Info

Double-stranded DNA

Single-stranded DNA

Double-stranded DNA

111.13 The slow heating of DNA causes the two strands to separate (denature).

at higher concentrations of DNA, because high concentrations increase the likelihood that the two complementary strands will collide. There will also be more renaturation with increasing time, because there are more opportunities for two complementary sequences to collide. These two factors together form a parameter called C0t, which equals the initial concentration multiplied by the renaturation time

A plot of the fraction of single-stranded DNA as a function of C0t during a renaturation reaction is called a C0t curve. A typical C0t curve for a prokaryotic organism is shown in FIGURE 11.14. The upper left-hand side of the curve represents the start of the renaturation reaction, when all of the DNA is single stranded, and so the proportion of single-stranded DNA is 1. As the reaction proceeds, single-stranded DNA pairs to form double-stranded DNA, represented by the decreasing fraction of single-stranded DNA. At the end of the reaction, the proportion of single-stranded DNA is 0, because all of the DNA is now double stranded. The value at which half of the DNA is reannealed is called C0t /

The rate of renaturation also depends on the size and complexity of the DNA molecules used. Consider the following analogy. Suppose we distribute 100 cards equally among the students in a class. We ask each student to write his or her name on the cards, and we put all the cards in a hat. We then randomly draw two cards from the hat and see if the names on the two cards match. If they don't match, we put them back in the hat; if they do match, we remove them, and we continue drawing until all the cards have been removed. If there are only four students in the class, each student will receive 25 cards. Because each student's name is on 25 cards, the chance of drawing two cards that match is high, and we will quickly empty the hat. If we do the same exercise in another class with 50 students, again using 100 cards, each student's name will appear on only two cards, and the chance of removing two cards with the same name is much lower. Thus, it will take longer to empty the hat.

This exercise resembles what occurs in the renaturation reaction. If we start with the same total amount of DNA, but there are only a few different sequences in the DNA, a chance collision between two complementary fragments is more likely to occur than if there were many different sequences. Therefore DNA from organisms with larger genomes will have a larger C0t / value.

Thus far, we have considered renaturation reactions in which each DNA sequence is present only once in each molecule. If some sequences are present in multiple copies, these sequences will be more likely to collide with a complementary copy, and renaturation of these sequences will be rapid. Think about our analogy of drawing names from a hat. Imagine that we have 50 students and 100 cards; each student gets two cards. This time, the students write only their first names on the cards. Again, we place the cards in the hat and draw out two cards at random. If

Denatured

^-Renatured

0 0

Post a comment