1. TOPICS IN (NANO) BIOTECHNOLOGY Lecture 7 5th May, 2006 PhD Course

2. Genes can be cloned in recombinant DNA vectors Cloning vector Procedure for cloning a eukaryotic gene in a bacterial plasmid 1. Isolation of vector and gene-source DNA 2. Insertion of DNA into the vector 3. Introduction of cloning vector into bacterial cells 4. Cloning of cells (and foreign gene) 5. Identification of cell clones carrying the gene of interest Nucleic acid hybridization Nucleic acid probe Genomic Clones

4. cDNA Clones

5. Cloned genes are stored in DNA libraries 1.genomic library – cloned set of rDNA fragments representing the entire genome of an organism 2.cDNA library - cloned set of rDNA fragments representing genes transcribed in a particular eukaryotic cell type (no introns, extrons etc) rDNA fragments generated, ligated & cloned The larger the fragments that are cloned, the smaller the size of the library Genomic and cDNA Libraries

6. Contains at least 1 copy of each fragment Screened using nucleic acid probes to identify specific genes Subcloning usually necessary for detailed analysis of genes N = ln (1-P)/ln (1-f) e.g. Human genome = 3.2 x 10 9 bp Lambda vector can accommodate 17kbp inserts N = ln(1-0.99)/ln(1-(1.7x10 4 bp insert/ 3.2 x 10 9 bp genome)) N = 8.22 x 10 5 plaques required in library Genomic Libraries

7. mRNA represents genes that are actively transcribed (or expressed) Eukaryotic mRNA – introns have been removed mRNA – converted into a DNA copy (cDNA) Size of library depends on number of ‘messages’ More complex than genomic library cDNA Libraries

8. Genomic Libraries

9. Libraries searched using specific probe Specificity extremely important Single-stranded nucleic acid fragments Radioactive vs non-radioactive Radioisotopes serve as tag - autoradiography Chemiluminescence, colorimetric, fluorescence Sources of probes Heterologous (other species) cDNA (genomic sequences with introns/promoter elements) Probe based on protein sequence 18-21 bases sufficient (ssDNA, RNA, antibodies) ID of specific DNA sequences

10. Expression Library Detect protein product of clone using antibodies Microarray technology ID of specific DNA sequences Chromosome walking If nearby sequences have been cloned, this can be used as starting point for isolation of adjacent genes

11. The PCR clones DNA entirely in vitro Polymerase chain reaction 1.Denaturation (heat to ~94 o C) 2.Annealing (37-72 o C) 3.Extension (72 o C) Polymerase Chain Reaction

13. Class 7_ Video 1 Polymerase Chain Reaction Class 7_ Video 1a

14. Separation of DNA fragments based on size, charge and shape differences Standardised MW markers run on the same gel for size comparison Agarose gel electrophoresis

15. Gel electrophoresis Video

16. DNA digested with restriction enzymes and separated by gel electrophoresis Gel treated with NaOH to denature DNA to ssDNA DNA transferred from gel to DNA binding filter DNA ‘fixed’by baking membranes/UV Incubate with ssDNA probe Autoradiography/chemiluminescence Southern blotting

17. Southern Blotting

18. DNA sequencing

19. Sequencing_movie_1Sequencing_movie_2

20. DNA sequencing http://www.dnalc.org/shockwave/cycseq.html

21. Isolation, amplification & sequencing Class 7_Isolation Class 7_Amplification Class 7_Sequencing