PCR保护碱基的设计-连接方法.doc

Cleavage Close to the End of DNA Fragments
(linearized vector)

Linearized vectors were incubated with the indicated enzymes (10 units/µg) for 60 minutes at the recommended incubation temperature and NEBuffer for each enzyme. Following ligation and transformation, cleavage efficiencies were determined by dividing the number of transformants from the digestion reaction by the number obtained from religation of the linearized DNA (typically 100-500 colonies) and subtracting from 100%. "Base Pairs from End" refers to the number of double-stranded base pairs between the recognition site and the terminus of the fragment; this number does not include the single-stranded overhang from the initial cut. Since it has not been demonstrated whether these single-stranded nucleotides contribute to cleavage efficiency, 4 bases should be added to the indicated numbers when designing PCR primers. Average efficiencies were rounded to the nearest whole number; experimental variation was typically within 10%. The numbers in parentheses refer to the number of independent trials for each enzyme tested (from Moreira, R. and Noren, C. (1995), Biotechniques, 19, 56-59).
Note: As a general rule, enzymes not listed below require 6 bases pairs on either side of their recognition site to cleave efficiently.
| A | B | E | H | K | M | N | P | S | X | 
Enzyme
Base pairs
from End
%Cleavage
Efficiency
Vector
Initial Cut
Aat II
3
2
1 
88 (2)
100 (2)
95 (2) 
LITMUS 29
LITMUS 28
LITMUS 29 
Nco I
Nco I
PinA I 
Acc65 I
2
1 
99 (2)
75 (3) 
LITMUS 29
pNEB193 
Spe I
Sac I 
Afl II
1
13 (2)
LITMUS 29
Stu I
Age I
1
1 
100 (1)
100 (2) 
LITMUS 29
LITMUS 29 
Xba I
Aat II 
Apa I
2
100 (1)
LITMUS 38
Spe I
Asc I
1
97 (2)
pNEB193
BamH I
Avr II
1
100 (2)
LITMUS 29
Sac I
BamH I
1
97 (2)
LITMUS 29
Hind III
Bgl II
3
100 (2)
LITMUS 29
Nsi I
BsiW I
2
100 (2)
LITMUS 29
BssH II
BspE I
2
1 
100 (1)
8 (2) 
LITMUS 39
LITMUS 38 
BsrG I
BsrG I 
BsrG I
2
1 
99 (2)
88 (2) 
LITMUS 39
LITMUS 38 
Sph I
BspE I 
BssH II
2
100 (2)
LITMUS 29
BsiW I
Eag I
2
100 (2)
LITMUS 39
Nhe I
EcoR I
1
1
1 
100 (1)
88 (1)
100 (1) 
LITMUS 29
LITMUS 29
LITMUS 39 
Xho I
Pst I
Nhe I 
EcoR V
1
100 (2)
LITMUS 29
Pst I
Hind III
3
2
1 
90 (2)
91 (2)
0 (2) 
LITMUS 29
LITMUS 28
LITMUS 29 
Nco I
Nco I
BamH I 
Kas I
2
1 
97 (1)
93 (1) 
LITMUS 38
LITMUS 38 
NgoM IV
Hind III 
Kpn I
2
2
1 
100 (2)
100 (2)
99 (2) 
LITMUS 29
LITMUS 29
pNEB193 
Spe I
Sac I
Sac I 
Mlu I
2
99 (2)
LITMUS 39
Eag I
Mun I
2
100 (1)
LITMUS 39
NgoM IV
Nco I
2
100 (1)
LITMUS 28
Hind III
NgoM IV
2
100 (1)
LITMUS 39
Mun I
Nhe I
1
2 
100 (1)
82 (1) 
LITMUS 39
LITMUS 39 
EcoR I
Eag I 
Not I
7
4
1 
100 (2)
100 (1)
98 (2) 
Bluescript SK-
Bluescript SK-
Bluescript SK- 
Spe I
Ksp I
Xba I 
Nsi I
3
3
2 
100 (2)
77 (4)
95 (2) 
LITMUS 29
LITMUS 29
LITMUS 28 
BssH II
Bgl II
BssH II 
Pac I
1
76 (3)
pNEB193
BamH I
Pme I
1
94 (2)
pNEB193
Pst I
Pst I
3
2
1 
98 (1)
50 (5)
37 (3) 
LITMUS 29
LITMUS 39
LITMUS 29 
EcoR V
Hind III
EcoR I 
Sac I
1
99 (2)
LITMUS 29
Avr II
Sal I
3
2
1 
89 (2)
23 (2)
61 (3) 
LITMUS 39
LITMUS 39
LITMUS 38 
Spe I
Sph I
Sph I 
Spe I
2
2 
100 (2)
100 (2) 
LITMUS 29
LITMUS 29 
Acc65 I
Kpn I 
Sph I
2
2
1 
99 (1)
97 (1)
92 (2) 
LITMUS 39
LITMUS 39
LITMUS 38 
Sal I
BsrG I
Sal I 
Xba I
1
1 
99 (2)
94 (1) 
LITMUS 29
LITMUS 29 
Age I
PinA I 
Xho I
1
97 (2)
LITMUS 29
EcoR I
Xma I
2
2 
98 (1)
92 (1) 
pNEB193
pNEB193 
Asc I
BssH II 

