Investigation 9 A “whodunit” Biotechnology: Restriction Enzyme Analysis of DNA Big Idea 3 Connections to Big Idea 1

Investigation 9 LO’s LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly used technologies. LO3.13 The student is able to pose questions about ethical, social, or medical issues surrounding human genetic disorders

Safety Note: Power supply on last and off first! manmonthly.com.au Power supply on last and off first! Don’t handle gels with bare hands.

Informational videos Preparing and pouring a gel. http://bcove.me/mweu5eru Loading a gel http://bcove.me/z1gm7u7j

Concentration Matters Boiling agarose and concentration. Gels are made with buffer!

Heat, cool to 60°, and pour smoothly.

Keep reagents cold

Avoid errors when you pipette. Practice with 10 drops of glycerol or corn syrup with 50 drops of water and one drop of blue food coloring. Practice gels 3X gelatin. Blow out with plastic pipette.

Load, add water to empty wells then fill right below top of gel

No budget? Make your own.

Pause when DNA has begun moving through gel, add buffer.

Staining…troublesome area. Destaining may take large volumes of water

Restriction Enzymes

DNA Profiling Foods Paternity Inherited disease Historical questions Question to ponder: Who owns your DNA?

Restriction Enzymes Restriction enzymes cut DNA at very specific locations. They are very predictable, each enzyme always cutting the same way. This characteristic is used in genetic engineering. Model with velcro pieces to emphasize the sticky ends. Restriction Enzyme Cut from EcoRI

Restriction Enzymes Cut at palindromes EcoRI GAATTC CTTAAG HindIII AAGCTT TTCGAA PstI CTGCAG GACGTC These are molecular tools! It is really useful when they leave “sticky ends.” Try the exercise on page S113. Named for the bacteria they were isolated from. http://asymptotia.com/wp-images/2008/08/e_coli.jpg

Recombinant DNA Is to “recombine.” Yep, a new piece of DNA from knitting pieces together. Restriction Enzymes cut Ligase “glues”

Restriction Mapping Makes a DNA fingerprint. The fragments, cut by restriction enzymes are RFLP’s or restriction fragment length polymorphisms.

RFLP’s separate by size.

About Lambda Lambda DNA is from a bacteriophage A bacteriophage is a virus which infects bacteria. The DNA piece is 48,502 base pairs long. Within this strand are locations which can be cut by restriction enzymes. These are specific locations.

Uncut PstI EcoRI HindIII 1 2 3 4 Band # Base pair size HindIII 23,130 9,416 6,557 4,361 2,322 2,027 Hind III sample size is known and will serve as the DNA Standard.

Use semi-log paper. The size in base pairs is graphed logarithmically. Size, base pairs Plot size vs. distance. Distance mm

Can you figure out “whodunit?” Use the provided samples and give it a shot. Motive, means, opportunity, DNA evidence.

Thinking About Your Results Choose one ethical problem from page S123. Research and write about it.