Plasmid copy number by method other than PCR

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exec
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Plasmid copy number by method other than PCR

Hello,

Is there any other method for plasmid copy number determination other than PCR based method. we are in search of a simple rapid method for determiantion of plasmid ocpy number.

Kristina Holmberg
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Hi,

Hi,

you can use both both UV spectrophotometry at 260 nm and quantitative analysis on an agarose gel. For reliable spectrophotometric DNA quantification, A260 readings should lie between 0.1 and 1.0.

More details at Qiagen's web

http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000229&r=1884

Jason King
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exec wrote:

exec wrote:

Hello,

Is there any other method for plasmid copy number determination other than PCR based method. we are in search of a simple rapid method for determiantion of plasmid ocpy number.

There is a new technology being developed for quantification of mRNA which involves the use of "color coded probes". It is a very smart method of analysing up to 16,000 different gene expression levels without having to do cDNA and traditional chip hybridizations. It is supposed to have the sensitivity and quantitation levels of Taqman qPCR.

The technique is explained in Nature Biotechnology March 2008 p293 by Fortina and Surry.

Although it is designed for RNA it could easily be adapted for detecting plasmid DNA. It would be simple and fast, though initially I guess the machinary for reading the chips will be expensive.

Tony Rook
Tony Rook's picture
Refer to the following

Refer to the following articles which describe several methods other than PCR, including using an agarose gel assay for the quantitation of plasmid copy number.

Torsten Schmidt, Karl FriehsCorresponding Author Contact Information, E-mail The Corresponding Author and Erwin Flaschel. Rapid determination of plasmid copy number. Journal of Biotechnology. Volume 49, Issues 1-3, 20 August 1996, Pages 219-229
doi:10.1016/0168-1656(96)01519-2

Abstract:
Plasmid copy number, the number of expression vectors per host cell, is a key variable in recombinant microbial cultivation. Therefore, it would be very helpful, if the plasmid copy number could be determined during the operating process period. A rapid quantification of this important process variable would even open the possibility of its use in process control. However, current assays like gel electrophoresis, CsCl-gradient centrifugation, HPLC and other methods are time consuming and difficult to quantify. Indirect methods, like the correlation of copy number with e.g. the activity of an enzyme, coded on the plasmid, are prone to errors due to the production kinetics, turnover rate and protein denaturation. Here, a method is presented, which enables the plasmid copy number to be determined in less than 30 min. This novel procedure based on plasmid isolation by means of a commercial DNA-isolation kit and quantification by capillary electrophoresis, should allow the copy number to be used in process control.

Elena A. Pushnova1, Michael Geier and Yu Sheng Zhu. An Easy and Accurate Agarose Gel Assay for Quantitation of Bacterial Plasmid Copy Numbers. Analytical Biochemistry
Volume 284, Issue 1, 15 August 2000, Pages 70-76

Abstract:
An assay for quantitation of plasmid copy numbers in bacterial cell cultures has been developed and validated. The method combines isolation of total bacterial DNA (including both plasmid and genomic DNA), running a series of twofold dilutions of total DNA in an agarose gel followed by ethidium bromide staining, and subsequent scanning of the gel picture negatives. We have developed a novel set of rules for integration of the scan data that allows us to achieve high assay precision, accuracy, and sensitivity. The assay validation results were as follows: intra- and interassay precision with %CV of 8.2–9.9 and 7.1–9.8%, respectively; ruggedness with %CV of 9.3–17.5%; spike recovery of 80–102%; and sensitivity of 1 plasmid copy per genome.

Steven J. Projan, Stephen Carleton and Richard P. Novick Determination of plasmid copy number by fluorescence densitometry. Plasmid
Volume 9, Issue 2, March 1983, Pages 182-190
doi:10.1016/0147-619X(83)90019-7

Abstract:
A simple and reliable method for the determination of plasmid copy numbers by direct fluorescence densitometry of ethidium bromide-stained electrophoretic gels was developed. In developing the method, the following parameters were evaluated and controlled: plasmid DNA trapping in the linear chromosomal DNA, staining-destaining kinetics for ethidium bromide, linearity of the fluorescence response, and the effect of the molecular topology of DNA on ethidium bromide binding to DNA in agarose.