New England Biolabs Technical Literature - Updated  03/05/2004

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 g-[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.
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
Acc I
GGTCGACC
CGGTCGACCG
CCGGTCGACCGG
8
10
12
0
0
0
0
0
0
Afl III
CACATGTG
CCACATGTGG
CCCACATGTGGG
8
10
12
0
>90
>90
0
>90
>90
Asc I
GGCGCGCC
AGGCGCGCCT
TTGGCGCGCCAA
8
10
12
>90
>90
>90
>90
>90
>90
Ava I
CCCCGGGG
CCCCCGGGGG
TCCCCCGGGGGA
8
10
12
50
>90
>90
>90
>90
>90
BamH I
CGGATCCG
CGGGATCCCG
CGCGGATCCGCG
8
10
12
10
>90
>90
25
>90
>90
Bgl II
CAGATCTG
GAAGATCTTC
GGAAGATCTTCC
8
10
12
0
75
25
0
>90
>90
BssH II
GGCGCGCC
AGGCGCGCCT
TTGGCGCGCCAA
8
10
12
0
0
50
0
0
>90
BstE II
GGGT(A/T)ACCC
9
0
10
BstX I
AACTGCAGAACCAATGCATTGG
AAAACTGCAGCCAATGCATTGGAA
CTGCAGAACCAATGCATTGGATGCAT
22
24
27
0
25
25
0
50
>90
Cla I
CATCGATG
GATCGATC
CCATCGATGG
CCCATCGATGGG
8
8
10
12
0
0
>90
50
0
0
>90
50
EcoR I
GGAATTCC
CGGAATTCCG
CCGGAATTCCGG
8
10
12
>90
>90
>90
>90
>90
>90
Hae III
GGGGCCCC
AGCGGCCGCT
TTGCGGCCGCAA
8
10
12
>90
>90
>90
>90
>90
>90
Hind III
CAAGCTTG
CCAAGCTTGG
CCCAAGCTTGGG
8
10
12
0
0
10
0
0
75
Kpn I
GGGTACCC
GGGGTACCCC
CGGGGTACCCCG
8
10
12
0
>90
>90
0
>90
>90
Mlu I
GACGCGTC
CGACGCGTCG
8
10
0
25
0
50
Nco I
CCCATGGG
CATGCCATGGCATG
8
14
0
50
0
75
Nde I
CCATATGG
CCCATATGGG
CGCCATATGGCG
GGGTTTCATATGAAACCC
GGAATTCCATATGGAATTCC
GGGAATTCCATATGGAATTCCC
8
10
12
18
20
22
0
0
0
0
75
75
0
0
0
0
>90
>90
Nhe I
GGCTAGCC
CGGCTAGCCG
CTAGCTAGCTAG
8
10
12
0
10
10
0
25
50
Not I
TTGCGGCCGCAA
ATTTGCGGCCGCTTTA
AAATATGCGGCCGCTATAAA
ATAAGAATGCGGCCGCTAAACTAT
AAGGAAAAAAGCGGCCGCAAAAGGAAAA
12
16
20
24
28
0
10
10
25
25
0
10
10
90
>90
Nsi I
TGCATGCATGCA
CCAATGCATTGGTTCTGCAGTT
12
22
10
>90
>90
>90
Pac I
TTAATTAA
GTTAATTAAC
CCTTAATTAAGG
8
10
12
0
0
0
0
25
>90
Pme I
GTTTAAAC
GGTTTAAACC
GGGTTTAAACCC
AGCTTTGTTTAAACGGCGCGCCGG
8
10
12
24
0
0
0
75
0
25
50
>90
Pst I
GCTGCAGC
TGCACTGCAGTGCA
AACTGCAGAACCAATGCATTGG
AAAACTGCAGCCAATGCATTGGAA
CTGCAGAACCAATGCATTGGATGCAT
8
14
22
24
26
0
10
>90
>90
0
0
10
>90
>90
0
Pvu I
CCGATCGG
ATCGATCGAT
TCGCGATCGCGA
8
10
12
0
10
0
0
25
10
Sac I
CGAGCTCG
8
10
10
Sac II
GCCGCGGC
TCCCCGCGGGGA
8
12
0
50
0
>90
Sal I
GTCGACGTCAAAAGGCCATAGCGGCCGC
GCGTCGACGTCTTGGCCATAGCGGCCGCGG
ACGCGTCGACGTCGGCCATAGCGGCCGCGGAA
28
30
32
0
10
10
0
50
75
Sca I
GAGTACTC
AAAAGTACTTTT
8
12
10
75
25
75
Sma I
CCCGGG
CCCCGGGG
CCCCCGGGGG
TCCCCCGGGGGA
6
8
10
12
0
0
10
>90
10
10
50
>90
Spe I
GACTAGTC
GGACTAGTCC
CGGACTAGTCCG
CTAGACTAGTCTAG
8
10
12
14
10
10
0
0
>90
>90
50
50
Sph I
GGCATGCC
CATGCATGCATG
ACATGCATGCATGT
8
12
14
0
0
10
0
25
50
Stu I
AAGGCCTT
GAAGGCCTTC
AAAAGGCCTTTT
8
10
12
>90
>90
>90
>90
>90
>90
Xba I
CTCTAGAG
GCTCTAGAGC
TGCTCTAGAGCA
CTAGTCTAGACTAG
8
10
12
14
0
>90
75
75
0
>90
>90
>90
Xho I
CCTCGAGG
CCCTCGAGGG
CCGCTCGAGCGG
8
10
12
0
10
10
0
25
75
Xma I
CCCCGGGG
CCCCCGGGGG
CCCCCCGGGGGG
TCCCCCCGGGGGGA
8
10
12
14
0
25
50
>90
0
75
>90
>90

