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1、-酶切位点保护碱基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
2、 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
3、 unit of oligonucleotide was phosphorylated using T4 polynucleotide kinase and g-32P ATP. 1 g of 5 32P-labeled oligonucleotide was incubated at 20C 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 re
4、quirement 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 effi
5、ciency 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 | EnzymeOligo SequenceChain Length% Cleavage2 hr20 hrAcc IGGTCGACC CGGTCGACCG CCGGTCGACCGG8 10 120 0 00 0 0Afl IIICACATGTG CCACATGTGG CCCACATGTGGG8
6、10 120 90 900 90 90Asc IGGCGCGCC AGGCGCGCCT TTGGCGCGCCAA8 10 1290 90 9090 90 90Ava ICCCCGGGG CCCCCGGGGG TCCCCCGGGGGA8 10 1250 90 9090 90 90BamH ICGGATCCG CGGGATCCCG CGCGGATCCGCG8 10 1210 90 9025 90 90Bgl IICAGATCTG GAAGATCTTC GGAAGATCTTCC8 10 120 75 250 90 90BssH IIGGCGCGCC AGGCGCGCCT TTGGCGCGCCAA8
7、10 120 0 500 0 90BstE IIGGGT(A/T)ACCC9010BstX IAACTGCAGAACCAATGCATTGG AAAACTGCAGCCAATGCATTGGAA CTGCAGAACCAATGCATTGGATGCAT22 24 270 25 250 50 90Cla ICATCGATG GATCGATC CCATCGATGG CCCATCGATGGG8 8 10 120 0 90 500 0 90 50EcoR IGGAATTCC CGGAATTCCG CCGGAATTCCGG8 10 1290 90 9090 90 90Hae IIIGGGGCCCC AGCGGCC
8、GCT TTGCGGCCGCAA8 10 1290 90 9090 90 90Hind IIICAAGCTTG CCAAGCTTGG CCCAAGCTTGGG8 10 120 0 100 0 75Kpn IGGGTACCC GGGGTACCCC CGGGGTACCCCG8 10 120 90 900 90 90Mlu IGACGCGTC CGACGCGTCG8 100 250 50Nco ICCCATGGG CATGCCATGGCATG8 140 500 75Nde ICCATATGG CCCATATGGG CGCCATATGGCG GGGTTTCATATGAAACCC GGAATTCCATA
9、TGGAATTCC GGGAATTCCATATGGAATTCCC8 10 12 18 20 220 0 0 0 75 750 0 0 0 90 90Nhe IGGCTAGCC CGGCTAGCCG CTAGCTAGCTAG8 10 120 10 100 25 50Not ITTGCGGCCGCAA ATTTGCGGCCGCTTTA AAATATGCGGCCGCTATAAA ATAAGAATGCGGCCGCTAAACTAT AAGGAAAAAAGCGGCCGCAAAAGGAAAA12 16 20 24 280 10 10 25 250 10 10 90 90Nsi ITGCATGCATGCA C
10、CAATGCATTGGTTCTGCAGTT12 2210 9090 90Pac ITTAATTAA GTTAATTAAC CCTTAATTAAGG8 10 120 0 00 25 90Pme IGTTTAAAC GGTTTAAACC GGGTTTAAACCC AGCTTTGTTTAAACGGCGCGCCGG8 10 12 240 0 0 750 25 50 90Pst IGCTGCAGC TGCACTGCAGTGCA AACTGCAGAACCAATGCATTGG AAAACTGCAGCCAATGCATTGGAA CTGCAGAACCAATGCATTGGATGCAT8 14 22 24 260
11、10 90 90 00 10 90 90 0Pvu ICCGATCGG ATCGATCGAT TCGCGATCGCGA8 10 120 10 00 25 10Sac ICGAGCTCG81010Sac IIGCCGCGGC TCCCCGCGGGGA8 120 500 90Sal IGTCGACGTCAAAAGGCCATAGCGGCCGC GCGTCGACGTCTTGGCCATAGCGGCCGCGG ACGCGTCGACGTCGGCCATAGCGGCCGCGGAA28 30 320 10 100 50 75Sca IGAGTACTC AAAAGTACTTTT8 1210 7525 75Sma I
