Enzyme And Oligo Sequence Chart
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1
2
3Cleavage 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 A
unit of oligonucleotide was phosphorylated using T4 polynucleotide
260
32
32
kinase and γ-[
P] ATP. 1 µg of 5´ [
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 MgCl
, 5 mM DTT and NaCl or KCl depending on the
2
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
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B
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C
|
E
|
H
|
K
|
M
|
N
|
P
|
S
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X
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Enzyme
Oligo Sequence
Chain
% Cleavage
Length
2 hr
20 hr
AccI
GGTCGACC
8
0
0
CGGTCGACCG
10
0
0
CCGGTCGACCGG
12
0
0
AflIII
CACATGTG
8
0
0
CCACATGTGG
10
>90
>90
CCCACATGTGGG
12
>90
>90
AscI
GGCGCGCC
8
>90
>90
AGGCGCGCCT
10
>90
>90
TTGGCGCGCCAA
12
>90
>90
AvaI
CCCCGGGG
8
50
>90
CCCCCGGGGG
10
>90
>90
TCCCCCGGGGGA
12
>90
>90
BamHI
CGGATCCG
8
10
25
CGGGATCCCG
10
>90
>90
CGCGGATCCGCG
12
>90
>90
BglII
CAGATCTG
8
0
0
GAAGATCTTC
10
75
>90
GGAAGATCTTCC
12
25
>90
BssHII
GGCGCGCC
8
0
0
AGGCGCGCCT
10
0
0
TTGGCGCGCCAA
12
50
>90
BstEII
GGGT(A/T)ACCC
9
0
10
BstXI
AACTGCAGAACCAATGCATTGG
22
0
0
AAAACTGCAGCCAATGCATTGGAA
24
25
50
CTGCAGAACCAATGCATTGGATGCAT
27
25
>90
ClaI
CATCGATG
8
0
0
GATCGATC
8
0
0
CCATCGATGG
10
>90
>90
CCCATCGATGGG
12
50
50
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