Ivan G. Ivanov and Dimcho R. Bachvarov. Determination of plasmid copy number by the “boiling” method. Analytical Biochemistry
Volume 165, Issue 1, 15 August 1987, Pages 137-141
doi:10.1016/0003-2697(87)90211-9

Abstract:
A fast and reliable approach for determination of plasmid copy number in Escherichia coli is proposed, based on the “boiling” method (5) for separation of plasmid and chromosomal DNA. The method includes in vivo uniform labeling of total bacterial DNA, separation of DNA into plasmid and chromosomal DNA fractions, and quantitation of DNA in the two fractions by radioactivity measurement. No isolation and purification of native DNA are necessary.

Carys A. Wrigley-Jonesa, Hilary Richardsb, Colin R. Thomasd and John M. Ward. A method for plasmid copy number determination in recombinant Streptomyces. Journal of Microbiological Methods
Volume 16, Issue 1, August 1992, Pages 69-80
doi:10.1016/0167-7012(92)90026-Z

Abstract:
A method for the measurement of plasmid copy number in Streptomyces mycelia is described. It is based on the preparation (on a mini-scale) of the total DNA present in the mycelium followed by gel electrophoresis with ethidium bromide, photography and densitometric scanning of photographic negatives. The method is suitable for pIJ101-derived multi-copy plasmids and is shown to have comparable accuracy to similar methods developed for unicellular host organisms. A novel ‘copy number’ reconstitution approach was employed to define the sensitivity of the method. We recommend the optimisation of plasmid topoisomer separation since we have found evidence for transient changes in their relative proportions for certain Streptomyces plasmids. Various plasmid topoisomers present in the extracts were identified by progressive DNasel nicking of pure plasmid DNA.

H. Michael Shepard and Barry Polisky. Measurement of plasmid copy number. Methods in Enzymology
Volume 68, 1979, Pages 503-513
Recombinant DNA
doi:10.1016/0076-6879(79)68039-4

Determining plasmid copy number. Trends in Biotechnology
Volume 5, Issue 8, August 1987, Page 213
doi:10.1016/0167-7799(87)90042-4

Carmen Coronado, M. Enrique Vázquez, Angel Cebolla and Antonio J. Palomares. Use of Firefly Luciferase Gene for Plasmid Copy Number Determination. Plasmid
Volume 32, Issue 3, November 1994, Pages 336-341
doi:10.1006/plas.1994.1074

Abstract:
A simple and rapid method for determining plasmid copy number is described. The eukaryotic luc gene is used as a marker to tag plasmid derivatives of several well-known vectors, and by measuring light activity plasmid copy number is determined. A comparative analysis using a standard hybridization procedure to estimate plasmid copy number by densitometry is also described.

K. Korpela, P. Buchert and H. Söderlund. Determination of plasmid copy number with nucleic acid sandwich hybridization. Journal of Biotechnology
Volume 5, Issue 4, May 1987, Pages 267-277
doi:10.1016/0168-1656(87)90024-1

Abstract:
Sandwich hybridization is a convenient method for the detection and quantification of specific genes. Here we have used the sandwich hybridization method for determining the copy number of cloning vectors. The Saccharomyces cerevisiae expression vector pAAH5 was present in 10 copies per yeast cell. Derivatives of the Escherichia coli plasmid pBR322 had copy numbers of around 15 increasing to 250 upon amplification with chloramphenicol as detected per genome equivalent. The mutant pHCl, derived from pBR322, was found in up to 2500 copies per bacterial chromosome in an unamplified culture.

Eugene Seneta* and Simon Tavaré. Some stochastic models for plasmid copy number. Theoretical Population Biology
Volume 23, Issue 2, April 1983, Pages 241-256
doi:10.1016/0040-5809(83)90016-3

Abstract:
Some stochastic models for the copy number of plasmids in a cell line are studied. When considering the behavior of copy number in the whole cell line, the theory of multitype branching processes is appropriate. Attention is paid to the cure rate in the cell line, and the asymptotic fractions of cells containing a given number of plasmids. These quantities are used to compare the models numerically.

exec
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Hi,

Hi,

Thanks everyone for all the suggestions. i would definitely try some of them and revert back giving the feedback.

meanwhile thanks a ton for all the replies

Tony Rook
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exec-

exec-

No problem - definitely try to post some feedback on which methods you found worked best for your application. As I first began trying to determine plasmid copy number within bacteria, I found that there was relatively little information out there. Any feedback on what works best and why would be very helpful for the rest of us.

Good luck!