Plasmid preparation

Last updated
Plasmid miniprep. 0.8% agarose gel ethidium bromide-stained. Plasmid miniprep.jpg
Plasmid miniprep. 0.8% agarose gel ethidium bromide-stained.

A plasmid preparation is a method of DNA extraction and purification for plasmid DNA. It is an important step in many molecular biology experiments and is essential for the successful use of plasmids in research and biotechnology. [1] [2] Many methods have been developed to purify plasmid DNA from bacteria. [1] [3] During the purification procedure, the plasmid DNA is often separated from contaminating proteins and genomic DNA.

Contents

These methods invariably involve three steps: growth of the bacterial culture, harvesting and lysis of the bacteria, and purification of the plasmid DNA. [4] Purification of plasmids is central to molecular cloning. A purified plasmid can be used for many standard applications, such as sequencing and transfections into cells.

Growth of the bacterial culture

Plasmids are almost always purified from liquid bacteria cultures, usually E. coli , which have been transformed and isolated. [5] [6] Virtually all plasmid vectors in common use encode one or more antibiotic resistance genes as a selectable marker, for example a gene encoding ampicillin or kanamycin resistance, which allows bacteria that have been successfully transformed to multiply uninhibited. [7] [8] [9] 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. [5] [9] [10] Bacteria are grown under favourable conditions.

Harvesting and lysis of the bacteria

There are several methods for cell lysis, including alkaline lysis, mechanical lysis, and enzymatic lysis. [11] [12] [13] [14]

Alkaline lysis

The most common method is alkaline lysis, which involves the use of a high concentration of a basic solution, such as sodium hydroxide, to lyse the bacterial cells. [15] [16] [17] When bacteria are lysed under alkaline conditions (pH 12.0–12.5) both chromosomal DNA and protein are denatured; the plasmid DNA however, remains stable. [16] [17] 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 to lower the pH to around 7, the large and less supercoiled chromosomal DNA and proteins form large complexes and precipitate; but the small bacterial DNA plasmids stay in solution. [17] [14]

Mechanical lysis

Mechanical lysis involves the use of physical force, such as grinding or sonication, to break down bacterial cells and release the plasmid DNA. There are several different mechanical lysis methods that can be used, including French press, bead-beating, and ultrasonication. [11] [12] [13] [14]

Enzymatic lysis

Enzymatic lysis, also called Lysozyme lysis, involves the use of enzymes to digest the cell wall and release the plasmid DNA. [11] The most commonly used enzyme for this purpose is lysozyme, which breaks down the peptidoglycan in the cell wall of Gram-positive bacteria. Lysozyme is usually added to the bacterial culture, followed by heating and/or shaking the culture to release the plasmid DNA. [11] [12] [13] [14]

Preparations by size

Plasmid preparation can be divided into five main categories based on the scale of the preparation: minipreparation, midipreparation, maxipreparation, megapreparation, and gigapreparation. The choice of which method to use will depend on the amount of plasmid DNA required, as well as the specific application for which it will be used. [18] [19]

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 they are: 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. [2]

Minipreparation

Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. [20] [21] Commonly used miniprep methods include alkaline lysis and spin-column based kits. [3] [22] 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 to analyze bacterial clones. 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. [21]

Midipreparation

The starting E. coli culture volume is 15-25 mL of Lysogeny broth (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

It is important to consider the downstream applications of the plasmid DNA when choosing a purification method. For example, if the plasmid is to be used for transfection or electroporation, a purification method that results in high purity and low endotoxin levels is desirable. Similarly, if the plasmid is to be used for sequencing or PCR, a purification method that results in high yield and minimal contaminants is desirable. [2] However, multiple methods of nucleic acid purification exist. [23] [24] [25] 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. [15] [23]

Ethanol precipitation

Ethanol precipitation is a widely used method for purifying and concentrating nucleic acids, including plasmid DNA. [26] The basic principle of this method is that nucleic acids are insoluble in ethanol or isopropanol but soluble in water. Therefore, it works by using ethanol as an antisolvent of DNA, causing it to precipitate out of solution and then it can be collected by centrifugation. The soluble fraction is discarded to remove other biomolecules. [27]

