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The experiment was performed as follows: 0.1 A260 unit of oligonucleotide was phosphorylated using T4 polynucleotide kinase and γ-[32P] ATP. 1 µg of 5' [32P]-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.
12
CTAGACTAGTCTAG
14
Sph I Stu I Xba I
GGCATGCC
8
CATGCATGCATG
12
ACATGCATGCATGT
14
AAGGCCTT
8
GAAGGCCTTC
10
AAAAGGCCTTTT
12
CTCTAGAG
8
GCTCTAGAGC
10
TGCTCTAGAGCA
12
CTAGTCTAGACTAG
>90
8
0
0
10
0
0
12
50
>90
9
0
10
22
0
0
24
25
>90
27
25
>90
8
0
0
8
0
0
10
>90 >90
12
50
50
8
>90 >90
10
>90 >90
12
>9wk.baidu.com >90
8
>90 >90
10
>90 >90
12
>90 >90
8
0
0
10
0
0
12
10
75
8
0
0
10
>90 >90
12
>90 >90
8
0
0
10
Enzyme Acc I Afl III Asc I Ava I BamH I
Oligo Sequence
GGTCGACC CGGTCGACCG CCGGTCGACCGG
CACATGTG CCACATGTGG CCCACATGTGGG
GGCGCGCC AGGCGCGCCT TTGGCGCGCCAA
CCCCGGGG CCCCCGGGGG TCCCCCGGGGGA
CGGATCCG CGGGATCCCG CGCGGATCCGCG
% Cleavage Chain Length 2 hr 20 hr
8
0
0
10
0
0
12
0
0
8
0
0
10
>90 >90
12
>90 >90
8
>90 >90
10
>90 >90
12
>90 >90
8
50
>90
10
>90 >90
12
>90 >90
8
10
10
CCTTAATTAAGG
12
Pme I
GTTTAAAC
8
GGTTTAAACC
10
GGGTTTAAACCC
12
AGCTTTGTTTAAACGGCGCGCCGG
24
Pst I
GCTGCAGC
8
TGCACTGCAGTGCA
14
AACTGCAGAACCAATGCATTGG
22
AAAACTGCAGCCAATGCATTGGAA
25
50
8
0
0
14
50
75
8
0
0
10
0
0
12
0
0
18
0
0
20
75
>90
22
75
>90
8
0
0
10
10
25
12
10
50
12
0
0
16
10
10
20
10
10
24
25
90
28
25
>90
Nsi I Pac I
TGCATGCATGCA
12
CCAATGCATTGGTTCTGCAGTT
22
TTAATTAA
8
GTTAATTAAC
New England Biolabs Technical Literature - Updated 04/04/00
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.
GGGT(A/T)ACCC
AACTGCAGAACCAATGCATTGG AAAACTGCAGCCAATGCATTGGAA CTGCAGAACCAATGCATTGGATGCAT
CATCGATG GATCGATC CCATCGATGG CCCATCGATGGG
GGAATTCC CGGAATTCCG CCGGAATTCCGG
GGCTAGCC CGGCTAGCCG CTAGCTAGCTAG
TTGCGGCCGCAA ATTTGCGGCCGCTTTA AAATATGCGGCCGCTATAAA ATAAGAATGCGGCCGCTAAACTAT AAGGAAAAAAGCGGCCGCAAAAGGAAAA
8
0
0
10
75
>90
12
25
25
10
>90 >90
12
>90 >90
Bgl II BssH II BstE II BstX I Cla I
EcoR I Hae III Hind III Kpn I Mlu I Nco I Nde I
Nhe I Not I
CAGATCTG GAAGATCTTC GGAAGATCTTCC
GGCGCGCC AGGCGCGCCT TTGGCGCGCCAA
GGGGCCCC AGCGGCCGCT TTGCGGCCGCAA
CAAGCTTG CCAAGCTTGG CCCAAGCTTGGG
GGGTACCC GGGGTACCCC CGGGGTACCCCG
GACGCGTC CGACGCGTCG
CCCATGGG CATGCCATGGCATG
