture 7 – cDNA libraries
cDNA populations have to be cloned. When they have been
inserted into plasmids which are used to transform bacterial
cells, you have a population of transformed bacterial cells
each of which contains a cDNA molecule.
This is called a cDNA Library.
An excellent cloning vector is
TOPO TA (Invitrogen)
BSL2016 / 2018 – Lecture 7 – cDNA libraries
3´ T-overhangs for direct ligation of PCR products
EcoR I sites flanking PCR product insertion site.
Kanamycin and ampicillin resistance genes for selection in E. coli
LacZa fragment for easy blue/white colony screening
BSL2016 / 2018 – Lecture 7 – cDNA libraries
Blue/White selection (lacz inactivation) allows the easy
identification of recombinants.
BSL2016 / 2018 – Lecture 7 – cDNA libraries
Once a cDNA library has been created, it must be screened
for the gene of interest to identify a particular cDNA clone
Standard cDNA libraries are screened by colony hybridisation
using homologous or heterologous DNA probes.
Homologous probes:
Probes which are 100% identical to the cDNA you are screening for.
Isolate protein, sequence it, deduce DNA sequence from the aa seq.
CYS-CYS-ARG-GLU-VAL-THR
TGC-TGC-CGC-GAA-GCC-ACA
BSL2016 / 2018 – Lecture 7 – cDNA libraries
Heterologous DNA probes are not 100% identical to the cDNA you
are trying to isolate, but sufficiently similar to form a DNA/DNA hybrid
Heterologous probes are usually sequences which have been cloned
from one species and which are similar to genes from other species
A cysteine rich seed gene from soybean will be similar in sequence
to a cysteine rich gene from pea – both are legumes and the proteins
in the seeds of both are very similar.
So, a cysteine rich gene from soybean will hybridise to its homologue
in the pea cDNA library.
BSL2016 / 2018 – Lecture 7 – cDNA libraries
For probes to hybridise to their targets in a colony hybridisation,
the probes have to be labelled (radioactive or non-radioactive)
Two main labelling techniques random priming and nick translation:
BSL2016 / 2018 – Lecture 7 – cDNA libraries
BSL2016 / 2018 – Lecture 7 – cDNA libraries
Colony
hybridisation is a
technique where
radioactive probe
sequences are
used to identify
bacterial colonies
carrying the gene
of interest.
BSL2016 / 2018 – Lecture 7 – cDNA libraries
An actual colony hybridisation result :
BSL2016 / 2018 – Lecture 7 – cDNA libraries
If you have already
isolated the protein,
another way of finding the
gene which codes for it is
to perform an “Expression
screen”.
First, you need to raise
antibodies against your
protein.
Antibodies will bind to
bacterial colonies expressing
“your” protein.