Lec4 - Lecture 4 Molecular Biology Methods 1 (Chapters...

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Unformatted text preview: Lecture 4 Molecular Biology Methods 1 (Chapters 5.2-5.3, Lodish 6th. Ed.) (Chapters 9.2- 9.4, Lodish 5th. Ed.) 1972 - Recombinant DNA technology Generating new artificial DNA molecules (genes) by combining DNA molecules from different organisms (“Genetic Engineering”) Paul Berg assembled the first recombinant DNA molecules by combining genes from a mammalian tumor virus (SV40) and from lambda bacteriophage. His experiments, published in 1972, represented crucial first steps in the subsequent development of recombinant genetic engineering . By stepwise methods such as he devised (illustrated hereafter), individual genes could be isolated and inserted into mammalian cells or into such rapidly growing organisms as bacteria. The genes themselves could then be studied, and their protein products expressed and even manufactured in large quantity. The discovery of restriction enzymes and DNA ligase and other enzymes, captured from nature allowed for the first time to “cut and paste” DNA molecules together to generate new “recombinant” ones. In 1980 Paul Berg received the Nobel Prize in Chemistry with Walter Gilbert and Frederick Sanger, for "his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA." 1973 - Recombinant DNA “Cloning” Genetically engineered DNA molecules may be introduced and replicated (i.e., “cloned”) in foreign cells. They inserted DNA from Xenopus (a toad) into a bacterial plasmid DNA (by “cut and paste” techniques see below) and transformed bacteria with the recombinant plasmid. Bacteria replicated and made multiple copies of the recombinant plasmid. Their experiments dramatically demonstrated how recombinant DNA engineering can be used to generate artificial genes and genetically-modified organisms and its potential impact on medicine, pharmacology, industry, and agriculture. Within several years, enhanced by new techniques for mapping and rapidly sequencing genes (see further below), genetic engineering became the basis for an explosion in biotechnology that continues unabated to the present. Herbert Boyer (at UCSF) and Stanley Cohen (at Stanford University) reported in 1973 the construction of functional organisms that combined and replicated genetic information (DNA) from different species. H. Boyer S. Cohen Artificial plasmids as vehicles to carry and replicate (clone) foreign DNA fragments - Cloning vectors Origin of replication Selection marker: gene confers resistance to an antibiotic (ampicilin) Segment of DNA containing multiple restriction sites That are unique - i.e., not present anywhere else in the plasmid - and can be used to insert foreign DNA Separation of DNA fragments according to their size by gel electrophoresis Specific band can be excised and DNA purified 100 200 300 400 500 350 bp M a r k e r D N A s D N A f r a g m e n t Agarose gel Stained with Ethidium Bromide Mixture of 1....
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Lec4 - Lecture 4 Molecular Biology Methods 1 (Chapters...

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