cDNA libraries

1
cDNA libraries represent the mRNAs synthesised
by a cell type. Eukaryotic genes consist of
exons (functional sequences) and introns (useless
intervening sequences). Introns are removed from
primary RNA transcripts by splicing.
2
Transcription
Splicing
AAAAAAA
A poly A tail is added to the 3 end. (A
different modification occurs at the 5 end).
3
Making cDNA libraries
Purified mRNAs are converted to DNA by reverse
transcriptase (RT).
AAAAAAA
mRNA
TTTTTTT
dNTPs Reverse transcriptase
DNA
TTTTTTT
4
Degrade RNA strand with RNAse
The 3 end can fold back and prime synthesis of
2nd DNA strand by RT
Process ends using single strand nuclease
Clone cDNA into a vector to obtain library
5
Screening libraries for positive
clones Specific clones can be identified by
1. Hybridisation 2. Immunochemical
selection 3. Screening for new biological
activity conferred by cloned gene product.
6
Selection by hybridisation Libraries can be
screened by hybridisation if a DNA probe is
available that is homologous to the target
gene. To make DNA probes, DNA polymerases can
be used to replicate a DNA fragment in vitro and
incorporate labelled nucleotides.
7
Labelling probes by random priming
DNA fragment
Heat denature
5
3
5
3
Add random primers all 4096 possible
hexanucleotides
8
5
3
3
5
5
3
3
5
Add DNA polymerase dNTPs one labelled dNTP
9
Denature
Numerous labelled copies of the fragment
sequences are synthesised.
10
32P dATP is used to make radioactively labelled
probes. These can be detected by autoradiography.
b-particles blacken X-ray film
DNA immobilised on membrane
11
Digoxygenin (DIG)-dUTP is used to make
non-radioactive probes. These can be detected
using enzyme-linked anti-DIG antibodies.
Enzyme cleaves substrate. Coloured product
precipitates around hybridising DNA.
Antibody against DIG
12
Screening a library by hybridisation.
13
To clone genes for specific proteins,
oligonucleotide probes can be designed from
partial amino acid sequence data. If a purified
protein has the N-terminal sequence His-Phe-Pro-
Met-Met-Trp An oligonucleotide can be chemically
synthesised to select clones carrying the gene
for the protein. H F P M
M W 5 CAT TTC CCC ATG ATG TGG 3
C C T
A G
14
Since the genetic code is degenerate, there are
16 different oligonucleotides that could code for
this peptide.
5 CAT TTC CCC ATG ATG TGG 3 5 CAC TTC CCC ATG
ATG TGG 3 5 CAT TTT CCC ATG ATG TGG 3 5 CAC
TTT CCC ATG ATG TGG 3 5 CAT TTC CCG ATG ATG TGG
3 5 CAT TTC CCA ATG ATG TGG 3 5 CAT TTC CCT
ATG ATG TGG 3 5 CAC TTC CCG ATG ATG TGG 3 5
CAC TTC CCA ATG ATG TGG 3 5 CAC TTC CCT ATG ATG
TGG 3 5 CAT TTT CCG ATG ATG TGG 3 5 CAT TTT
CCA ATG ATG TGG 3 5 CAT TTT CCT ATG ATG TGG
3 5 CAC TTT CCG ATG ATG TGG 3 5 CAC TTT CCA
ATG ATG TGG 3 5 CAC TTT CCT ATG ATG TGG 3
A mixed probe is synthesised.
15
T4 polynucleotide kinase transfers a radioactive
phosphate from ATP to the 5 end of a
polynucleotide. This enzyme can be used to label
oligonucleotides with 32P-phosphate. Labelled
oligonucleotides are then used as probes to
identify positive clones.
16
Immunological selection Colonies or plaques are
transferred to a nylon membrane and treated with
detergent to expose proteins. The membrane is
treated with antibody specific for the protein
expressed from the target gene. Positive clones
bind antibody. The membrane is treated with
enzyme-linked secondary antibody. The enzyme
cleaves a chromogenic (colour-generating)
substrate. This results in staining of positive
clones.
17
Screening for biological activity If a cloned
gene is expressed to give a functional
protein, clones showing a new biological activity
can be identified.
For example, clones expressing an enzyme gene
can be detected using chromogenic substrates.