Spin column

Spin column-based nucleic acid purification is a method of purifying DNA, RNA or plasmid from a sample using a spin column filter. [25] The method is based on the principle of selectively binding nucleic acids to a solid matrix in the spin column, while other contaminants, such as proteins and salts, are washed away. The conditions are then changed to elute the purified nucleic acid off the column using a suitable elution buffer. [25]

Phenol–chloroform extraction

The basic principle of the phenol-chloroform extraction is that DNA and RNA are relatively insoluble in phenol and chloroform, while other cellular components are relatively soluble in these solvents. The addition of a phenol/chloroform mixture will dissolve protein and lipid contaminants, leaving the nucleic acids in the aqueous phase. It also denatures proteins, like DNase, which is especially important if the plasmids are to be used for enzyme digestion. Otherwise, smearing may occur in enzyme restricted form of plasmid DNA. [24]

Beads-based extraction

In beads-based extraction, addition of a mixture containing magnetic beads commonly made of iron ions binds to plasmid DNA, separating them from unwanted compounds by a magnetic rod or stand. [25] 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 digestion. This form of miniprep can also be automated, which increases the conveniency while reducing mechanical error.

Related Research Articles

Molecular biology is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions.

<span class="mw-page-title-main">Plasmid</span> Small DNA molecule within a cell

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the internet.

DNA primase is an enzyme involved in the replication of DNA and is a type of RNA polymerase. Primase catalyzes the synthesis of a short RNA segment called a primer complementary to a ssDNA template. After this elongation, the RNA piece is removed by a 5' to 3' exonuclease and refilled with DNA.

Lysis is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a lysate. In molecular biology, biochemistry, and cell biology laboratories, cell cultures may be subjected to lysis in the process of purifying their components, as in protein purification, DNA extraction, RNA extraction, or in purifying organelles.

<span class="mw-page-title-main">Genetic transformation</span> Genetic alteration of a cell by uptake of genetic material from the environment

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.

Phenol extraction is a laboratory technique that purifies nucleic acid samples using a phenol solution. Phenol is common reagent in extraction because its properties allow for effective nucleic acid extraction, particularly as it strongly denatures proteins, it is a nucleic acid preservative, and it is immiscible in water.

Alkaline lysis or alkaline extraction is a method used in molecular biology to isolate plasmid DNA from bacteria.

<span class="mw-page-title-main">Acid guanidinium thiocyanate-phenol-chloroform extraction</span>

Acid guanidinium thiocyanate-phenol-chloroform extraction is a liquid–liquid extraction technique in biochemistry. It is widely used in molecular biology for isolating RNA. This method may take longer than a column-based system such as the silica-based purification, but has higher purity and the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short RNA species, such as siRNA, miRNA, gRNA and tRNA.

<span class="mw-page-title-main">Colicin</span> Type of bacteriocin produced by and toxic to some strains of Escherichia coli

A colicin is a type of bacteriocin produced by and toxic to some strains of Escherichia coli. Colicins are released into the environment to reduce competition from other bacterial strains. Colicins bind to outer membrane receptors, using them to translocate to the cytoplasm or cytoplasmic membrane, where they exert their cytotoxic effect, including depolarisation of the cytoplasmic membrane, DNase activity, RNase activity, or inhibition of murein synthesis.

fis E. coli gene

fis is an E. coli gene encoding the Fis protein. The regulation of this gene is more complex than most other genes in the E. coli genome, as Fis is an important protein which regulates expression of other genes. It is supposed that fis is regulated by H-NS, IHF and CRP. It also regulates its own expression (autoregulation). Fis is one of the most abundant DNA binding proteins in Escherichia coli under nutrient-rich growth conditions.

Transformation efficiency refers to the ability of a cell to take up and incorporate exogenous DNA, such as plasmids, during a process called transformation. The efficiency of transformation is typically measured as the number of transformants per microgram of DNA added to the cells. A higher transformation efficiency means that more cells are able to take up the DNA, and a lower efficiency means that fewer cells are able to do so.

RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. Several methods are used in molecular biology to isolate RNA from samples, the most common of these is guanidinium thiocyanate-phenol-chloroform extraction. The filter paper based lysis and elution method features high throughput capacity.

<span class="mw-page-title-main">Spin column-based nucleic acid purification</span>

Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.

