Some Basic Tools: How to Cut, Paste, Copy, Measure, Visualize, and Clone DNA - Insect Molecular Genetics - page 188

Table 5.3: Target Sites for Selected Restriction Endonucleases.

Organism from which endonuclease
was isolated

Abbreviation Recognition sequences indicates

cleavage site (| indicates cleavage site)

Bacillus amyloliquefaciens H Bam HI G | GATCC

Bacillus globigii Bgl II A | GATCT

Escherichia coli RY13 Eco RI G | AATTC

Haemophilus aegyptius Hae III GG | CC

Klebsiella pneumoniae Kpn II GGTAC | C

Nocaardia otitidis-caviarum Not I GC | GGCCGC

Providencia stuartii Pst I CTGCA | G

Serratia marcescens Sma I CCC | GGG

Recognition sequences are written from 5 ′ to 3 ′ with only one strand given.
(Modified from Kessler and Manta 1990.)

Table 5.4: Three Types of Termini are Created by Restriction Enzyme Cleavage of Double-
Stranded DNA: a 5 ′ Overhang, Blunt Ends, or a 3 ′ Overhang.

Eco RI Pvu II Kpn I

G | AATTC CAG | CTG GGTAC | C

CTTAA | G GTC | GAC C | CATGG

G 3 ′ 5 ′ AATTC CAG 3 ′ 5 ′ CTG GGTAC 3 ′ 5 ′ C

CTTAA 5 ′ 3 ′ G GTC 5 ′ 3 ′ GAC C 5 ′ 3 ′ CATGG

5 ′ overhang Blunt end 3 ′ overhang

The | indicates where cleavage occurs when DNA is restricted, or cut, by three representative enzymes.

3 ′ -hydroxyl termini on each strand ( Table 5.3 ). Endonucleases that produce stag-
gered breaks generate either 5 ′ -phosphate extensions or 3 ′ -hydroxyl extensions.
Other endonucleases produce “blunt” breaks ( Table 5.4 ).

It is often desirable to generate DNA fragments of a specific length, with a
specific sequence, and with a particular type of end. This precision is possible
with well-characterized DNA that has been sequenced. Such precision is not
possible with uncharacterized DNA, except to predict whether the ends will be
blunt, or with 5 ′ or 3 ′ overhangs. It is difficult to predict precisely the length
of the DNA fragments that will be generated after digesting unknown DNA
sequences with a particular restriction endonuclease, although we can predict
the sequences at the ends of each fragment ( Table 5.4 ). [Why is that?]

Predictable fragment lengths would occur if all DNA sequences contained
50% guanine and cytosine (G + C) base pairs, and if all bases were distributed
randomly in the DNA. Under these conditions, a four-base sequence that is
recognized by the restriction endonuclease would occur approximately every