Abstract
Escherichia coli and Saccharomyces cerevisiae are currently the two most important organisms in synthetic biology. E.coli is almost always used for fundamental DNA manipulation while yeast is the simplest host system for studying eukaryotic gene expression and performing large scale DNA assembly. Yeast expression studies may also require altering of the chromosomal DNA by homologous recombination. All these studies require the verification of the expected DNA sequence and the fastest method of screening is colony PCR, which is direct PCR of DNA in cells without prior DNA purification. Colony PCR is hampered by the difficulty of releasing DNA into the PCR mix and the presence of PCR inhibitors. We hereby present one protocol for E. coli and two protocols for S. cerevisiae differing in efficiency and complexity as well as an overview of past and possible future developments of efficient S. cerevisiae colony PCR protocols.
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References
Güssow D, Clackson T (1989) Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res 17:4000
Gibson DG, Benders GA, Axelrod KC, Zaveri J, Algire MA, Moodie M, Montague MG, Venter JC, Smith HO, Hutchison CA (2008) One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic mycoplasma genitalium genome. Proc Natl Acad Sci U S A 105:20404–20409
Pereira F, Azevedo F, Parachin NS, Hahn-Hägerdal B, Gorwa-Grauslund MF, Johansson B (2016) Yeast pathway kit: a method for metabolic pathway assembly with automatically simulated executable documentation. ACS Synth Biol 5:386–394
Flávio Azevedo Humberto Pereira (2016) Online yeast colony PCR protocols. In: Public Github Gist. https://gist.github.com/BjornFJohansson/490ca933976d286cbaef37a07df486b8. Accessed 1 Jul 2016
Sathe GM, O’Brien S, McLaughlin MM, Watson F, Livi GP (1991) Use of polymerase chain reaction for rapid detection of gene insertions in whole yeast cells. Nucleic Acids Res 19:4775
Ling M, Merante F, Robinson BH (1995) A rapid and reliable DNA preparation method for screening a large number of yeast clones by polymerase chain reaction. Nucleic Acids Res 23:4924–4925
Wang H, Kohalmi SE, Cutler AJ (1996) An improved method for polymerase chain reaction using whole yeast cells. Anal Biochem 237:145–146
Bourke MT, Scherczinger CA, Ladd C, Lee HC (1999) NaOH treatment to neutralize inhibitors of Taq polymerase. J Forensic Sci 44:1046–1050
Akada R, Murakane T, Nishizawa Y (2000) DNA extraction method for screening yeast clones by PCR. BioTechniques 28:668–670. 672, 674
Linke B, Schröder K, Arter J, Gasperazzo T, Woehlecke H, Ehwald R (2010) Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption. BioTechniques 49:655–657
Lõoke M, Kristjuhan K, Kristjuhan A (2011) Extraction of genomic DNA from yeasts for PCR-based applications. BioTechniques 50:325–328
Rossen L, Nørskov P, Holmstrøm K, Rasmussen OF (1992) Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol 17:37–45
Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34
Pham TA, Kawai S, Murata K (2011) Visualization of the synergistic effect of lithium acetate and single-stranded carrier DNA on Saccharomyces cerevisiae transformation. Curr Genet 57:233–239
Harju S, Fedosyuk H, Peterson KR (2004) Rapid isolation of yeast genomic DNA: bust n’ grab. BMC Biotechnol 4:8
Blount BA, Driessen MRM, Ellis T (2016) GC Preps: fast and easy extraction of stable yeast genomic DNA. Sci Rep 6:26863
Walsh PS, Metzger DA, Higuchi R (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10:506–513
Kermekchiev MB, Kirilova LI, Vail EE, Barnes WM (2009) Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples. Nucleic Acids Res 37:e40
Wang Y, Prosen DE, Mei L, Sullivan JC, Finney M, Vander Horn PB (2004) A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res 32:1197–1207