<span class="mw-page-title-main">Bacterial one-hybrid system</span> Method for identifying the sequence-specific target site of a DNA-binding domain

The bacterial one-hybrid (B1H) system is a method for identifying the sequence-specific target site of a DNA-binding domain. In this system, a given transcription factor (TF) is expressed as a fusion to a subunit of RNA polymerase. In parallel, a library of randomized oligonucleotides representing potential TF target sequences are cloned into a separate vector containing the selectable genes HIS3 and URA3. If the DNA-binding domain (bait) binds a potential DNA target site (prey) in vivo, it will recruit RNA polymerase to the promoter and activate transcription of the reporter genes in that clone. The two reporter genes, HIS3 and URA3, allow for positive and negative selections, respectively. At the end of the process, positive clones are sequenced and examined with motif-finding tools in order to resolve the favoured DNA target sequence.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

Phenol–chloroform extraction is a liquid-liquid extraction technique in molecular biology used to separate nucleic acids from proteins and lipids.

Diagnostic microbiology is the study of microbial identification. Since the discovery of the germ theory of disease, scientists have been finding ways to harvest specific organisms. Using methods such as differential media or genome sequencing, physicians and scientists can observe novel functions in organisms for more effective and accurate diagnosis of organisms. Methods used in diagnostic microbiology are often used to take advantage of a particular difference in organisms and attain information about what species it can be identified as, which is often through a reference of previous studies. New studies provide information that others can reference so that scientists can attain a basic understanding of the organism they are examining.

DH5-Alpha Cells are E. coli cells engineered by American biologist Douglas Hanahan to maximize transformation efficiency. They are defined by three mutations: recA1, endA1 which help plasmid insertion and lacZΔM15 which enables blue white screening. The cells are competent and often used with calcium chloride transformation to insert the desired plasmid. A study of four transformation methods and six bacteria strains showed that the most efficient one was the DH5 strain with the Hanahan method.

NAIL-MS is a technique based on mass spectrometry used for the investigation of nucleic acids and its modifications. It enables a variety of experiment designs to study the underlying mechanism of RNA biology in vivo. For example, the dynamic behaviour of nucleic acids in living cells, especially of RNA modifications, can be followed in more detail.

