1. Molecular genetics (cloning) by E. Börje Lindström This learning object has been funded by the European Commissions FP6 BioMinE project

2. Definitions Genetic recombination: - DNA from two different molecules are combined – in vivo - homology is necessary Recombinant DNA: - combination of two DNA molecules in vitro - complete homology is not necessary Molecular cloning: - isolation of a bacterial population (a clone), that all cell have a specific gene sequence.

3. Why cloning? The specific gene moved from a complex environment to a less complex one easier to study 1) Structure and function of genes 2) Regulations 3) Sequencing of DNA 4) Production of proteins/enzymes/ metabolites

4. Methodology The cloning can be divided into the following steps: 1) Selection of the gene to clone:- isolation and fragmentation 2) Selection of a vector 3) Construction of a recombinant-molecule:- chromosomal DNA/plasmid 4) Introduction into a host cell 5) Selection of the ’right’ clone 6) Amplification 7) Analysis of the received DNA molecule

5. 1 0 Selection of the gene to clone Suppose that the gene is on a bacterial chromosome (genomic) chromosome gene  -isolate the chromosome - cell debris  -fragment the chromosome - enzymatic/ mechanically (shearing) 123

6. Restriction and Modification - restriction enzymes/ endonucleases - acting at specific sequences (4-6 bp) Foreign DNA entering the bacteria are destroyed by: The own bacterial DNA is protected by: -modification of the bases in these sequences - often through methylation (CH 3 -)

7. Some properties of these enzymes Restriction enzymes: -cut within the DNA-molecule - cut ds-DNA - perpendicularly (straight) or - symmetrically around the mid- point - works on any type of DNA - normally 1-5 ’cuts’ per DNA- molecule

8. Some properties of these enzymes, cont. Modification enzymes: -the mehylases recognise the same sequence as its equivalent restriction enzyme - the methylase travel along the DNA-molecule with a speed of ~ 150 bp/min - must act perfectly otherwise the cell will dye Summary: Important for the cell:-protect its own DNA - destroy foreign DNA Can be used in cloning of genes and analysis of DNA-fragments There are several different restriction-modification systems in E. coli

9. 2 0 Selection of a vector Viruses: - e.g. - phage Cosmids: - the cos-sites of the - phage Plasmids: - Some properties that make plasmids good candidates - Low molecular weight –easy to transfer - Replicate independent of the chromosome - Several copies/ cell - Closed molecules  stable  easy to isolate - Often only 1 restr. site/ enzyme - Have selection traits (antibiotic resistance)

10. Where is the vector stored? Normally the vector (plasmid) is stored in the bacterial cell! Chromosome Vector/plasmids  - isolation of the vector - linearization with restriction enzymes

11. 3 0 Construction of a recombinant- molecule In vitro -use the same restriction enzyme for the genomic DNA and the vector - here BamH1 1 2 3  + BamH1 Genomic DNA Plasmid BamH1 Tc r Ap r  + BamH1 Ap r ½ Tc Mix + DNA ligase

12. 3 0 Construction of a recombinant- molecule, cont.vector Ap r 1 3 2 Re-ligated vector Ap r Tc r 4 1 2 3 Ap r ½ Tc

13. 4 0 Introduction into a host cell Mechanism: -Transformation (free recombinant-DNA) - (Electroporation) Ap r 1 2 3 Tc r 4

14. 4 0 Introduction into a host cell, cont. Conclusion: -The original chromosome with the special gene is distributed into small pieces in the bacterial clone. - The special gene is always in some of the bacteria (a transformant)

15. 5 0 How to find the bacterium with the wanted gene? Cultivate the transformed culture Spread the culture on a nutrient plate and select for transformants! Nutrient agar + ampicillin  Replica plating Nutrient agar + ampicillin + tetracycline

16. 5 0 How to find the bacterium with the wanted gene, cont.? Among those not growing on tetracycline are those with the gene inserted! How to find? 1) Suppose that the gene is expressed  new property - Selection is possible

17. 5 0 How to find the bacterium with the wanted gene, cont.? 2) The gene is not expressed (no product) - use some types of ’probes’ (radioactive, antibodies or stained) - screening necessary

18. Colony/ plaque hybridization Master plate  Filter paper Transfer  - lyse the bacteria - denature the DNA - add the probe - wash out unbound probe  - put the filter on a X-ray film  -The searched colony! - Pick on master plate!

19. 6 0 Amplification The copy number can be increase by blocking the protein synthesis Through PCR (polymerase chain reaction)