CCATATGG CCCATATGGG CGCCATATGGCG GGGTTTCATATGAAACCC GGAATTCCATATGGAATTCC GGGAATTCCATATGGAATTCCC
24
CTGCAGAACCAATGCATTGGATGCAT
26
Pvu I
CCGATCGG
8
ATCGATCGAT
10
TCGCGATCGCGA
12
Sac I
CGAGCTCG
8
Sac II
GCCGCGGC
8
TCCCCGCGGGGA
12
Sal I
GTCGACGTCAAAAGGCCATAGCGGCCGC
28
GCGTCGACGTCTTGGCCATAGCGGCCGCGG
0
0
>90 >90
75
>90
75
>90
Xho I Xma I
CCTCGAGG CCCTCGAGGG CCGCTCGAGCGG
CCCCGGGG CCCCCGGGGG CCCCCCGGGGGG TCCCCCCGGGGGGA
8
0
0
10
10
25
12
10
75
8
0
0
10
25
75
12
50
>90
14
>90 >90
14
10
>90
>90 >90
0
0
0
25
0
>90
0
0
0
25
0
50
75
>90
0
0
10
10
>90 >90
>90 >90
0
0
0
0
10
25
0
10
10
10
0
0
50
>90
0
0
10
50
10
75
10
25
75
75
0
10
0
10
10
50
>90 >90
10
>90
10
>90
0
50
0
50
0
0
0
25
10
50
>90 >90 >90 >90 >90 >90
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.
30
ACGCGTCGACGTCGGCCATAGCGGCCGCGGAA 32
Sca I
GAGTACTC
8
AAAAGTACTTTT
12
Sma I
CCCGGG
6
CCCCGGGG
8
CCCCCGGGGG
10
TCCCCCGGGGGA
12
Spe I
GACTAGTC
8
GGACTAGTCC
10
CGGACTAGTCCG
12
CTAGACTAGTCTAG
14
Sph I Stu I Xba I
GGCATGCC
8
CATGCATGCATG
12
ACATGCATGCATGT
14
AAGGCCTT
8
GAAGGCCTTC
10
AAAAGGCCTTTT
12
CTCTAGAG
8
GCTCTAGAGC
10
TGCTCTAGAGCA
12
CTAGTCTAGACTAG
>90
8
0
0
10
0
0
12
50
>90
9
0
10
22
0
0
24
25
>90
27
25
>90
8
0
0
8
0
0
10
>90 >90
12
50
50
8
>90 >90
10
>90 >90
12
>9wk.baidu.com >90
8
>90 >90
10
>90 >90
12
>90 >90
8
0
0
10
0
0
12
10
75
8
0
0
10
>90 >90
12
>90 >90
8
0
0
10
Enzyme Acc I Afl III Asc I Ava I BamH I
Oligo Sequence
GGTCGACC CGGTCGACCG CCGGTCGACCGG
CACATGTG CCACATGTGG CCCACATGTGGG
GGCGCGCC AGGCGCGCCT TTGGCGCGCCAA
CCCCGGGG CCCCCGGGGG TCCCCCGGGGGA
CGGATCCG CGGGATCCCG CGCGGATCCGCG
% Cleavage Chain Length 2 hr 20 hr
8
0
0
10
0
0
12
0
0
8
0
0
10
>90 >90
12
>90 >90
8
>90 >90
10
>90 >90
12
>90 >90
8
50
>90
10
>90 >90
12
>90 >90
8
10
10
CCTTAATTAAGG
12
Pme I
GTTTAAAC
8
GGTTTAAACC
10
GGGTTTAAACCC
12
AGCTTTGTTTAAACGGCGCGCCGG
24
Pst I
GCTGCAGC
8
TGCACTGCAGTGCA
14
AACTGCAGAACCAATGCATTGG
22
AAAACTGCAGCCAATGCATTGGAA
25
50
8
0
0
14
50
75
8
0
0
10
0
0
12
0
0
18
0
0
20
75
>90
22
75
>90
8
0
0
10
10
25
12
10
50
12
0
0
16
10
10
20
10
10
24
25
90
28
25
>90
Nsi I Pac I
TGCATGCATGCA
12
CCAATGCATTGGTTCTGCAGTT
22
TTAATTAA
8
GTTAATTAAC
New England Biolabs Technical Literature - Updated 04/04/00
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.
