酶切位点所加保护碱基.doc

寡核苷酸近末端位点的酶切(Cleavage Close to the End of DNA Fragments (oligonucleotides) 为了解不同内切酶对识别位点以外最少保护碱基数目的要求，NEB采用了一系列含识别序列的短双链寡核苷酸作为酶切底物进行实验。实验结果对于确定双酶切顺序将会有帮助（比如在多接头上切割位点很接近时），或者当切割位点靠近DNA末端时也很有用。在本表中没有列出的酶，则通常需在识别位点两端至少加上6个保护碱基，以确保酶切反应的进行。实验方法：用-32PATP在T4多聚核苷酸激酶的作用下标记0.1A260单位的寡核苷酸。取1 g已标记了的寡核苷酸与20单位的内切酶，在20C条件下分别反应2小时和20小时。反应缓冲液含70 mM Tris-HCl (pH 7.6), 10 mM MgCl2 , 5 mM DTT及适量的NaCl或KCl（视酶的具体要求而定）。20%的PAGE（7 M尿素）凝胶电泳分析，经放射自显影确定酶切百分率。本实验采用自连接的寡核苷酸作为对照。若底物有较长的回文结构，切割效率则可能因为出现发夹结构而降低。酶寡核苷酸序列链长切割率%2 hr20 hrAcc IGGTCGACC CGGTCGACCG CCGGTCGACCGG 8 10 120 0 00 0 0Afl IIICACATGTG CCACATGTGG CCCACATGTGGG8 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 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 AGCGGCCGCT 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 GGAATTCCATATGGAATTCC 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 CCAATGCATTGGTTCTGCAGTT12 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 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 ICCCGGG 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 TGCTCTAGAGCA 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 90克隆PCR产物的方法之一，是在PCR产物两端设计一定的限制酶切位点，经酶切后克隆至用相同酶切的载体中。但实验证明，大多数限制酶对裸露的酶切位点不能切断。必须在酶切位点旁边加上一个至几个保护碱基，才能使所定的限制酶对其识别位点进行有效切断。下表列举了15种限制酶，分别比较了各种限制酶在其酶切位点旁边分别加0、1、2、3个保护碱基后的切断情况。表中的：(-)为不能切断；()为不能完全切断；(+)为能完全切断。结果显示，基本上所有限制酶，在其酶切位点旁边加上3个以上的保护碱基后，可以对其酶切位点进行有效切断。Linearized vectors were incubated with the indicated 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 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 | EnzymeBase pairsfrom End%CleavageEfficiencyVectorInitial CutAatII321 88 (2)100 (2)95 (2) LITMUS 29LITMUS 28LITMUS 29 NcoINcoIPinAI Acc65I2199 (2)75 (3)LITMUS 29pNEB193SpeISacIAflII113 (2)LITMUS 29StuIAgeI11100 (1)100 (2)LITMUS 29LITMUS 29XbaIAatIIApaI2100 (1)LITMUS 38SpeIAscI197 (2)pNEB193BamHIAvrII1100 (2)LITMUS 29SacIBamHI197 (2)LITMUS 29HindIIIBglII3100 (2)LITMUS 29NsiIBsiWI2100 (2)LITMUS 29BssHIIBspEI21100 (1)8 (2)LITMUS 39LITMUS 38BsrGIBsrGIBsrGI2199 (2)88 (2)LITMUS 39LITMUS 38SphIBspEIBssHII2100 (2)LITMUS 29BsiWIEagI2100 (2)LITMUS 39NheIEcoRI111100 (1)88 (1)100 (1)LITMUS 29LITMUS 29LITMUS 39XhoIPstINheIEcoRV1100 (2)LITMUS 29PstIHindIII32190 (2)91 (2)0 (2)LITMUS 29LITMUS 28LITMUS 29NcoINcoIBamHIKasI2197 (1)93 (1)LITMUS 38LITMUS 38NgoMIVHindIIIKpnI221100 (2)100 (2)99 (2)LITMUS 29LITMUS 29pNEB193SpeISacISacIMluI299 (2)LITMUS 39EagIMunI2100 (1)LITMUS 39NgoMIVNcoI2100 (1)LITMUS 28HindIIINgoMIV2100 (1)LITMUS 39MunINheI12100 (1)82 (1)LITMUS 39LITMUS 39EcoRIEagINotI741100 (2)100 (1)98 (2)Bluescript SK-Blu