The Myth of the Dinosaurs’ Broken DNA

恐龍基因破碎之謎 (B4)

Diocesan Girls’ School

Tracy Law, Sindy Wong, Polly Yu, Queenie Lai

Hong Kong Student Science Project Competition 2004

Observation

Advanced biotechnology and cloning technology

There are no dinosaurs cloned

DNA molecules of dinosaurs are broken

Oxidation may be a major cause in breaking down the DNA molecules

Oxidation of DNA molecules can be brought about by oxidizing agents and free radicals

Problem

Why was the dinosaur DNA broken into fragments as found in the fossils?

Hypothesis

Free radicals have a stronger action on DNA molecules than oxidizing agents.

Aim

To compare the effectiveness of free radicals and oxidizing agents in breaking down the DNA molecules.

PrinciplesBreaking Down of DNA

Nucleotides are linked together by phospho-ester bonds

Breaking down of DNA is due to the destruction of phospho-ester bonds of the sugar-phosphate backbone of the molecules

DNA is a giant chain of nucleotides

Source of DNA

We use DNA from pig liver

because of the following reasons:

Liver cells have relatively bigger nuclei, it would be easier to extract DNA

DNA molecules are all similar in structure

Pig liver is easily available

Free radicals will be formed from hydrogen peroxide in the presence of iron (III) ions.

Fe 2+ + H2O2 ----> Fe 3+ + OH - + . OH

Fe 3+ + H2O2 ----> Fe 2+ + . OOH + H+

We propose that these free radicals react with and break the DNA chains

Source of Free Radicals

Source of Oxidizing Agents

Hydrogen peroxide and acidified potassium permanganate are used in this experiment because they are well-known oxidizing agents.

DNA Gel ElectrophoresisPoles

The negatively charged DNA molecules will move to the anode side of the gel

Gel

Anode

Cathode

Gel

Contains pores so that DNA molecules can pass through

The negatively charged DNA molecules will move to the anode side of the gel

Design of the experiment

Setup ReactantsA Pure pig liver DNA

B DNA+ 6% H2O2

C DNA + 0.05M KMnO4/H+

D DNA + 0.1 M FeCl3

E

DNA + 6% H2O2 + 0.1M FeCl3

Independent variable:

Various treatments of DNA

Dependent variable:

The DNA pattern on the gel

Control variables:

Time of running the gel, source of DNA extracts (pig), temperature, pH

Assumption

If pig’s DNA is broken down by the attack of free radicals, dinosaurs' DNA can be broken down by the same mechanism.

ProceduresPreparation of DNA samples

Cut pig liver tissue into smaller pieces

Purify DNA by adding chemicals and enzymes, followed by centrifugation

Add TE buffer into the gel tank

Load DNA samples into the wells

Connect the gel tank to a

150V power pack

DNA gel electrophoresis

Visualization of DNA

Stain the gel overnight using methylene blue

Destain the gel in distilled water

Front

Setup A Setup C Setup E

Setup B Setup D

Bulges

Result

Bulges

Findings

Ferric chloride forms crypto-hydroxyl radicals with DNA molecules, and the mechanism causes DNA to break into smaller pieces

The oxidizing power of hydrogen peroxide and acidified potassium permanganate is not strong enough to break the DNA strands

Free radicals have a stronger breaking action on DNA molecules

Ferric chloride and hydrogen peroxide react with each other to form hydroxyl radicals, which then react with DNA molecules and break them down into smaller pieces

Conclusion

Free radicals were a more likely cause for breaking down the dinosaurs’ DNA than oxidizing agents.