Isolation of DNA Fragments
Following cleavage of DNA by restriction enzymes or other manipulations to be discussed later, DNA fragments frequently must be isolated. Fortunately, fractionation according to size is particularly easy because, as discussed earlier, DNA possesses a constant charge-to-mass ratio and double-stranded DNA fragments of the same length have the same shape and therefore migrate during electrophoresis at a rate nearly inde-pendent of their sequence. Generally, the larger the DNA, the slower it migrates.
resolution is obtainable in electrophoresis. With care, two fragments whose
sizes differ by 0.5% can be separated if they lie within a range of 2 to 50,000
base pairs. No single electrophoresis run could possess such high resolution
over this entire range. A typical range for adequate size separation might be 5
to 200 base pairs or 50 to 1,000 base pairs, and so on. The material through
which the DNA is electro - phoresed must possess special properties. It should
be inexpensive, easily used, uncharged, and it should form a porous network.
Two materials meet the requirements: agarose and polyacrylamide.
electrophoresis, bands formed by the different-sized frag-ments may be located
by autoradiography if the DNA had been radio-labeled before the separation.
Usually 32PO4 is a convenient label because phosphate is
found in RNA and DNA, 32P emits particularly energetic electrons
making them easily detectable, and finally 32P has a short half-life
so that most of the radioactive atoms in a sample will decay in a reasonable time.
The isotope 33P is also used. Its beta decay is weaker, and it has a
half-life of 90 days. Often, sufficient DNA is present that it may be detected
directly by staining with ethidium bromide. The enhanced fluorescence from
ethidium bromide interca-lated in the DNA compared to its fluorescence in
solution permits detection of as little as 5 ng of DNA in a band. After the
electrophoretic separation and detection of the DNA, the desired fragments can be isolated from the gel.