Biology Reference
In-Depth Information
256 bases (4
4
), a six-base sequence would occur approximately every 4kb (4
6
or
4096 bases), and an eight-base sequence would occur approximately every 65 kb.
However, many segments of DNA are not random in their G
+
C content. For
example, highly repetitive DNA may have several nucleotides repeated millions
of times, which obviously would bias restriction site frequencies significantly. The
percentage of G
+
C in DNA from different sources may vary from 22 to 73%.
Many different restriction endonucleases are commercially available either in
native or cloned form. Most manufacturers provide standardized buffers (high-,
medium-, or low-salt) for optimizing the reaction conditions and protocols for
carrying out the digestions. Among those available, some recognize identical
sequences, although they may vary with respect to their sensitivity to methyla-
tion and cleavage rates (
Roberts et al. 2005
). Restriction endonucleases can be
degraded if not properly stored at
−
20°C and should be subdivided into small
amounts so they do not undergo multiple freeze-thaw cycles before being used.
Choosing which endonuclease to use is determined by the goals of the proj-
ect, and a computer program is available to assist in designing experiments
using restriction enzymes (
Martin et al. 2006
). Enzymes that produce small seg-
ments of a few hundred bases are useful for restriction mapping or for sequenc-
ing. Enzymes that produce fragments of 1
−
10kb are useful for mapping
large DNA regions and for cloning whole genes with their introns and control
sequences. Generating even larger fragments (5-50kb) is necessary for cloning
into some vectors or for genome walking (discussed in Chapter 6).
Digestion reactions with restriction endonucleases contain: the DNA sub-
strate, the restriction endonuclease(s), Tris buffer, Mg
2
+
, NaCl, 2-mercaptoetha-
nol, and bovine serum albumin. All endonucleases require Mg
2
+
as a cofactor,
and most are active at pH values ranging from 7.2 to 7.6. The major difference
among the endonucleases is their dependence on ionic strength and their tem-
perature optima. Most digestions are done at 37°C, but a few restriction endo-
nucleases perform better at lower temperatures. The manufacturer usually
measures endonuclease activity with bacteriophage lambda (
λ
) DNA as a sub-
strate, but activity of the endonuclease varies with different DNA substrates and
also can be modified by the neighboring sequences. Activity rates can vary by a
factor of 10- to 50-fold in your laboratory experiments.
The number and variety of endonucleases available for genetic manipula-
tions continue to increase. Endonucleases that recognize longer recognition
sequences are useful if large DNA fragments are to be separated by pulsed-field
gel electrophoresis. New microbial sources of enzymes are being sought, espe-
cially those that tolerate high temperatures. Catalogs obtained from suppliers