12、CCCGGG CCCCGGGG CCCCCGGGGG TCCCCCGGGGGA6 8 10 120 0 10 9010 10 50 90Spe IGACTAGTC GGACTAGTCC CGGACTAGTCCG CTAGACTAGTCTAG8 10 12 1410 10 0 090 90 50 50Sph IGGCATGCC CATGCATGCATG ACATGCATGCATGT8 12 140 0 100 25 50Stu IAAGGCCTT GAAGGCCTTC AAAAGGCCTTTT8 10 1290 90 9090 90 90Xba ICTCTAGAG GCTCTAGAGC TGCT
13、CTAGAGCA CTAGTCTAGACTAG8 10 12 140 90 75 750 90 90 90Xho ICCTCGAGG CCCTCGAGGG CCGCTCGAGCGG8 10 120 10 100 25 75Xma ICCCCGGGG CCCCCGGGGG CCCCCCGGGGGG TCCCCCCGGGGGGA8 10 12 140 25 50 900 75 90 90Cleavage Close to the End of DNA Fragments(linearized vector)Linearized vectors were incubated with the ind
14、icated enzymes (10 units/g) 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 o
15、f 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
16、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 numb
17、ers 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 |
18、 H | K | M | N | P | S | X | EnzymeBase pairs from End%Cleavage EfficiencyVectorInitial CutAat II3 2 188 (2) 100 (2) 95 (2)LITMUS 29 LITMUS 28 LITMUS 29Nco I Nco I PinA IAcc65 I2 199 (2) 75 (3)LITMUS 29 pNEB193Spe I Sac IAfl II113 (2)LITMUS 29Stu IAge I1 1100 (1) 100 (2)LITMUS 29 LITMUS 29Xba I Aat
19、IIApa I2100 (1)LITMUS 38Spe IAsc I197 (2)pNEB193BamH IAvr II1100 (2)LITMUS 29Sac IBamH I197 (2)LITMUS 29Hind IIIBgl II3100 (2)LITMUS 29Nsi IBsiW I2100 (2)LITMUS 29BssH IIBspE I2 1100 (1) 8 (2)LITMUS 39 LITMUS 38BsrG I BsrG IBsrG I2 199 (2) 88 (2)LITMUS 39 LITMUS 38Sph I BspE IBssH II2100 (2)LITMUS 2
20、9BsiW IEag I2100 (2)LITMUS 39Nhe IEcoR I1 1 1100 (1) 88 (1) 100 (1)LITMUS 29 LITMUS 29 LITMUS 39Xho I Pst I Nhe IEcoR V1100 (2)LITMUS 29Pst IHind III3 2 190 (2) 91 (2) 0 (2)LITMUS 29 LITMUS 28 LITMUS 29Nco I Nco I BamH IKas I2 197 (1) 93 (1)LITMUS 38 LITMUS 38NgoM IV Hind IIIKpn I2 2 1100 (2) 100 (2
21、) 99 (2)LITMUS 29 LITMUS 29 pNEB193Spe I Sac I Sac IMlu I299 (2)LITMUS 39Eag IMun I2100 (1)LITMUS 39NgoM IVNco I2100 (1)LITMUS 28Hind IIINgoM IV2100 (1)LITMUS 39Mun INhe I1 2100 (1) 82 (1)LITMUS 39 LITMUS 39EcoR I Eag INot I7 4 1100 (2) 100 (1) 98 (2)Bluescript SK- Bluescript SK- Bluescript SK-Spe I
22、 Ksp I Xba INsi I3 3 2100 (2) 77 (4) 95 (2)LITMUS 29 LITMUS 29 LITMUS 28BssH II Bgl II BssH IIPac I176 (3)pNEB193BamH IPme I194 (2)pNEB193Pst IPst I3 2 198 (1) 50 (5) 37 (3)LITMUS 29 LITMUS 39 LITMUS 29EcoR V Hind III EcoR ISac I199 (2)LITMUS 29Avr IISal I3 2 189 (2) 23 (2) 61 (3)LITMUS 39 LITMUS 39
23、 LITMUS 38Spe I Sph I Sph ISpe I2 2100 (2) 100 (2)LITMUS 29 LITMUS 29Acc65 I Kpn ISph I2 2 199 (1) 97 (1) 92 (2)LITMUS 39 LITMUS 39 LITMUS 38Sal I BsrG I Sal IXba I1 199 (2) 94 (1)LITMUS 29 LITMUS 29Age I PinA IXho I197 (2)LITMUS 29EcoR IXma I2 298 (1) 92 (1)pNEB193 pNEB193Asc I BssH II-第 8 页Cleavage Close to the End of DNA Fragments(oligonucleotides)