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Genetic Engineering 1 Lecture 18 Pages 323 - 340 The Tools of Molecular Biology Old fashioned way was to breed for what you wanted 10_01_experiment.DNA .jpg Man has been doing this since the Agricultural revolution, some 10,000 years ago… The Chinese have been doing it! Mendel did it too! What do we do NOW… • Breeding is still used. • However, quicker and more results orientated methods now exist due to technology. • First, we may need to filter the cell types… • Label target cells with florescent dye attached to characteristic cell specific proteins – Brain cells have some unique proteins – Liver cells have other unique proteins, etc. Still used - an automated cell sorter 10_02_cell_sorter.jpg Once you have the cells what next? • OPTIONS • Issue was to examine the DNA in a consistent manner • Best method (Restriction Digestion) is to use restriction enzymes (REs) – Come mainly from bacteria • They are bacterial immune systems against foreign DNA – They are all Proteins – Many types have been isolated from different species of bacteria – Scientists use REs individually or in a mix 10_04_Restrict.nuclease.jpg What do you do with these digested fragments of DNA? • Isolate those that you want to work on • How? – Best method is to use gel electrophoresis • Agarose • Polyacrylamide 10_05_gel.electrophor.jpg Then what do you do with this piece of DNA? • EITHER – Clone – Sequence • Rely on the use of dideoxy nucleotides 10_07_1_enzym.dideoxy.jpg 10_07_2_enzym.dideoxy.jpg 10_08_DNA.sequencing .jpg Other methods used Shotgun cloning In the Dark… 10_09_Shotgun.sequenc.jpg Use computer software to assemble Problems with this method What can they be? 10_10_Repetit.sequence.jpg YACs BACs Cloning Techniques used to obtain DNA sequence information 10_11_BAC.clones.jpg 10_12_de_renaturation.jpg 10_13_hybridization.jpg Blotting Purpose = to make a permanent record of the results of a gel electrophoresis. The compass • Southern blots - DNA • Northern blots - RNA • Western blots - Proteins • Eastern blots - ? 10_14_1_Southrn.blotting.jpg 10_14_2_Southrn.blotting.jpg DNA microassays New techniques Many uses DNA Chips 10_15_DNA.microarrays .jpg
