Lecture 4, 04/09/18, tue Today's lecture covered important and widely used laboratory methods and techniques in molecular biology. These techniques are important because they are majorly involved in producing the data that many algorithmic methods in bioinformatics are being applied to. Important concepts include: - restriction enzymes, blunt vs. sticky ends - gel electrophoresis - restriction mapping - pcr (polymerase chain reaction) amplification, primers, dna polymerase, - southern, northern, western blotting - cloning, plasmid vectors, ligation, antibiotic resistance, blue white selection, - dna sequencing: maxam & gilbert method (chemical), sanger method (enzymatic); shotgun sequencing - protein sequencing: possible, but more difficult and involved than dna sequencing Note: - electrophoresis separates molecules (e.g., DNA, RNA) based on size and electrical charge - pcr is sensitive to primer-dependent reaction conditions, it does not always work perfectly - in cloning, many things can go wrong - specific mechanisms (such as selection for antibiotic resistance, restriction mapping, ...) are used to recognise and correct for these errors (cells without plasmid, plasmid that don't incorporate gene, ...) - most modern dna sequencing techniques are based on the sanger method - computational methods play a key role in assembly of sequenced genome fragments - the sanger method is based on replication with random chain-termination, one reaction for each A,C,G,T - dna sequencing is not error free Resources: - Garret and Grisham: Biochemistry. Saunders College Publishing, 1995 (Parts of Chapters 1-7) - Alberts, Bray, Lewis, Raff, Roberts, Watson: Molecular Biology of the Cell. Garland Publishing, Inc., 1994 (Parts of Chapters 1-7) - Benjamin Lewin: Genes V. Oxford University Press, 1994. - Griffiths, Miller, Suzuki, Lewontin, Gelbart: An Introduction to Genetic Analysis. Freeman and Company, 1993. - Watson, Gilman, Witkowsi, Zoller: Recombinant DNA. Scientific American Books, 1992.