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Genetic Engineering 1

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!

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Genetic Engineering 1

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  1. Genetic Engineering 1 Lecture 18 Pages 323 - 340

  2. The Tools of Molecular Biology

  3. 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!

  4. 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.

  5. Still used - an automated cell sorter 10_02_cell_sorter.jpg

  6. 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

  7. 10_04_Restrict.nuclease.jpg

  8. 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

  9. 10_05_gel.electrophor.jpg

  10. Then what do you do with this piece of DNA? • EITHER • Clone • Sequence • Rely on the use of dideoxy nucleotides

  11. 10_07_1_enzym.dideoxy.jpg

  12. 10_07_2_enzym.dideoxy.jpg

  13. 10_08_DNA.sequencing .jpg

  14. Other methods used Shotgun cloning In the Dark…

  15. 10_09_Shotgun.sequenc.jpg Use computer software to assemble

  16. Problems with this method What can they be?

  17. 10_10_Repetit.sequence.jpg

  18. YACsBACs Cloning Techniques used to obtain DNA sequence information

  19. 10_11_BAC.clones.jpg

  20. 10_12_de_renaturation.jpg

  21. 10_13_hybridization.jpg

  22. 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 - ?

  23. 10_14_1_Southrn.blotting.jpg

  24. 10_14_2_Southrn.blotting.jpg

  25. DNA microassays New techniques Many uses DNA Chips

  26. 10_15_DNA.microarrays .jpg

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