DNA experiments Gels cyclization and Cryoelectron microscopy 26052004

DNA experiments Gels, cyclization, and Cryo-electron microscopy 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Molecular Biology l 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) l Often in practice : modify the genome of a cell or an organism, by adding a gene. 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Molecular Biology l 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. l To check that the restriction went as they planed, they check the length of the DNA fragment by gel electrophoresis. 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Gel Electrophoresis l. A gel is composed of flexible fibers and pores. l An electric field is applied on a gel containing loaded 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. 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Gel Electrophoresis 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Deoxyribo-Nucleic Acid A goes only with T, C goes only with G. Bases are linked together by a sugar backbone. The sugars are linked together by the 5’ and 3’ carbons. Hence, a non-circular strand of DNA has a 5’ carbon free at one end, and a 3’ at the other end. It provides an orientation to a strand of DNA. Figures : Mathews & van Holde, “Biochemistry” 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Sequence l. A sequence of DNA is written from the 5’ end to the 3’ end. l In double stranded DNA, the two strand have always opposite directions. Example : CGCGAATTCACG is 5’ CGCGAATTCACG 3’ |||||| 3’ GCGCTTAAGTGC 5’ l Polymerases 26/05/2004 read DNA only from 5’ to 3’. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Annealing l Two strands of DNA can “stick” their sequence are complementary. l It is often used to link two double strand together : CGATCGCG 3’ 5’ CGCGAT TAGCGC 5’ 3’ GCGCTAGC l The 26/05/2004 5’ CGCGATCGCG 3’ 3’ GCGCTAGCGC 5’ extremities are called sticky ends. Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Cyclization l. A cyclized DNA is a molecule of DNA with both ends links together. It is called plasmid. l 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. 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Probability of Cyclization l. A minicircle is more difficult to cyclize than a plasmid because of the DNA stiffness. l Experiment : put a molecule of doublestranded DNA, with sticky ends, in solution and wait a given time. l Discriminate between cyclised and linear DNA by gel electrophoresis, and count : you get a probability of cyclization for this molecule. 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Boltzmann law l The energy needed for cyclization can be related to probability by the Boltzmann law. l Thus, this experiment allows to measure the free energy of the minicircle formation reaction. 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Minicircles sequences c 11 T 15 (158 bp): GATGAATTCACGGATCCGGTTTTTTGCCCGTTTTTTGCCCGTTTTTTCCGGATCCGTACAGGAATTCTA GACCTAGGGTGCCTAATGAGCTAACTCACATTAATTGCGCC ATGGAATC t 11 T 15 (158 bp): GATGAATTCACGGATCCGGTTTTTTGCCCGTTTTTTGCCCGTTTTTTCCGGATCCGTACAGGAATTCTA GACCTAGGGTGCCTAATGAGTGCCCTTTTATAGCTTAAACGCGTTGCGCC ATGGAATC 26/05/2004 Restriction site Cla I : AT/CGAT TATA box : TTTTATA A-tracts : TTTTTT Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Cyro-electro-microscopy 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne

Stereo pairs 82. tiff 26/05/2004 One minicircle Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne 83. tiff

3 d reconstruction 26/05/2004 Arnaud Amzallag, Ecole polytechnique Fédérale de Lausanne