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Lab 305: DNA Fingerprinting A Simulation

Background

Many of the revolutionary changes that have occurred in biology over the past fifteen years can be attributed directly to the ability to manipulate DNA in defined ways. The principal tools for this recombinant DNA technology are enzymes that can cut, mend, wind, unwind, transcribe, repress, and replicate DNA. Restriction enzymes are the "chemical scissors" of the molecular biologist; these enzymes cut DNA at specific nucleotide sequences. A sample of someone's DNA, incubated with restriction enzymes, is reduced to millions of DNA fragments of varying sizes. A DNA sample from a different person would have a different nucleotide sequence and would thus be enzymatically "chopped up" into a very different collection of fragments. The DNA fragments are separated by electricity -- agarose gel electrophoresis -- and tagged or stained in some fashion so that they can be visualized and studied. The resulting pattern of restriction fragments resembles the pricing bar code used on supermarket products -- small bars lined up in a column, the largest fragments closest to the beginning of the gel, the smallest at the end of the gel.

Methods of DNA identification have been applied to many branches of science and technology, including medicine (prenatal tests, genetic screening), conservation biology (guiding captive breeding programs for endangered species), and forensic science. In the latter discipline, analysis of the pattern of DNA fragments in a restriction digest, loosely called a DNA fingerprint,enables us to discriminate between suspects accused of a crime or potential fathers in a paternity suit.

In DNA fingerprinting, radioactive pieces of DNA called probes are added to the separated DNA fragments. The probes are designed to bind to specific sequences on the DNA, thus marking some of the fragments. X-ray film placed on top of the gel will become exposed by the radioactivity, and black bands will become visible on the film. The variation in DNA from one person to the next is so great that the probability of two people sharing the same DNA fingerprint is essentially zero. And unlike conventional fingerprints, which are often difficult to gather at a crime scene, a DNA fingerprint can be made from a very small sample of blood, skin or semen -- or even a single hair!


Purpose

In this laboratory, to prepare and analyze a simulated DNA fingerprint, the student will:
  • cut a DNA sample -- the evidence! --by incubating it with restriction enzymes,
  • load an agarose gel with the restriction digest,
  • conduct gel electrophoresis to spread out the mixture of DNA fragments in the digest,
  • stain and photograph the gel to visualize the DNA,
  • analyze the resulting banding pattern or "DNA fingerprint," and use it to "solve" an imaginary crime.

Materials (per team)
gel electrophoresis box             agarose, [0.8%], liquid held at 65°C (25-30 mL)
power supply restriction enzymes, on ice
casting tray and comb DNA samples, on ice
P-20 micropipet and tips electrophoresis buffer, 300 mL (1X TBE or 1X TAE)
racks for 1.5 mL reaction tubes restriction buffer [2X]
1.5 mL reaction tubes (5) loading dye
plastic trays for staining sterile water
Ziploctm baggie marking pen
400 mL beaker for buffer container for waste
beaker or graduate cylinder for agarose     mini ice bucket (Styrofoam cup)


Class use: water bath, crushed ice containers, microcentrifuges, documentation stations (camera, film, filter, hood, UV transilluminator) ethidium bromide staining solution [1 µg/mL].





Lab 305: Gel Electrophoresis Apparatus Diagram



ITEMS TO LABEL:

On power supply On each gel box
digital display anode
on/off switch cathode
voltage select dial (+) lead
switch between Volts and milliAmps (-) lead
(-) and (+) channels for one of the gel boxes lid
lo/hi range switch (on Lo 20-160 V; on Hi, 165-300 V)  
platform for casting tray  
light indicating "POWER SUPPLY IS ON"  
light indicating "CHECK FUSE"
and
red & black wires connecting power supply with each gel box  




Lab 305: Gel Electrophoresis Apparatus Diagram



ITEMS TO LABEL:

On power supply On each gel box
digital display anode
on/off switch cathode
voltage select dial (+) lead
switch between Volts and milliAmps (-) lead
(-) and (+) channels for one of the gel boxes lid
platform for casting tray  
light indicating "CHECK FUSE"  
and
red & black wires connecting power supply with each gel box  


Part I     Part II & III     Part IV     Part V     Part VI

Crime Scenario     Historical Mystery     Teacher's Guide



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