Transcript Southern Blotting. - +* Tudalenau Bangor Pages

Lecture 3 – Selection of Recombinants & clone analysis
The white colonies will all be recombinants, but only one
of these many colonies will contain the gene you are interested in.
To identify a colony containing a specific cloned gene,
you can use:
1) Hybridisation of colonies to specific probe sequences
2) Expression screening where you detect the product of the
cloned gene.
Lecture 3 – Selection of Recombinants & clone analysis
Remember that to identify a colony containing a specific gene,
you need to know SOMETHING about the gene.
DNA sequence
Hybridisation screening
Polymerase Chain Reaction (PCR)
PROTEIN sequence
Antibody screening
Lecture 3 – Selection of Recombinants & clone analysis
Hybridisation techniques rely on a probe sequence which is
complementary to the cloned gene, or to a sequence in the genome.
How do you get the probe???
In order to get a probe, you need to know SOMETHING
about the gene you are trying to find.
1) Protein sequence - you might have isolated the protein
and sequenced it.
From the protein sequence, you can deduce the DNA sequence:
Glu---Asp--Met--Trp--Tyr
GAA-GAT-ATG-AGG-TAT
Lecture 3 – Selection of Recombinants & clone analysis
The DNA sequence can be artificially made in a
DNA synthesiser and used as a probe
Applied
Biosystems
DNA
synthesiser
Lecture 3 – Selection of Recombinants & clone analysis
DNA hybridisation is based on the fact that the 2 strands
of the double helical DNA are complementary:
Lecture 3 – Selection of Recombinants & clone analysis
The two strands can be separated by heating or alkali –
the hydrogen bonds between the bases are broken, making
two single stranded DNA molecules:
Lecture 3 – Selection of Recombinants & clone analysis
Complementary (probe) sequences can bind to the single strands:
How do you make DNA radioactive?
Lecture 3 – Selection of Recombinants & clone analysis
Lecture 3 – Selection of Recombinants & clone analysis
If the complementary probe sequences are radioactively
tagged, the hybrid formed between the probe and the target
will also be radioactive :
You now need to detect this radioactive hybrid, so that you can
identify the clone – this is done using Colony Hybridisation:
Lecture 3 – Selection of Recombinants & clone analysis
Lecture 3 – Selection of Recombinants & clone analysis
An actual colony hybridisation result :
B1007 – Identifying and Studying Cloned Genes – Lecture 4
Lecture 3 – Selection of Recombinants & clone analysis
Once a clone has been identified as hybridising to the probe
sequence, It has to be further characterised, by isolating plasmid
DNA and mapping the insert. This procedure is called
Restriction mapping, and identifies restriction enzyme sites.
Lecture 3 – Selection of Recombinants & clone analysis
By analysing the number and size of fragments produced
by restriction enzyme cleavage, a “map” of the DNA fragment
can be produced.
This map is unique, and
defines the sequence
which has been cloned.
Lecture 3 – Selection of Recombinants & clone analysis
Within the cloned sequence, there will be a part which contains
the gene of interest, and a segment which does not. The easiest
way of finding out which segment of the cloned sequence carries
a gene is to use a technique called Southern blotting.
Southern Blotting was invented by Prof Ed. Southern of
Edinburgh University and is a way of transferring DNA from a
gel to a membrane, wherethey can be hybridised to radioactively
tagged probe sequences
It allows you to precisely locate the fragment in your cloned
sequence which contains the gene you are trying to isolate.
Lecture 3 – Selection of Recombinants & clone analysis
Southern Blotting.
Lecture 3 – Selection of Recombinants & clone analysis
Gel photograph
Southern blot
Lecture 3 – Selection of Recombinants & clone analysis
Southern blotting also allows you to detect specific genes
in a genome.
It is so sensitive that you can identify one gene out of the whole
genome.
Restricted genomic DNA
Autorad of genomic blot