A plasmid preparation is a method of
DNA extraction
The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. Currently, it is a routine procedure in molecular biology or forensic analyses. For the chemical method, many different kits are used for extraction, and s ...
and purification for
plasmid DNA. Many methods have been developed to purify plasmid
DNA from
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typically a few micrometr ...
that directly exist in the environment or bacteria that grown at laboratory level.
These methods invariably involve three steps:
* Growth of the bacterial culture
* Harvesting and
lysis of the bacteria
* Purification of plasmid DNA
Plasmid purification and preparation from the environment
Plasmid purification directly from the environment is an important tool in
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
and
biotechnology
Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used ...
, and helps to advance our understanding of the role of plasmids in the
biology
Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary i ...
of different organisms.
In recent years, the use of cultivation-independent methods such as total community DNA (TC-DNA) extraction has allowed for a more comprehensive understanding of the prevalence and distribution of plasmids in the environment.
This approach involves extracting DNA directly from environmental samples, allowing for the detection and quantification of plasmid occurrence and abundance without the need for cultivation.
Several screening methods exist, such as the use of
PCR-based screening combined with
Southern blot hybridization.
PCR amplification, by the use of specific primers targeting specific plasmid groups, is a powerful that tool can help to identify the presence and abundance of certain types of plasmids in environmental samples.
Growth of the bacterial culture
Plasmids are almost always purified from liquid
bacteria cultures, usually ''
E. coli'', which have been
transformed and isolated.
Virtually all plasmid vectors in common use encode one or more
antibiotic resistance genes as a
selectable marker A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, a ...
, for example a gene encoding ampicillin or kanamycin resistance, which allows bacteria that have been successfully transformed to multiply uninhibited.
Bacteria that have not taken up the plasmid vector are assumed to lack the resistance gene, and thus only colonies representing successful transformations are expected to grow.
Bacteria are grown under favourable conditions.
Harvesting and lysis of the bacteria
When bacteria are lysed under
alkaline conditions (pH 12.0–12.5) both chromosomal DNA and
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
are denatured; the plasmid DNA however, remains stable. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of
ssDNA. After the addition of
acetate-containing neutralization
buffer
Buffer may refer to:
Science
* Buffer gas, an inert or nonflammable gas
* Buffer solution, a solution used to prevent changes in pH
* Buffering agent, the weak acid or base in a buffer solution
* Lysis buffer, in cell biology
* Metal ion buffer
* ...
the large and less
supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids stay in solution.
Preparations by size
Kits are available from varying manufacturers to purify plasmid DNA, which are named by size of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit.
Minipreparation
Minipreparation of plasmid DNA is a rapid, small-scale isolation of
plasmid DNA from
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typically a few micrometr ...
. It is based on the
alkaline lysis method. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep".
Minipreps are used in the process of
molecular cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their DNA replication, replication within Host (biology), host organisms. The use of the word ''cloning'' re ...
to analyze bacterial
clones
Clone or Clones or Cloning or Cloned or The Clone may refer to:
Places
* Clones, County Fermanagh
* Clones, County Monaghan, a town in Ireland
Biology
* Clone (B-cell), a lymphocyte clone, the massive presence of which may indicate a pathologi ...
. A typical plasmid DNA yield of a miniprep is 5 to 50 µg depending on the cell strain.
Miniprep of a large number of plasmids can also be done conveniently on filter paper by lysing the cell and eluting the plasmid on to filter paper.
Midipreparation
The starting E. coli culture volume is 15-25 mL of
Lysogeny broth
Lysogeny broth (LB) is a nutritionally rich medium primarily used for the growth of bacteria. Its creator, Giuseppe Bertani, intended LB to stand for lysogeny broth, but LB has also come to colloquially mean Luria broth, Lennox broth, life br ...
(LB) and the expected DNA yield is 100-350 µg.
Maxipreparation
The starting E. coli culture volume is 100-200 mL of LB and the expected DNA yield is 500-850 µg.
Megapreparation
The starting E. coli culture volume is 500 mL – 2.5 L of LB and the expected DNA yield is 1.5-2.5 mg.
Gigapreparation
The starting E. coli culture volume is 2.5-5 L of LB and the expected DNA yield is 7.5–10 mg.
Purification of plasmid DNA
Multiple methods of nucleic acid purification exist. All work on the principle of generating conditions where either only the nucleic acid precipitates, or only other
biomolecules precipitate, allowing the nucleic acid to be separated.
Ethanol precipitation
Ethanol precipitation Ethanol precipitation is a method used to purify and/or concentrate RNA, DNA, and polysaccharides such as pectin and xyloglucan from aqueous solutions by adding ethanol as an antisolvent.
DNA precipitation Theory
DNA is polar due to its ...
works by using
ethanol
Ethanol (abbr. EtOH; also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound. It is an alcohol with the chemical formula . Its formula can be also written as or (an ethyl group linked to a ...
as an
antisolvent
Salting out (also known as salt-induced precipitation, salt fractionation, anti-solvent crystallization, precipitation crystallization, or drowning out) is a purification technique that utilizes the reduced solubility of certain molecules in a s ...
of DNA, causing it to precipitate out of solution. The soluble fraction is discarded to remove other biomolecules.
Spin column
Spin column-based nucleic acid purification precipitates nucleic acid such that it binds a solid matrix and other components flow through. The conditions are then changed to elute the purified nucleic acid.
Phenol–chloroform extraction
In a
phenol–chloroform extraction Phenol–chloroform extraction is a liquid-liquid extraction technique in molecular biology used to separate nucleic acids from proteins and lipids.
Process
Aqueous samples, lysed cells, or homogenised tissue are mixed with equal volumes of a pheno ...
, addition of a
phenol
Phenol (also called carbolic acid) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile. The molecule consists of a phenyl group () bonded to a hydroxy group (). Mildly acidic, it ...
/
chloroform mixture will dissolve protein and lipid contaminants, leaving the nucleic acids in the aqueous phase. It also denatures proteins, like
DNase Deoxyribonuclease (DNase, for short) refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells ...
, which is especially important if the plasmids are to be used for
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
digestion. Otherwise, smearing may occur in enzyme restricted form of plasmid DNA.
Beads-based extraction
In beads-based extraction, addition of a mixture containing magnetic beads commonly made of
iron
Iron () is a chemical element with Symbol (chemistry), symbol Fe (from la, Wikt:ferrum, ferrum) and atomic number 26. It is a metal that belongs to the first transition series and group 8 element, group 8 of the periodic table. It is, Abundanc ...
ions binds to plasmid DNA, separating them from unwanted compounds by a magnetic rod or stand. The plasmid-bound beads are then released by removal of the magnetic field and extracted in an elution solution for down-stream experiments such as
transformation or
restriction digest
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed ''DNA fragmentation'' (this term is used for other procedures as well). Hartl and Jones describe it this way:
...
ion. This form of miniprep can also be automated, which increases the conveniency while reducing mechanical error.
References
Further reading
*
{{refend
External links
*http://www.protocol-online.org/prot/Molecular_Biology/Plasmid/Miniprep/
A miniprep procedure using diatomaceous earth to bind DNA during purification and washing.
Biological techniques and tools
Genetics techniques
Molecular biology