GGGT(A/T)ACCC
AACTGCAGAACCAATGCATTGG AAAACTGCAGCCAATGCATTGGAA CTGCAGAACCAATGCATTGGATGCAT
CATCGATG GATCGATC CCATCGATGG CCCATCGATGGG
GGAATTCC CGGAATTCCG CCGGAATTCCGG
GGCTAGCC CGGCTAGCCG CTAGCTAGCTAG
TTGCGGCCGCAA ATTTGCGGCCGCTTTA AAATATGCGGCCGCTATAAA ATAAGAATGCGGCCGCTAAACTAT AAGGAAAAAAGCGGCCGCAAAAGGAAAA
8
0
0
10
75
>90
12
25
25
10
>90 >90
12
>90 >90
Bgl II BssH II BstE II BstX I Cla I
EcoR I Hae III Hind III Kpn I Mlu I Nco I Nde I
Nhe I Not I
CAGATCTG GAAGATCTTC GGAAGATCTTCC
GGCGCGCC AGGCGCGCCT TTGGCGCGCCAA
GGGGCCCC AGCGGCCGCT TTGCGGCCGCAA
CAAGCTTG CCAAGCTTGG CCCAAGCTTGGG
GGGTACCC GGGGTACCCC CGGGGTACCCCG
GACGCGTC CGACGCGTCG
CCCATGGG CATGCCATGGCATG
CCATATGG CCCATATGGG CGCCATATGGCG GGGTTTCATATGAAACCC GGAATTCCATATGGAATTCC GGGAATTCCATATGGAATTCCC
24
CTGCAGAACCAATGCATTGGATGCAT
26
Pvu I
CCGATCGG
8
ATCGATCGAT
10
TCGCGATCGCGA
12
Sac I
CGAGCTCG
8
Sac II
GCCGCGGC
8
TCCCCGCGGGGA
12
Sal I
GTCGACGTCAAAAGGCCATAGCGGCCGC
28
GCGTCGACGTCTTGGCCATAGCGGCCGCGG
0
0
>90 >90
75
>90
75
>90
Xho I Xma I
CCTCGAGG CCCTCGAGGG CCGCTCGAGCGG
CCCCGGGG CCCCCGGGGG CCCCCCGGGGGG TCCCCCCGGGGGGA
8
0
0
10
10
25
12
10
75
8
0
0
10
25
75
12
50
>90
14
>90 >90
14
10
>90
>90 >90
0
0
0
25
0
>90
0
0
0
25
0
50
75
>90
0
0
10
10
>90 >90
>90 >90
0
0
0
0
10
25
0
10
10
10
0
0
50
>90
0
0
10
50
10
75
10
25
75
75
0
10
0
10
10
50
>90 >90
10
>90
10
>90
0
50
0
50
0
0
0
25
10
50
>90 >90 >90 >90 >90 >90
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.
30
ACGCGTCGACGTCGGCCATAGCGGCCGCGGAA 32
Sca I
GAGTACTC
8
AAAAGTACTTTT
12
Sma I
CCCGGG
6
CCCCGGGG
8
CCCCCGGGGG
10
TCCCCCGGGGGA
12
Spe I
GACTAGTC
8
GGACTAGTCC
10
CGGACTAGTCCG