DNA experiments

1. DNA experiments Gels, cyclization, and Cryo-electron microscopy Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

2. Molecular Biology • branch of biology dealing with the ultimate physicochemical organization of living matter and especially with the molecular basis of inheritance and protein synthesis(source : Merriam-Webster dictionary) • Often in practice : modify the genome of a cell or an organism, by adding a gene. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

3. Molecular Biology • Experimentalists need to replicate, cut and ligate pieces of DNA. They use special enzymes for that purpose. Restriction enzymes recognize particular sequences of DNA and cut it. • To check that the restriction went as they planed, they check the length of the DNA fragment by gel electrophoresis. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

4. Gel Electrophoresis • A gel is composed of flexible fibers and pores. • An electric field is applied on a gel containing loaded DNA. DNA is negatively charged : it will move toward the positive electrode. Its speed depends on the concentration of the gel, and on the size of the molecule. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

5. Gel Electrophoresis Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

6. Deoxyribo-Nucleic Acid Figures : Mathews & van Holde, “Biochemistry” Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

7. Sequence • A sequence of DNA is written from the 5’ end to the 3’ end. • In double stranded DNA, the two strand have always opposite directions. Example : CGCGAATTCACG is 5’CGCGAATTCACG 3’ | | | | | | | | | | | 3’ GCGCTTAAGTGC 5’ • Polymerases read DNA only from 5’ to 3’. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

8. Annealing • Two strands of DNA can “stick” their sequence are complementary. • It is often used to link two double strand together : CGATCGCG 3’ 5’CGCGAT TAGCGC 5’ 5’CGCGATCGATCGCG 3’ 3’ GCGCTAGC 3’ GCGCTAGCTAGCGC 5’ • The extremities are called sticky ends. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

9. Cyclization • A cyclized DNA is a molecule of DNA with both ends links together. It is called plasmid. • A plasmid is used to insert new genes in cells. It from several hundreds to few thousands bases. When it is below 200 or 300 bp, it is called a minicircle. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

10. Probability of Cyclization • A minicircle is more difficult to cyclize than a plasmid because of the DNA stiffness. • Experiment : put a molecule of double-stranded DNA, with sticky ends, in solution and wait a given time. • Discriminate between cyclised and linear DNA by gel electrophoresis, and count : you get a probability of cyclization for this molecule. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

11. Boltzmann law • The energy needed for cyclization can be related to probability by the Boltzmann law. • Thus, this experiment allows to measure the free energy of the minicircle formation reaction. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

12. Minicircles sequences c11T15 (158 bp): GATGAATTCACGGATCCGGTTTTTTGCCCGTTTTTTGCCGTTTTTTGCCC GTTTTTTGCCGTTTTTTGCCCGTTTTTTCCGGATCCGTACAGGAATTCTA GACCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCC ATGGAATC t11T15 (158 bp): GATGAATTCACGGATCCGGTTTTTTGCCCGTTTTTTGCCGTTTTTTGCCC GTTTTTTGCCGTTTTTTGCCCGTTTTTTCCGGATCCGTACAGGAATTCTA GACCTAGGGTGCCTAATGAGTGCCCTTTTATAGCTTAAACGCGTTGCGCC ATGGAATC Restriction site Cla I : AT/CGAT TATA box :TTTTATA A-tracts :TTTTTT Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

13. Cyro-electro-microscopy Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

14. Stereo pairs 82.tiff 83.tiff One minicircle Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

15. 3d reconstruction Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne