DNA Fingerprinting Analysis

Goal: Amplify certain region of DNA sample obtained, then generate DNA Fingerprint to  find the perpetrator of a crime.

This is general procedure including PCR and DNA Electrophoresis.  Please follow any specific procedures that might have come with your ordered DNA kit.  Chelex® or any other reagent may not be used in actual experiment.

        A. Extraction of DNA

1.      Your instructor will give you isolated cheek cells of suspects, and victim, unknown person.

2.      Dissolve cheek cells into the saline solution, then transfer 1.0ml of solution to 1.5ml microcentrifuge tube for individual DNA.  Label the tubes as victim (V), unknown (U), the first suspect (S1), the second suspect 2 (S2), and so on.

3.      Place the samples into the microcentrifuge. Spin for 10 minute at 3000 RPM.   

4.      Carefully pour off supernatant without loosing any cell pellet at the bottom of the microcentrifuge tubes.

5.      Resuspend the cell pellets by adding saline solution, then pipetting liquid in and out several times.

6.      Mix 30 ul of cell suspension and 100 ul of Chelex in a clean tube.  Shake well to mix.

7.      By using a boiling water bath, heat the tube for 10 minutes. Then cool the tube at room temperature. (or in an ice bath)

8.      Shake tube vigorously, place in a microcentrifuge, and spin for 1 minute at 3000 RPM.

9.      Transfer 30 ul of supernatant which contains the DNA into a clean 1.5 ml tube. Be careful, so any cell debris and Chelex beads are not transferred.

10.  Store your sample on ice or in a freezer in prior to PCR

B.     Polymerase Chain Reaction

1.      Use a micropipette with a fresh tip to add appropriate primer/loading buffer mix to a test tube.

2.      Add DNA you just extracted from the cheek cells into the reaction tubes, and then add primer/loading buffer mix from step one.  Tap tubes with fingers to mix.

3.      Add appropriate enzymes as such as Taq DNA Polymerase, PCR buffer, deoxynucleotides into the tubes.  For ease of experiment, Ready-To-Go™ PCR bead can be used.

4.      By now, students should have a number of the reaction tubes that match with suspects’, victim’s and unknown.

5.      Add one to two drops of mineral oil to the reaction tubes.

6.      If laboratory is equipped with a thermal cycler, you need to transfer PCR mixtures into appropriate size of autoclave tubes. However, experiment can carry out using three different temperature setting of water baths.

7.      All three sequences are considered as one PCR cycle.  Thus, one student will keep track of time and temperature, and rest of the group should hold tubes in the water baths.  Repeat for 30 cycles.

C.     DNA Electrophoresis

1.      Students will receive prepared agarose gel, which is ready to load the samples.

2.      Remove the comb, then carefully load 10 ul of DNA samples into each well. Make a map indicating what went into each well.

3.      Make sure that wells are near the negative charge. (The negative charge is usually black and positive charge is red.)

4.      Since many factors determine the overall time to run the gel, your instructor will decide how long the DNA samples will run.

5.      Stain your gel with methylene blue or any commercial stain which works better than conventional stain.

Analysis and Conclusion:

·        You have used 1% agarose gel in this experiment.  If you have used 0.5% or 1.5% agarose gel, what difference in results would you find?  Why?

·        What other applications of DNA Fingerprinting are used in our society?

·        Explain the major role of restriction enzyme in DNA fingerprinting.  

·        The experiment performed above, is it 100% accurate that it can be applied in a real crime investigation? If not, what else can be done to improve the method?  

·        What is the probability of finding an identical DNA match with yours sample in the population?

·        During electrophoresis, observe the ends of the electrophoresis chamber.   Do you see bubbles?  Do you see bubbles at both ends?  Is there the same amount of bubbles at each end? Use your best knowledge of chemistry and draw conclusion of what you just have observed.

·        James Watson and Francis Crick deduce the structure of DNA by x-ray diffraction.  Please, further your research regarding to mechanism of x-ray diffraction.

·        Recent claim about cloned baby by CLONAID™ probably shocked many people. However, it would not be a surprise for scientists who have been involved with human genome project or genetic engineering. What do you think about the issue of human cloning?  What would be the pros and cons if government, employers, and insurance companies use DNA profiles as ID cards?

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