Molecular Biology II Gene Cloning 4: Advanced Gene

Cloning

Screening – Nucleic Acid Hybridisation:

Any gene sequence can be isolated if a suitable nucleic probe is available


Colony Hybridisation

How to make labelled probes?

1. Plate out the cDNA library (1000s of colonies) of the organism of interest
2. Carry out colony hybridisation, using a probe appropriate for the gene of study.
3. Identify the radioactive colonies. These colonies will contain DNA that the probe can hybridise
with.
4. The colony can then be identified on the original agar plate and a culture can be grown
5. Plasmid DNA can be prepared

Ways to check culture contains the correct cDNA:

 Ensure all radioactive colonies contain identical inserts (PCR then gel)
 Sequence inserts
 Check for sequence homology to gene of study

Can be used to look for organ specific genes.

1. Plate cDNA libraries for the organs being studied.
2. Probe all plates with all of the cDNA for that organ. All colonies should be labelled

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Cloning

3. Now probe a plate with the cDNA for another organ. Any colonies that do not hybridise contain
tissue specific genes.
Subtractive Hybridisation:
A technique for constructing tissue specific libraries

1. Use RT-PCR to generate kidney ss-cDNA from mRNA

2. Anneal with excess liver mRNA
3. This will leave kidney specific ss-cDNA only
4. Separate hybrids from ss-cDNA
 Use hydroxyapatite chromatography – ds will bind to column, ss will flow through
5. Convert to ds-cDNA
6. Construct a cDNA library (commercially available, £600)

Cross Hybridisation:
Isolating homologous (but different) genes
Cross hybridise a gene library with a probe for YFG at reduced stringency
High GC content is more stable and makes this easier
Requires lower temperature
May be an artefact (not entirely homologous)

Gene orthologs – the same gene in two species (human & mouse  globin)
Gene paralogs – related genes in the same species (human  &  globins)

Proteomics:
Sometimes we can identify a phenotype, but not the genotype
We can use a protein (the final gene product) to work our way back to the gene
Protein  Protein Identity (primary sequence)  Gene Identity (DNA sequence)

I) Edman Degradation

Requires a well purified protein or a clear band on a SDS-PAGE (band can be transferred to a
membrane using western blotting, protein is eluted from the membrane)

a) Chemical modification of N-terminal residue

b) Cleavage of N-terminal residue
c) Identification of released derivative
d) Repeat a) - c) for up to 30 residues

This will provide a short N-terminal sequence

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Cannot be used on ‘spots’ on 2D-gels

II) Electrospray Ionisation Mass Spectrometry (ESI)

a) A solution of protein is sprayed into a mass spec machine in a volatile solvent

b) Solvent evaporates and protein molecules remain suspended
c) Molecules are separated by mass spec
d) Mass/charge ratio is determined

Determines the exact mass of a protein with very

high accuracy (within 1 D of a 50,000 D protein)
Requires a pure protein, and cannot use bands from
gels

III) MALDI–TOF (MS)

Matrix Assisted Laser Desorption Ionisation Time Of Flight
Determines the exact masses of a series of fragments derived from a very small protein sample

a) Proteins are separated by 1D or 2D gel electrophoresis

b) Individual spots / bands are picked and the protein is eluted from the gel
 There will be many spots or bands / cell
 Each will contain one unique protein
c) Proteins are digested by trypin (resulting in 5-70AA peptides)
d) Digest is mixed with matrix and mass spec is carried out
e) Mass/charge ratio is determined
f) The mass of each peptide is calculated
g) A computer then compares the mass of each peptide to the mass of every predicted protein
generated in silico (by the computer).
The chance of having several peptides of exact mass matching is very low
By working out the protein sequence we can determine the gene

Also known as peptide fingerprinting

IV) Tandem Mass Spectrometry (MS/MS)

Determines the AA sequence of a series of fragments derived from a very small protein sample
a) Digest protein using trypsin
b) Use mass spec to yield a spectrum
c) Individual fragments are fragmented. Fragments differ by one AA

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d) AA sequence can be determined

Gel spots can be analysed
Using the data from I – IV we need to work out the gene.

If the genome sequence is already available then we can search the database for the sequences
that we have found. The N-terminal sequence alone can be sufficient. Having both N-terminal
sequence and some internal sequences allows us to make the identity more certain.

If the genome sequence is not available then this is a lot more difficult

For any AA sequence we can design PCR primers that will encode that sequence
These primers will bind anywhere within the gene library that contains the sequence
One of these will contain the gene for the protein

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