Nucleic Acids And Isolation Flashcards
Why are ribonuclease inhibitors used in RNA work areas?
Because ribonucleases can easily damage RNA and are commonly found on skin flakes.
The purification of pure nuclei acids from a sample containing lysed cells and tissue fragments falls into two general approaches. What are they?
1) . Silica/ion exchange resin adsorption
2) . Organic solvent extractions
Why are silica/ion exchange purification methods so popular?
They offer speed, ease of use and the ability to completely automate the process.
Describe the principle of the ion exchange method.
The principle of the ion exchange method is that negatively charged nucleic acids will interact with surfaces presenting positively charged groups (often DEAE) coated onto beads.
At low salt concentrations and appropriate pH many cellular components including DNA and RNA will bind to the resin beads. Therefore if you flow a lysed cellular sample through such a column many cellular components including nucleic acids will be captured.
Subsequent washing of the column with increasing salt concentrations will elute non-nucleic acid components and eliminate them.
Washing of the column with additional medium and salt buffers can allow the selective elution of RNA and DNA, which can be subsequently precipitated to allow the buffer to be changed.
Ion exchange methods this offer ‘turntable’ elution dependant on need, however the high salt elution buffers necessitate the precipitation or dialysis of the nucleic acid for subsequent uses.
Describe how silica based methods of nucleic acid purification work.
Silica based gels or membranes work on adsorption of nucleic acids to the surface of silica under high salt concentration (which is also enhanced in the presence of ethanol).
These high salt conditions include chaotropic salts and so this method presents an ideal purification method for any lysis method involving chaotropes (which is pretty much most quick prep methods).
The rapidity of binding is emphasised by the fact that loading of the columns can be performed by centrifugation or vacuum (forcing the sample through the silica gel at high g or application of a vacuum to the column). Proteins and other contaminants are not bound under these conditions and are immediately lost from the column. Washes with ethanolic salt solution removes further impurities.
Once the column is vacuumed or centrifuged to remove the remaining wash solution a low salt (water or weak buffer) elution is performed. This frees the nucleic acid from the matrix in a relatively small volume, which can be obtained by centrifugation or the application of vacuum around suitable collection tubes. The low salt elution is directly applicable to many analytical techniques.
Rapid DNA extractions can be performed in 10-15 mins or less. Fully automated purification is achievable at the level of 96-well microtitre plates.
Recent advances in this technology include the use of magnetic silica beads to allow the use of magnetic isolation, rather than columns, a technique which is relatively easy to automate and can retain the nucleic acids in a single tube throughout the entire process.
Describe how organic solvent extractions work.
Mixing an organic solution (usually beginning with a mixture of phenol, chloroform and isoamyl alcohol) with your lysed sample and shaking it well will cause the lipids and proteins to partition into the organic phase. Proteins have largely hydrophobic cores which when revealed will encourage this partitioning.
Those proteins that are unlikely to enter the organic phase are likely to be further denatured by contact with it and precipitate within the aqueous phase.
Centrifugation resolves the upper aqueous and lower (denser) organic phases, with a white interface layer of precipitated proteins and cellular debris.
Recovering the aqueous phase and sequentially repeating the process with chloroform:isoamyl alcohol will remove any residual phenol from the DNA and RNA containing aqueous phase.
Recovery of the nucleic acid is then usually achieved by precipitation with sodium acetate and ethanol, followed by centrifugation.
Variations of the extractions solutions and procedures, including addition of chaotropes in the aqueous phase, will work equally well for RNA extraction.
One benefit of the organic extraction methods over silica based methods is their indifference to the size of the nucleic acids being isolated. Silica based techniques are poor for small (100bp or less) DNA and RNA, whereas organic extraction methods work well for large and small nucleic acids.
What is one benefit of organic nucleic acids over silica based methods?
One benefit of the organic extraction methods over silica based methods is their indifference to the size of the nucleic acids being isolated. Silica based techniques are poor for small (100bp or less) DNA and RNA, whereas organic extraction methods work well for large and small nucleic acids.
Why have organic nucleic acid extraction methods largely fallen out of favour?
Phenol requires many precautions to work with.
How can mRNAs be isolated?
Eukaryotic mRNA structure lends itself to a unique method of isolation from a cell lysate.
