All Title Author
Keywords Abstract

PLOS ONE  2012 

Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

DOI: 10.1371/journal.pone.0053360

Full-Text   Cite this paper   Add to My Lib


We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.


[1]  Smith HO, Wilcox KW (1970) A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J Mol Biol 51: 379–391.
[2]  Danna K, Nathans D (1971) Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc Natl Acad Sci U S A 68: 2913–2917.
[3]  Mertz JE, Davis RW (1972) Cleavage of DNA by R1 restriction endoculease generates cohesive ends. Proc Natl Acad Sci U S A 69: 3370–3374.
[4]  Cohen SN, Chang AC, Boyer HW, Helling RB (1973) Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A 70: 3240–3244.
[5]  Sgaramella V, van de Sande JH, Khorana HG (1970) Studies on Polynucleotides, C. A novel joining reaction catalyzed by the T4-polynucleotide ligase. Proc Natl Acad Sci U S A 67: 1468–1475.
[6]  Heyneker HL, Shine J, Goodman HM, Boyer HW, Rosenberg J, et al. (1976) Synthetic lac operator DNA is functional in vivo. Nature 263: 748–752.
[7]  Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, et al. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1354.
[8]  Mullis K, Faloona F, Scharf S, Saiki R, Horn G, et al. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51: Pt 1, 263–273.
[9]  Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491.
[10]  Marchuk D, Drumm M, Saulino A, Collins FS (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res 19: 1154.
[11]  Walhout AJ, Temple GF, Brasch MA, Hartley JL, Lorson MA, et al. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Meth Enzymol 328: 575–592.
[12]  Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10: 1788–1795.
[13]  Farmer AA, Clontech Laboratories Inc. (2002) Patent US-6410317.
[14]  Lu Q (2005) Seamless cloning and gene fusion. Trends Biotechnol 23: 199–207.
[15]  Yang XW, Model P, Heintz N (1997) Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol 15: 859–865.
[16]  Bubeck P, Winkler M, Bautsch W (1993) Rapid cloning by homologous recombination in vivo. Nucleic Acids Res 21: 3601–3602.
[17]  Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20: 123–128.
[18]  Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18: 6069–6074.
[19]  Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4: 251–256.
[20]  Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6: 343–345.
[21]  Chen GJ, Qiu N, Karrer C, Caspers P, Page MG (2000) Restriction site-free insertion of PCR products directionally into vectors. Biotechniques 28: 498–500, 504–505.
[22]  van den Ent F, L?we J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67: 67–74.
[23]  Unger T, Jacobovitch Y, Dantes A, Bernheim R, Peleg Y (2010) Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172: 34–44.
[24]  Picard V, Ersdal-Badju E, Lu A, Bock SC (1994) A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Res 22: 2587–2591.
[25]  Barik S (1997) Mutagenesis and gene fusion by megaprimer PCR. Methods Mol Biol 67: 173–182.
[26]  Chen JR, Lü JJ, Wang HF (2008) Rapid and efficient gene splicing using megaprimer-based protocol. Mol Biotechnol 40: 224–230.
[27]  He Q, Marjamaki M, Soini H, Mertsola J, Viljanen MK (1994) Primers are decisive for sensitivity of PCR. Biotechniques 17: 82–87.
[28]  Apte A, Daniel S (2009) PCR primer design. Cold Spring Harb Protoc doi:10.1101/pdb.ip65.
[29]  Mammedov TG, Pienaar E, Whitney SE, TerMaat JR, Carvill G, et al. (2008) A fundamental study of the PCR amplification of GC-rich DNA templates. Comput Biol Chem 32: 452–457.
[30]  Hansen LL, Justesen J (2006) PCR amplification of highly GC-rich regions. Cold Spring Harb Protoc doi:10.1101/pdb.prot4093.
[31]  McPherson MJ, M?ller SG (2000) PCR basics: from background to bench. New York: Springer-Verlag. 276 p.
[32]  R?dstr?m P, L?fstr?m C, L?venklev M, Knutsson R, Wolffs P (2008) Strategies for overcoming PCR inhibition. Cold Spring Harb Protoc doi:10.1101/pdb.top20.
[33]  Tindall KR, Kunkel TA (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27: 6008–6013.
[34]  Lundberg KS, Shoemaker DD, Adams MW, Short JM, Sorge JA, et al. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated form Pyrococcus furiosus. Gene 108: 1–6.
[35]  Eckert KA, Kunkel TA (1991) DNA polymerase fidelity and the polymerase chain reaction. Genome Res 1: 17–24.
[36]  Frey B, Suppmann B (1995) Demonstration of the expand PCR system’s greater fidelity and higher yields with a lacI-based PCR fidelity assay. Biochemica 2: 34–35.
[37]  Papworth C, Bauer JC, Braman J, Wright DA (1996) Site-directed mutagenesis in one day with >80% efficiency. Strategies 9: 3–4.
[38]  Bauer JC, Dowain AW, Braman JC, Geha RS, Strategene (1998) Circular site-directed mutagenesis, Patent US-5789166.
[39]  Hemsley A, Arnheim N, Toney MD, Cortopassi G, Galas DJ (1989) A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res 17: 6545–6551.


comments powered by Disqus