The Renaturation of DNA

Very valuable technique which can be used to:
1) show genetic relatedness between organisms
2) can detect particular sequences (i.e. particular genes) if you have a DNA or RNA probe.
3) Can determine the frequency with which certain sequences appear [highly repetitive sequences will reanneal very rapidly].

 Two conditions which must be met in order for Reannealing to occur:
1) 0.15m to 0.5M NaCl concentration (the salt concentration must be high enough so that the phosphate groups of the two DNA strands are not repulsed, preventing H bond formation between nitrogenous base pairs) The + charged Na+ ions will partially shield the negative charges of the phosphates in the backbone.

 2) The temperature of the sample must be 20-25C below the Tm. At this temperature range, random intrastrand H bonding is disrupted, favoring interstrand H bonding between complementary base pairs.
 

 Renaturation studies can be combined with electron microscopy which allows you to actually visualize the DNA strands

fragment #1:
        A-B-C-D-E-F-G-H
        A-B-C-D-E-F-G-H

fragment #2:
        A-B-C-J-K-F-G-H
        A-B-C-J-K-F-G-H

If you DENATURE, mix, then allow reannealing to occur between 1&2 it will look like the following heteroduplex (Fig. 1):

                  D-E
       A-B-C/     \F-G-H
       A-B-C\     /F-G-H
                  J-K
 

Study of the rate at which denatured DNA (ssDNA) renatures.

 Visualize a long DNA molecule 100 base pairs in length

1...ATC...ATC....ATC......ATC.....ATC....100
1...TAG...TAG....TAG......TAG....GAG...100

Interspersed sequence of 3 base pairs in length (ATC)
repeated sequence represents 15% of the total DNA [3% X 5 = 15%]

1. Now, if you cleave the DNA into very small segments
2. denature the segments into single strands
3. allow the strands to renature over time
4. moniter renaturation by measuring the change in absorbance at 260nm

A graph of the results is shown in Fig. 2 .

Interpretation:
2 different classes of fragments

1) fast renaturing repetitive fragments
~15% of the DNA

2) slow renaturing - unique sequences, non-repetitive
~85% of the DNA

Repeated DNA sequences reanneal faster than the unique sequences of DNA because they are present at a higher concentration.
Assumption in this type of analysis: the slowest fraction represents fragments of DNA for which there is only one copy.

 If you perform the experiment with DNA fragments in which there are no repeating sequences you get a single, slow reannealing curve as shown in Fig. 3.

 All renaturation will occur at the same rate
 

 1 class of DNA fragments means that fragments are present in only 1 copy

Repetitive sequences are common in multicellular eukaryotic organisms but not single-celled eukaryotes or prokaryotes.

 4 Classes of sequences in higher eukaryotes (Fig. 4):

unique [ 1 copy] (majority of gene sequences)
slightly repetitive [ 1-10 copies]
moderately repetitive [10-several 100 copies]
highly repetitive [several hundred - several million copies]

satellite DNA

Sequences of normal length but which occur in very high #s of copies
ex: genes which code for rRNAs (280 copies in humans) (450 copies in amphibians )