Cleavage Close to the End of DNA Fragments (oligonucleotides)
To test the varying requirements restriction endonucleases have for the number of bases flanking their recognition
sequences, a series of short, double-stranded oligonucleotides that contain the restriction endonuclease recognition sites
(shown in red) were digested. This information may be helpful when choosing the order of addition of two restriction
endonucleases for a double digest (a particular concern when cleaving sites close together in a polylinker), or when
selecting enzymes most likely to cleave at the end of a DNA fragment.
The experiment was performed as follows: 0.1 A260 unit of oligonucleotide was phosphorylated using T4 polynucleotide
kinase and γ-[32
P] ATP. 1 µg of 5´ [32
P]-labeled oligonucleotide was incubated at 20°C with 20 units of restriction
endonuclease in a buffer containing 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 5 mM DTT and NaCl or KCl depending on the
salt requirement of each particular restriction endonuclease. Aliquots were taken at 2 hours and 20 hours and analyzed by
20% PAGE (7 M urea). Percent cleavage was determined by visual estimate of autoradiographs.
As a control, self-ligated oligonucleotides were cleaved efficiently. Decreased cleavage efficiency for some of the longer
palindromic oligonucleotides may be caused by the formation of hairpin loops.
| A | B | C | E | H | K | M | N | P | S | X |
Enzyme Oligo Sequence Chain
Length
% Cleavage
2 hr 20 hr
AccI GGTCGACC
CGGTCGACCG
CCGGTCGACCGG
8
10
12
0
0
0
0
0
0
AflIII CACATGTG
CCACATGTGG
CCCACATGTGGG
8
10
12
0
>90
>90
0
>90
>90
AscI GGCGCGCC
AGGCGCGCCT
TTGGCGCGCCAA
8
10
12
>90
>90
>90
>90
>90
>90
AvaI CCCCGGGG
CCCCCGGGGG
TCCCCCGGGGGA
8
10
12
50
>90
>90
>90
>90
>90
BamHI CGGATCCG
CGGGATCCCG
CGCGGATCCGCG
8
10
12
10
>90
>90
25
>90
>90
BglII CAGATCTG
GAAGATCTTC
GGAAGATCTTCC
8
10
12
0
75
25
0
>90
>90
BssHII GGCGCGCC
AGGCGCGCCT
TTGGCGCGCCAA
8
10
12
0
0
50
0
0
>90
BstEII GGGT(A/T)ACCC 9 0 10
BstXI AACTGCAGAACCAATGCATTGG
AAAACTGCAGCCAATGCATTGGAA
CTGCAGAACCAATGCATTGGATGCAT
22
24
27
0
25
25
0
50
>90
ClaI CATCGATG
GATCGATC
CCATCGATGG
CCCATCGATGGG
8
8
10
12
0
0
>90
50
0
0
>90
50
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EcoRI GGAATTCC
CGGAATTCCG
CCGGAATTCCGG
8
10
12
>90
>90
>90
>90
>90
>90
HaeIII GGGGCCCC
AGCGGCCGCT
TTGCGGCCGCAA
8
10
12
>90
>90
>90
>90
>90
>90
HindIII CAAGCTTG
CCAAGCTTGG
CCCAAGCTTGGG
8
10
12
0
0
10
0
0
75
KpnI GGGTACCC
GGGGTACCCC
CGGGGTACCCCG
8
10
12
0
>90
>90
0
>90
>90
MluI GACGCGTC
CGACGCGTCG
8
10
0
25
0
50
NcoI CCCATGGG
CATGCCATGGCATG
8
14
0
50
0
75
NdeI CCATATGG
CCCATATGGG
CGCCATATGGCG
GGGTTTCATATGAAACCC
GGAATTCCATATGGAATTCC
GGGAATTCCATATGGAATTCCC
8
10
12
18
20
22
0
0
0
0
75
75
0
0
0
0
>90
>90
NheI GGCTAGCC
CGGCTAGCCG
CTAGCTAGCTAG
8
10
12
0
10
10
0
25
50
NotI TTGCGGCCGCAA
ATTTGCGGCCGCTTTA
AAATATGCGGCCGCTATAAA
ATAAGAATGCGGCCGCTAAACTAT
AAGGAAAAAAGCGGCCGCAAAAGGAAAA
12
16
20
24
28
0
10
10
25
25
0
10
10
90
>90
NsiI TGCATGCATGCA
CCAATGCATTGGTTCTGCAGTT
12
22
10
>90
>90
>90
PacI TTAATTAA
GTTAATTAAC
CCTTAATTAAGG
8
10
12
0
0
0
0
25
>90
PmeI GTTTAAAC
GGTTTAAACC
GGGTTTAAACCC
AGCTTTGTTTAAACGGCGCGCCGG
8
10
12
24
0
0
0
75
0
25
50
>90
PstI GCTGCAGC
TGCACTGCAGTGCA
AACTGCAGAACCAATGCATTGG
AAAACTGCAGCCAATGCATTGGAA
CTGCAGAACCAATGCATTGGATGCAT
8
14
22
24
26
0
10
>90
>90
0
0
10
>90
>90
0
PvuI CCGATCGG
ATCGATCGAT
TCGCGATCGCGA
8
10
12
0
10
0
0
25
10
SacI CGAGCTCG 8 10 10
SacII GCCGCGGC
TCCCCGCGGGGA
8
12
0
50
0
>90
CGGCCGEagI
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SalI GTCGACGTCAAAAGGCCATAGCGGCCGC
GCGTCGACGTCTTGGCCATAGCGGCCGCGG
ACGCGTCGACGTCGGCCATAGCGGCCGCGGAA
28
30
32
0
10
10
0
50
75
ScaI GAGTACTC
AAAAGTACTTTT
8
12
10
75
25
75
SmaI CCCGGG
CCCCGGGG
CCCCCGGGGG
TCCCCCGGGGGA
6
8
10
12
0
0
10
>90
10
10
50
>90
SpeI GACTAGTC
GGACTAGTCC
CGGACTAGTCCG
CTAGACTAGTCTAG
8
10
12
14
10
10
0
0
>90
>90
50
50
SphI GGCATGCC
CATGCATGCATG
ACATGCATGCATGT
8
12
14
0
0
10
0
25
50
StuI AAGGCCTT
GAAGGCCTTC
AAAAGGCCTTTT
8
10
12
>90
>90
>90
>90
>90
>90
XbaI CTCTAGAG
GCTCTAGAGC
TGCTCTAGAGCA
CTAGTCTAGACTAG
8
10
12
14
0
>90
75
75
0
>90
>90
>90
XhoI CCTCGAGG
CCCTCGAGGG
CCGCTCGAGCGG
8
10
12
0
10
10
0
25
75
XmaI CCCCGGGG
CCCCCGGGGG
CCCCCCGGGGGG
TCCCCCCGGGGGGA
8
10
12
14
0
25
50
>90
0
75
>90
>90
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