1. 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.

2. 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 PROTEIN sequence Hybridisation screening Antibody screening Polymerase Chain Reaction (PCR)

3. 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

4. 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

5. 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:

6. 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:

7. Lecture 3 – Selection of Recombinants & clone analysis Complementary (probe) sequences can bind to the single strands: How do you make DNA radioactive?

8. Lecture 3 – Selection of Recombinants & clone analysis

9. 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:

10. Lecture 3 – Selection of Recombinants & clone analysis

11. An actual colony hybridisation result :

12. Lecture 3 – Selection of Recombinants & clone analysis B1007 – Identifying and Studying Cloned Genes – Lecture 4 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.

13. 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.

14. 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.

15. Lecture 3 – Selection of Recombinants & clone analysis Southern Blotting.

16. Lecture 3 – Selection of Recombinants & clone analysis Gel photograph Southern blot

17. 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