Ghadessy FJ, Ong JL, Holliger P (2001) Directed evolution of polymerase function by compartmentalized self-replication. Proc Natl Acad Sci U S A 98:4552–4557
Baar C, d’Abbadie M, Vaisman A, Arana ME, Hofreiter M, Woodgate R, Kunkel TA, Holliger P (2011) Molecular breeding of polymerases for resistance to environmental inhibitors. Nucleic Acids Res 39:e51
Winship PR (1989) An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res 17:1266
Varadaraj K, Skinner DM (1994) Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. Gene 140:1–5
Henke W, Herdel K, Jung K, Schnorr D, Loening SA (1997) Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res 25:3957–3958
Hengen PN (1997) Optimizing multiplex and LA-PCR with betaine. Trends Biochem Sci 22:225–226
Mytelka DS, Chamberlin MJ (1996) Analysis and suppression of DNA polymerase pauses associated with a trinucleotide consensus. Nucleic Acids Res 24:2774–2781
Frackman S, Kobs G, Simpson D, Storts D et al (1998) Betaine and DMSO: enhancing agents for PCR. Promega Notes 65:27–29
Kang J, Lee MS, Gorenstein DG (2005) The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. J Biochem Biophys Methods 64:147–151
Hardjasa A, Ling M, Ma K, Yu H (2010) Investigating the effects of DMSO on PCR fidelity using a restriction digest-based method. J Exp Microbiol Immunol 14:161–164
Rees WA, Yager TD, Korte J, von Hippel PH (1993) Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry 32:137–144
Spiess A-N, Mueller N, Ivell R (2004) Trehalose is a potent PCR enhancer: lowering of DNA melting temperature and thermal stabilization of taq polymerase by the disaccharide trehalose. Clin Chem 50:1256–1259
Desai UJ, Pfaffle PK (1995) Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli. BioTechniques 19(780–782):784
Bachmann B, Lüke W, Hunsmann G (1990) Improvement of PCR amplified DNA sequencing with the aid of detergents. Nucleic Acids Res 18:1309
Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63:3741–3751
Li H, Huang J, Lv J, An H, Zhang X, Zhang Z, Fan C, Hu J (2005) Nanoparticle PCR: nanogold-assisted PCR with enhanced specificity. Angew Chem Int Ed 44:5100–5103
Yang W, Li X, Sun J, Shao Z (2013) Enhanced PCR amplification of GC-rich DNA templates by gold nanoparticles. ACS Appl Mater Interfaces 5:11520–11524
Khaliq RA, Sonawane PJ, Sasi BK, Sahu BS, Pradeep T, Das SK, Mahapatra NR (2010) Enhancement in the efficiency of polymerase chain reaction by TiO 2 nanoparticles: crucial role of enhanced thermal conductivity. Nanotechnology 21:255704
Jia J, Sun L, Hu N, Huang G, Weng J (2012) Graphene enhances the specificity of the polymerase chain reaction. Small 8:2011–2015
Musso M, Bocciardi R, Parodi S, Ravazzolo R, Ceccherini I (2006) Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences. J Mol Diagn 8:544–550
Ralser M, Querfurth R, Warnatz H-J, Lehrach H, Yaspo M-L, Krobitsch S (2006) An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun 347:747–751
Zhang Z, Kermekchiev MB, Barnes WM (2010) Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. J Mol Diagn 12:152–161
Dallas-Yang Q, Jiang G, Sladek FM (1998) Avoiding false positives in colony PCR. BioTechniques 24:580–582
Lee AB, Cooper TA (1995) Improved direct PCR screen for bacterial colonies: wooden toothpicks inhibit PCR amplification. BioTechniques 18:225–226
Colony Immunoblotting Assay for Detection of Bacterial Cell-surface or Extracellular Proteins —BIO-PROTOCOL. http://www.bio-protocol.org/e888. Accessed 26 Jul 2016
Acknowledgment
This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI).
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Azevedo, F., Pereira, H., Johansson, B. (2017). Colony PCR. In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 1620. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-7060-5_8
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DOI: https://doi.org/10.1007/978-1-4939-7060-5_8
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