Biology Reference
In-Depth Information
5.5 Shearing DNA
A variety of protocols are available to mechanically produce fragmented DNA.
DNA can be sheared by sonication or by high-speed stirring. Sonication with
ultrasound can produce DNA fragments of
≈
300 nucleotides. High-speed stirring
of cells in a blender at 1500 revolutions/minute for 30 minutes will produce DNA
molecules with a mean size of 8 kb. Breakage occurs essentially at random with
respect to DNA sequence, and the broken ends consist of short, single-stranded
regions. These single-stranded termini must be modified before the DNA can be
joined to a vector, so mechanically sheared DNA rarely is used in experiments.
More often, DNA is cut in a specific manner with a restriction endonuclease to
make it easier to manipulate.
5.6 Cutting DNA with Restriction Endonucleases
Most cloning projects use restriction endonucleases to cut DNA. Restriction
enzymes were discovered as an outcome of basic research aimed at under-
standing how bacteria control infections by
bacteriophages
(viruses that invade
bacteria). Most bacteria contain a variety of specific endonucleases that guard
against invasion of foreign DNA (
Frank 1994
). These endonucleases make cuts in
the double-stranded DNA invading the cell unless the DNA has been modified
in a specific manner by methylation. Thus, “foreign” DNA has an inappropri-
ate methylation pattern, but the bacterium's own DNA is protected because its
methylation pattern is recognized. More than 3681 restriction enzymes (restric-
tion endonucleases) have been identified, and 588 are commercially available
(
Roberts et al. 2005
). Endonucleases with at least 221 different sequence speci-
ficities are commercially available (
Kessler and Manta 1990, Pingoud et al. 1993,
Roberts et al. 2005
).
Restriction enzymes are identified by three-letter abbreviations for the par-
ent organism (for example,
Hin
for
H
aemophilus
in
luenzae
or
Bam
for
B
acillus
am
yloliquefaciens
). An additional letter is added, if needed, to identify a spe-
cific strain or serotype (
Hin
d
or
Bam
H
). A Roman numeral is added to reflect the
order of identification or characterization of the specific endonuclease (
Hind
III
or
Bam
H
I
,
Table 5.3
).
Restriction endonucleases recognize specific sequences in DNA. They
cleave the DNA in a precise manner, producing either blunt or staggered cuts
(
Table 5.3
). Most endonucleases recognize sequences of four to six nucleotides,
but some have seven- and eight-base recognition sites, and a very few recog-
nize 12-base recognition sites. [Which endonucleases will produce the larger
DNA fragments?] Restriction enzymes cleave DNA to form 5
′
-phosphate and
