cDNA synthesis results in the generation of 1000

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cDNA synthesis results in the generation of
1000s of cDNA molecules.
All these cDNA molecules are derived from
individual mRNAs
mRNAs 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
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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)
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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
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Blue/White selection (lacz inactivation) allows
the easy identification of recombinants.
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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
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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.
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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
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(No Transcript)
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Colony hybridisation is a technique where
radioactive probe sequences are used to identify
bacterial colonies carrying the gene of interest.
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An actual colony hybridisation result
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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.