The majority of eukaryotic RNA is polyadenylated at its 3’end. This columns of magnetic magnetic beads coated with the complimentary TTTT(n) sequence can be used to bind the mRNA, even whilst in a complex mixture such as cell lysate in a chaotropic call solution.
As with other affinity purification methods, washing out the column/beads removes contaminants and a low salt, high temperature elution yields purified mRNA.
Describe equilibrium density gradient (isopycnic) centrifugation.
A sample is centrifuged at 100,000g for hours in a solution containing CsCl. This will form a self-sustaining density gradient.
DNA, with some consideration of its GC content, will concentrate in a layer at the point of equivalent density withing the CsCl density gradient.
Once centrifugation is halted, the gradient remains intact for sufficient time to allow DNA to be precisely removed from the appropriate portion of the tube. Ethidium Bromide is usually added to allow visualisation of the DNA layer under UV illumination.
An adaptation of CsCl isopycnic centrifugation allows for the purification of covalently crossed circular (ccc) plasmid DNA away from genomic DNA and those circles with Nick or breaks. In this case saturating amounts of ethidium bromide are added before the centrifugation. Under such conditions ccc DNA (with intercalated ethidium bromide) has a distinct lower density than other forms of the plasmid and the resultant band can be easily visualised and isolated.
In all cases ethidium bromide can be later removed by organic solvent extraction, and CsCl by dialysis.
Issues with this method are the need for and operation of an expensive ultracentrifuge and the potential hazards of both CsCl and ethidium bromide. Isopycnic centrifugation does however generate extremely pure DNA.
What are the drawbacks of using isopycnic centrifugation methods?
Issues with this method are the need for and operation of an expensive ultracentrifuge and the potential hazards of both CsCl and ethidium bromide. Isopycnic centrifugation does however generate extremely pure DNA.
What is alkaline lysis DNA extraction used for?
Alkaline lysis is a DNA isolation methodology suited to the purification of covalently closed circular DNA molecules such as bacterial plasmids, away from other cellular components and chromosomal DNA.
Describe the alkaline lysis method of DNA extraction.
Bacterial cells containing a plasmid are grown in appropriate liquid culture overnight.
Cells from a portion of the culture (dependent on plasmid yield required) is pelleted by centrifugation.
The cell pellet is re suspended and left to rest for a short period in an ice bath, in a weakly buffered solution containing glucose, EDTA, ribonuclease A and lysozyme.
Glucose ensures an isosmotic solution, EDTA chelates divalent cations to reduce DNase activity, ribonuclease A aids degradation of RNA when the cell is lysed and lysozyme hells degrade the cell wall.
Solution II (NaOH and SDS) is added and the mixture is mixed gently. The solution becomes clear and viscous at this stage as the cells lyse. NaOH helps the cell wall lysis and denatures all double stranded DNA in the cell causing the two stands to separate. SDS is a strong detergent and dissolves the cell membrane, releasing the contents. In addition SDS binds to and denatures cellular proteins in the mixture reducing DNase damage and aiding protein removal later.
After a brief pause in an ice bath 3M potassium acetate solution is added. This neutralises the alkali, causing single stranded DNA to re-anneal to its partner strand if possible. Plasmid DNA is a covalently closed circle which can reform itself quickly, but genomic DNA is too complex to achieve this. The high salt concentration also encourages the precipitation of protein-SDS complexes. A white precipitate of SDS, protein and ss genomic DNA forms, which can be pelleted by centrifugation.
Double stranded plasmid DNA, (along with some small RNA molecules, RNase and remaining contaminants which are soluble in the high salt solution) is obtained by decanting the supernatant after centrifugation.
Plasmid DNA can then be further cleaned and concentrated by other methods.
How are nucleic acids often precipitated?
After purification away from the myriad of non-nucleic acids found in biological samples, there is often a requirement to concentrate the sample depending on yield.
Nucleic acids are readily precipitated in the presence of an alcohol, a process which is enhanced by moderate levels of salts and low temperatures.
It has been noted that occasionally plasmids obtained using the alkaline lysis method cut and behave poorly in subsequent manipulations. What is an a,tentative approach to purification of ccc DNA?
The rapid triton-boiling lysis method developed by Holmes and Quigley in 1981.
