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DNA Cloning

  1. Michael Andrew Quail

Published Online: 23 SEP 2005

DOI: 10.1038/npg.els.0005344

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Quail, M. A. 2005. DNA Cloning. eLS. .

Author Information

  1. The Wellcome Trust Sanger Institute, Cambridge, UK

Publication History

  1. Published Online: 23 SEP 2005

This is not the most recent version of the article. View current version (15 DEC 2010)

Table 1. A selection of restriction endonucleases commonly used in DNA cloning
EnzymeaSourceRecognition sitebEnds produced
  1. a

    Enzymes commonly get their names from abbreviations of the organism from which they were first isolated. Roman numerals after the name depict the order in which enzymes were discovered from an organism, for example, HindIII was the third enzyme to be isolated from Haemophilus influenzae.

  2. b

    The recognition sequence for one strand in the 5′–3′ direction is shown. Typically restriction sites have twofold dyad symmetry, so the same site is seen when reading in the 5′–3′ direction on the other strand. Enzymes cleave the DNA strand within each recognition site to give a 3′ hydroxyl group and a 5′ phosphate group, at the position denoted by ˆ.

BamHIBacillus amyloliquefaciens H5′ GˆGATCC 3′5′ overhang
EcoRIEscherichia coli5′ GˆAATTC 3′5′ overhang
HindIIIHaemophilus influenzae5′ AˆAGCTT 3′5′ overhang
NcoINocardia corallina5′ CˆCATGG 3′5′ overhang
NdeINeisseria denitrificans5′ CAˆTATG 3′5′ overhang
NotINocardia otitidis-caviarum5′ GCˆGGCCGC 3′5′ overhang
PstIProvidencia stuartii5′ CTGCAˆG 3′3′ overhang
Sau3AIStaphylococcus aureus 3A5′ ˆGATC 3′5′ overhang
SmaISerratia marcescens5′ CCCˆGGG 3′Blunt
Table 2. A summary of the properties of the various classes of vectors used in DNA cloning
VectorCloning capacityUseCommon examplesMethod of introduction into cellType of growth on agar plateCopy numberOriginal reference
PlasmidsUp to 12 kbMultiple (e.g. mutagenesis, overexpression, sequencing, screening, manipulation)pUC, pBR322, pGEMTransformation or electroporation into bacteria. Various methods for eukaryotes (see text)Colonies1–700Cohen et al. (1973) PNAS 70: 3240–3244
M13Normally up to 6 kbProbe generation, sequencing, in vitro mutagenesisM13mp18TransfectionPlaques100–200Messing et al. (1977) PNAS 74: 3642–3646
Bacteriophage lambda (λ)9–23 kbGenomic and cDNA libaries, expression screeningλZAP, λFIXPackaging of construct followed by infectionPlaquesN/AMurray and Murray (1974) Nature 251: 476–481
CosmidsUp to 47 kbGenomic libraries, genome mappingSupercos, lawrist, tropistPackaging of construct followed by infectionColoniesUp to 50Collins and Hohn (1978) PNAS 75: 4242–4246
FosmidsUp to 43 kbGenomic libraries, genome mappingpFOS1Packaging of construct followed by infectionColonies1–2Kim et al. (1992) NAR 20: 1083–1085
P1 bacteriophage30–100 kbGenomic libraries, genome mappingpAd10sacBIIPackaging of construct followed by infectionColonies1–2Sternberg PNAS 87: 103–107
BACsUp to 300 kbGenomic libraries, genome mappingpBACe3.6, pBeloBACIIElectroporationColonies1–2Shizuya et al. (1992) PNAS 89: 2629–2633
PACsUp to 300 kbGenomic libraries, genome mappingpCYPAC2ElectroporationColonies1–2Ioannou et al. (1994) Nature Genetics 6: 84–89
YACs20–2000 kbGenomic libraries, genome mappingpYAC4Electroporation, spheroplast absorption, or lithium-mediated transformationColonies1Burke et al. (1987) Science 236: 806–812