Abstract
The benefits of adding bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to PCR were evaluated with reaction mixtures containing substances that inhibit amplification. Whereas 10- to 1,000-fold more FeCl3, hemin, fulvic acids, humic acids, tannic acids, or extracts from feces, freshwater, or marine water were accommodated in PCR when either 400 ng of BSA per microl or 150 ng of gp32 per microl was included in the reactions, neither BSA nor gp32 relieved interference significantly when minimum inhibitory levels of bile salts, bilirubin, EDTA, NaCl, sodium dodecyl sulfate, or Triton X-100 were present. Use of BSA and gp32 together offered no more relief of inhibition than either alone at its optimal level, and neither protein had any noticeable effect on amplification in the absence of inhibitors.
Full Text
The Full Text of this article is available as a PDF (736.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Akane A., Shiono H., Matsubara K., Nakamura H., Hasegawa M., Kagawa M. Purification of forensic specimens for the polymerase chain reaction (PCR) analysis. J Forensic Sci. 1993 May;38(3):691–701. [PubMed] [Google Scholar]
- Boom R., Sol C. J., Salimans M. M., Jansen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. doi: 10.1128/jcm.28.3.495-503.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chase J. W., Williams K. R. Single-stranded DNA binding proteins required for DNA replication. Annu Rev Biochem. 1986;55:103–136. doi: 10.1146/annurev.bi.55.070186.000535. [DOI] [PubMed] [Google Scholar]
- Demeke T., Adams R. P. The effects of plant polysaccharides and buffer additives on PCR. Biotechniques. 1992 Mar;12(3):332–334. [PubMed] [Google Scholar]
- Höss M., Kohn M., Päbo S., Knauer F., Schröder W. Excrement analysis by PCR. Nature. 1992 Sep 17;359(6392):199–199. doi: 10.1038/359199a0. [DOI] [PubMed] [Google Scholar]
- Höss M., Päbo S. DNA extraction from Pleistocene bones by a silica-based purification method. Nucleic Acids Res. 1993 Aug 11;21(16):3913–3914. doi: 10.1093/nar/21.16.3913. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kreader C. A. Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution. Appl Environ Microbiol. 1995 Apr;61(4):1171–1179. doi: 10.1128/aem.61.4.1171-1179.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loomis W. D. Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods Enzymol. 1974;31:528–544. doi: 10.1016/0076-6879(74)31057-9. [DOI] [PubMed] [Google Scholar]
- McKeown B. J. An acetylated (nuclease-free) bovine serum albumin in a PCR buffer inhibits amplification. Biotechniques. 1994 Aug;17(2):246–248. [PubMed] [Google Scholar]
- Panaccio M., Lew A. PCR based diagnosis in the presence of 8% (v/v) blood. Nucleic Acids Res. 1991 Mar 11;19(5):1151–1151. doi: 10.1093/nar/19.5.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Romanowski G., Lorenz M. G., Wackernagel W. Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils. Appl Environ Microbiol. 1993 Oct;59(10):3438–3446. doi: 10.1128/aem.59.10.3438-3446.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rossen L., Holmstrøm K., Olsen J. E., Rasmussen O. F. A rapid polymerase chain reaction (PCR)-based assay for the identification of Listeria monocytogenes in food samples. Int J Food Microbiol. 1991 Nov;14(2):145–151. doi: 10.1016/0168-1605(91)90101-t. [DOI] [PubMed] [Google Scholar]
- Rossen L., Nørskov P., Holmstrøm K., Rasmussen O. F. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol. 1992 Sep;17(1):37–45. doi: 10.1016/0168-1605(92)90017-w. [DOI] [PubMed] [Google Scholar]
- Schwarz K., Hansen-Hagge T., Bartram C. Improved yields of long PCR products using gene 32 protein. Nucleic Acids Res. 1990 Feb 25;18(4):1079–1079. doi: 10.1093/nar/18.4.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Somerville C. C., Knight I. T., Straube W. L., Colwell R. R. Simple, rapid method for direct isolation of nucleic acids from aquatic environments. Appl Environ Microbiol. 1989 Mar;55(3):548–554. doi: 10.1128/aem.55.3.548-554.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsai Y. L., Olson B. H. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl Environ Microbiol. 1992 Jul;58(7):2292–2295. doi: 10.1128/aem.58.7.2292-2295.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weyant R. S., Edmonds P., Swaminathan B. Effect of ionic and nonionic detergents on the Taq polymerase. Biotechniques. 1990 Sep;9(3):308–309. [PubMed] [Google Scholar]
- Widjojoatmodjo M. N., Fluit A. C., Torensma R., Verdonk G. P., Verhoef J. The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples. J Clin Microbiol. 1992 Dec;30(12):3195–3199. doi: 10.1128/jcm.30.12.3195-3199.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]