References

  1. 1 2 Li JF, Li L, Sheen J (January 2010). "Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology". Plant Methods. 6 (1): 1. doi: 10.1186/1746-4811-6-1 . PMC   2829548 . PMID   20180960.
  2. 1 2 3 Prazeres DM, Monteiro GA (December 2014). Tolmasky ME, Alonso JC (eds.). "Plasmid Biopharmaceuticals". Microbiology Spectrum. 2 (6): 2.6.02. doi: 10.1128/microbiolspec.PLAS-0022-2014 . PMID   26104457.
  3. 1 2 Lezin G, Kosaka Y, Yost HJ, Kuehn MR, Brunelli L (2011). "A one-step miniprep for the isolation of plasmid DNA and lambda phage particles". PLOS ONE. 6 (8): e23457. Bibcode:2011PLoSO...623457L. doi: 10.1371/journal.pone.0023457 . PMC   3156146 . PMID   21858126.
  4. ↑ Bouchard R, et al. (2010). Laboratory Methods in Microbiology. Universal Scientific. pp. 119–126.
  5. 1 2 Suza W, Lee D (15 October 2021). "11. Recombinant DNA Technology; Ligase enzyme and gene cloning". Genetics, Agriculture, and Biotechnology. Iowa State University.
  6. ↑ Ismail R, Allaudin ZN, Lila MA (September 2012). "Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status". Vaccine. 30 (41): 5914–5920. doi: 10.1016/j.vaccine.2012.02.061 . PMID   22406276.
  7. ↑ "Plasmid". Genome.gov. Retrieved 2022-12-10.
  8. ↑ Batree L, Shriner W, Creech C (2017). "Biotechnology". Principles of biology. Open Oregon Educational Resources.
  9. 1 2 Bennett PM (March 2008). "Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria". British Journal of Pharmacology. 153 (Suppl 1): S347–S357. doi:10.1038/sj.bjp.0707607. PMC   2268074 . PMID   18193080.
  10. ↑ Smalla K, Jechalke S, Top EM (February 2015). "Plasmid Detection, Characterization, and Ecology". Microbiology Spectrum. 3 (1): PLAS–0038–2014. doi:10.1128/microbiolspec.PLAS-0038-2014. PMC   4480600 . PMID   26104560.
  11. 1 2 3 4 Rahman MM, Hosano N, Hosano H (April 2022). "Recovering Microalgal Bioresources: A Review of Cell Disruption Methods and Extraction Technologies". Molecules. 27 (9): 2786. doi: 10.3390/molecules27092786 . PMC   9104913 . PMID   35566139.
  12. 1 2 3 Weber S, Grande PM, Blank LM, Klose H (2022). "Insights into cell wall disintegration of Chlorella vulgaris". PLOS ONE. 17 (1): e0262500. Bibcode:2022PLoSO..1762500W. doi: 10.1371/journal.pone.0262500 . PMC   8759652 . PMID   35030225.
  13. 1 2 3 Wang D, Li Y, Hu X, Su W, Zhong M (April 2015). "Combined enzymatic and mechanical cell disruption and lipid extraction of green alga Neochloris oleoabundans". International Journal of Molecular Sciences. 16 (4): 7707–7722. doi: 10.3390/ijms16047707 . PMC   4425044 . PMID   25853267.
  14. 1 2 3 4 Borchers A, Pieler T (November 2010). "Programming pluripotent precursor cells derived from Xenopus embryos to generate specific tissues and organs". Genes. 1 (3): 413–426. doi: 10.3390/mi8030083 . PMC   6190294 . PMID   24710095.
  15. 1 2 Williams JA (June 2013). "Vector Design for Improved DNA Vaccine Efficacy, Safety and Production". Vaccines. 1 (3): 225–249. doi: 10.3390/vaccines1030225 . PMC   4494225 . PMID   26344110.
  16. 1 2 Zazilek G (2010-04-12). "Alkaline Lysis". askabiologist.asu.edu. Retrieved 2023-01-02.
  17. 1 2 3 Birnboim HC, Doly J (November 1979). "A rapid alkaline extraction procedure for screening recombinant plasmid DNA". Nucleic Acids Research. 7 (6): 1513–1523. doi:10.1093/nar/7.6.1513. PMC   342324 . PMID   388356.
  18. ↑ Serghini MA, Ritzenthaler C, Pinck L (May 1989). "A rapid and efficient 'miniprep' for isolation of plasmid DNA". Nucleic Acids Research. 17 (9): 3604. doi:10.1093/nar/17.9.3604. PMC   317816 . PMID   2726501.
  19. ↑ Kovalenko SA, Tanaka M, Ozawa T (December 1994). "Simple methods for preparation of plasmid DNA yielding long and accurate sequence data". Nucleic Acids Research. 22 (25): 5771–5772. doi:10.1093/nar/22.25.5771. PMC   310149 . PMID   7838738.
  20. ↑ Chowdhury K (May 1991). "One step 'miniprep' method for the isolation of plasmid DNA". Nucleic Acids Research. 19 (10): 2792. doi:10.1093/nar/19.10.2792. PMC   328215 . PMID   2041760.
  21. 1 2 "Plasmid Mini-Prep | College of Biological Sciences". cbs.umn.edu. Retrieved 2023-01-10.
  22. ↑ Zhang S, Cahalan MD (2007-07-29). "Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit". Journal of Visualized Experiments (6): 247. doi:10.3791/247. PMC   2557117 . PMID   18997895.
  23. 1 2 Tan SC, Yiap BC (2009). "DNA, RNA, and protein extraction: the past and the present". Journal of Biomedicine & Biotechnology. 2009: 574398. doi: 10.1155/2009/574398 . PMC   2789530 . PMID   20011662.
  24. 1 2 "Phenol-Chloroform Extraction | Herman Lab | Nebraska". hermanlab.unl.edu. Retrieved 2023-01-10.
  25. 1 2 3 4 Ali N, Rampazzo RC, Costa AD, Krieger MA (2017). "Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics". BioMed Research International. 2017: 9306564. doi: 10.1155/2017/9306564 . PMC   5529626 . PMID   28785592.
  26. ↑ Zeugin JA, Hartley JL (1985). "Ethanol Precipitation of DNA" (PDF). Focus. 7 (4): 1–2. Retrieved 2008-09-10.
  27. ↑ "Barrick Lab :: ProtocolsEthanolPrecipitation". barricklab.org. Retrieved 2023-01-10.

Further reading

This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.