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

7-38 p. 247

View animation on your cd-rom

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

Now you take each one of thosepieces and clone it (view plasmid cloning animation)

Often each of thesecloned cDNA is packagedinto a virus-you thenhave a cDNA library7-16,222

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

View Sequencing Animation

DNA microarrays (aka DNA chips)

A relatively new method to screen thousands ofgenes simultaneously

Fig. 7-39 p. 249

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?