The Green Phoenix: A History of Genetically Modified Plants

By Paul F. Lurquin | Go to book overview

APPENDIX 2
Hybridization Experiments
with DNA

The two strands of DNA are complementary, in the sense that an adenine (A) in one strand will always face and interact by hydrogen-bonding with a thymine (T) in the other strand. The same holds true for guanine (G) and cytosine (C). The strands of DNA can be separated by agents that destroy hydrogen bonds, such as high temperature (that of boiling water, 100°C, will denature DNA rapidly) or high pH (pH 11 is sufficient). Once the two strands of a DNA molecule are separated in solution, if the temperature (or pH) is brought down to a suitable point, the two strands can reassociate. They do this by [probing] one another for correct complementarity—that is, base-pairing. Perfect base-pairing between all the bases is ensured if reassociation is not too rapid and is allowed to occur several degrees below the temperature at which the double helix starts to dissociate. The rate at which the reassociation takes place can be measured. This is because singlestranded DNA absorbs more UV light at 260 nm than double-stranded DNA. What is measured is thus the drop in UV light absorbance as a function of time. If very small amounts of a test DNA are used, as in Pot curves (see chapter 1), radioactive DNA must be used for detection. In that case, aliquots of reassociating DNA are harvested at given time intervals and run through a hydroxylapatite column. Hydroxylapatite has a greater affinity for double-stranded DNA than for its denatured, single-stranded version, and it is thus possible to elute one before the other. Thus the fraction of doublestranded (reassociated) DNA can be easily measured as a function of time.

Britten and Kohne (1968) demonstrated that DNA reassociation kinetics follows a second-order (sigmoidal) function. This was expected, because DNA reassociation kinetics involves two molecules of single-stranded DNA,

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