182 Pingoud, Alves, and Geiger4.5. Cleavage of Single-Stranded DNA
The substrate of restriction endonucleases is double-stranded DNA,
but some virus DNAs are single stranded and some protocols in molecu-
lar biology, such as DNA sequencing and site-directed mutagenesis,
use single-stranded DNA. Because of internal sequence homologies,
these DNAs show a lot of secondary structures that can reconstitute
recognition sequences of restriction enzymes (see Section 2.4.3.).
Therefore, enzymes with 4- or 5-bp long recognition sites will often
cleave these single-stranded DNAs.
Type II restriction enzymes with nonpalindromic recognition sequences
cleaving more than 5 bp outside of this sequence (e.g., FokI, GsuI,
Eco57I, cfTable 11) can be used to cleave any specific DNA sequence
in single-stranded DNA. This reaction is based on the use of synthetic
oligodeoxynucleotides that form an adaptor comprising the double-
stranded recognition sequence linked to an overhanging single-stranded
sequence that is complementary to the desired cleavage site in the
target DNA. After hybridization of this adaptor to the target sequence,
the restriction enzyme binds to the recognition sequence within the
oligodeoxynucleotide and cleaves the target DNA as shown in Fig. 2
(192,193). In principle, this procedure should be applicable also to
double-stranded target DNA.
- Labeling Restriction Fragments at Their Ends
For a variety of analytical purposes, radioactive labeling of restric-
tion fragments is necessary (see Sections 3.5.3. and 3.5.4.). Normally,
ethidium bromide is used to stain double-stranded restriction frag-
ments for analytical applications. If the amount of a produced frag-
ment is less than about 10 ng, the fluorescence of this dye may not be
strong enough for the detection of the fragment. By radioactive label-
ing, the detection limit can be reduced up to 1000-fold. There are
several methods for labeling the 5' or 3' end of each strand in the DNA
fragment (for detailed protocols, cf [21,194,1951).
All restriction enzymes (according to Bennett [77] NciI is not an
exception to this rule as previously believed) generate 5'-phosphate
and 3'-OH groups at the termini of the DNA fragments (see note added
in proof at end of chapter). In order to obtain 5'-labeled termini, restric-
tion fragments must be dephosphorylated with alkaline phosphatase,
- Labeling Restriction Fragments at Their Ends