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Common Techniques Used in Cell Physiology

Common Techniques Used in Cell Physiology. First… a reminder. Nucleic Acids have polarity-comes from where next base can be added- “start” on the 5’ end- you can only add bases to the 3’ end 4-3 p. 102. Double stranded DNA (or RNA:DNA duplexes) are arranged antiparallel 5’ CAGT 3’

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Common Techniques Used in Cell Physiology

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  1. Common Techniques Used in Cell Physiology First… a reminder Nucleic Acids have polarity-comes from where next base can be added- “start” on the 5’ end- you can only add bases to the 3’ end 4-3 p. 102

  2. Double stranded DNA (or RNA:DNA duplexes) are arranged antiparallel 5’ CAGT 3’ 3’ GTCA 5’ Often, only one strand is listed (Genbank)- this is conventionally the top strand (sense strand) and it reads 5’ 3’ left to right.

  3. With this you can understand PCR-one of the most powerful and common techniques used in cell phys research today. Dr. Tom Brock- microbiologist looking for extremophiles- found Thermus aquaticus in Yellowstone hot springs-thriving at 70 C! Chien and Trella- Univ. Cincinnati first isolated DNA polymerase from Taq Kary Mullis- Cetus corporation-first figured out how to use Taq in PCR reaction Hoffman LaRoche- bought rights to PCR for 300 million

  4. 7-38 p. 247

  5. View animation on your cd-rom

  6. You can also use the same sort of idea to convert mRNA into DNA (cDNA) (7-15 p. 221)

  7. Now you take each one of those pieces and clone it (view plasmid cloning animation)

  8. Often each of these cloned cDNA is packaged into a virus-you then have a cDNA library 7-16,222

  9. What’s a cDNA library good for? Say you are looking for a protein that shows up in liver during cancer. Make a cDNA library and screen it for this protein. 7-18, 225

  10. If you are screening for DNA must have a probe (piece of complementary DNA that you think will stick to the right thing) You can also look for proteins in libraries 7-21, 228

  11. Many of these techniques require that you know how the sequence of DNA at some stage of the game. You determine that (usually) with Sanger (dideoxy) sequencing 7-29,234

  12. View Sequencing Animation

  13. DNA microarrays (aka DNA chips) Fig. 7-39 p. 249 A relatively new method to screen thousands of genes simultaneously

  14. Group Exercise #2 Look at the article I handed out (Lipshutz et. al, 1999. Nature Genetics. 21: 20-24 This is a complex article, but we can pull it apart In groups of 4: Group 1- How do you make a microarray? Group 2-How do you screen an array? Group 3-What are the applications for arrays?

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