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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’ - PowerPoint PPT Presentation
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Common Techniques Used in Cell Physiology
First… a reminder
Nucleic Acids havepolarity-comes fromwhere next base can be added-“start” on the 5’ end-you can only add bases to the3’ end4-3 p. 102
Double stranded DNA(or RNA:DNA duplexes)are arranged antiparallel
5’ CAGT 3’3’ GTCA 5’
Often, only one strandis listed (Genbank)-this is conventionally thetop strand (sense strand)and it reads5’ 3’ left to right.
With this you can understand PCR-one of the mostpowerful and common techniques used in cell physresearch today.
Dr. Tom Brock-microbiologist looking for extremophiles-found Thermus aquaticus in Yellowstonehot springs-thriving at 70 C!
Chien and Trella-Univ. Cincinnati first isolated DNA polymerase from Taq
Kary Mullis-Cetus corporation-first figured outhow to use Taq in PCR reaction
Hoffman LaRoche-bought rights to PCR for 300 million
What’s a cDNAlibrary good for?
Say you are lookingfor a protein thatshows up in liver duringcancer. Make a cDNAlibrary and screen it forthis protein.7-18, 225
If you are screeningfor DNA must havea probe (piece of complementary DNAthat you think willstick to the right thing)
You can also look for proteins in libraries7-21, 228
Many of these techniques requirethat you know howthe sequence of DNAat some stage of thegame.
You determine that(usually) withSanger (dideoxy)sequencing7-29,234
DNA microarrays (aka DNA chips)
A relatively new method to screen thousands ofgenes simultaneously
Fig. 7-39 p. 249