基因片段连接到质粒

载体上时，可有以下几种连接方式：①最常用的是粘端连接。若DNA插入片段与适当的

载体存在同源粘性末端，这将是最方便的克隆途径。同源粘性末端包括相同一种内切酶产生

的粘性末端和不同的内切酶产生的互补粘性末端，后者连接成的DNA不能再被原切割内切酶

识别，而不利于从重组子上完整地将插入片段重新切割下来。同源粘性末端连接效率高，但

也存在弊端，例如插入片段存在两个方向插入的可能性；插入片段间可相互连接，导致载体

中可能存在几个插入片段；载体自身环化机率高，故应在连接前，将酶切完全的质粒载体DN

A用碱性磷酸酶处理，使其5端去磷酸化以减少自身环化，降低假阳性重组子背景。②如

重组对象不适合粘端连接，则可以用平端连接进行重组。通常的做法是用产生平端的内切

酶切割载体。如用产生粘端的内切酶切割载体，其产生的粘端若是5端突出的，需要用DNA

聚合酶将粘端补平；若是3端突出的，需要用单链核酸酶或T4 DNA聚合酶(它有35

外切酶活性)将突出的3端削平。病毒DNA同样也需补平或削平，将其修饰成平端。然后再

用连接酶将它们连接在一起。平端连接的特点是可以恢复一个原始的，甚至产生一个新的酶

切位点。位点的恢复或创建是十分有用的，它提供了一条简捷的重组子筛选鉴定途径，并可

方便地使插入片段重新从重组子中回收出来。平端连接存在的弊端是它的连接效率比粘端连

接低得多，连接时需要高浓度的连接酶和高浓度的DNA末端(大于1μmol),而且插入方向不确

定。③也可以一端是粘端，另一端是平端进行连接。例如要把〖ＷＴ５ＢＸ〗Eco〖ＷＴ

５ＢＺ〗RI/〖ＷＴ５ＢＸ〗Hae〖ＷＴ５ＢＺ〗Ⅲ的目的基因克隆到pBR322上时，首先用〖

ＷＴ５ＢＸ〗Hin〖ＷＴ５ＢＺ〗dⅢ切开质粒DNA，用大肠杆菌DNA多聚酶I的Klenow片段，

修补〖ＷＴ５ＢＸ〗Hin〖ＷＴ５ＢＺ〗dⅢ消化后的5端突出部分，使之成为平端，再用

〖ＷＴ５ＢＸ〗Eco〖ＷＴ５ＢＺ〗RI消化，电泳纯化，所得载体DNA的一端为平端，另一端

为〖ＷＴ５ＢＸ〗Eco〖ＷＴ５ＢＺ〗RI粘端。这样就可以与〖ＷＴ５ＢＸ〗Eco〖ＷＴ５Ｂ

Ｚ〗RI/〖ＷＴ５ＢＸ〗Hae〖ＷＴ５ＢＺ〗Ⅲ的目的基因进行连接。连接中，外源DNA片段

的插入只有一种可能性，有助于病毒DNA与载体的定向克隆。
④将病毒DNA修饰成平端后，可用酶促法在二端加上适当的接头(linker)或适配子(adaptor)

，将其修饰成粘端，然后再与相同粘端的载体连接。这种连接虽然有助于病毒DNA片段的

回收，但是修饰过程复杂，效率也低，转化细菌后非重组背景高，且可能有多拷贝插入及双

向插入等。