Nucleic acid sample preparation involves a range of techniques to transform a sample which cannot be directly analyzed into one that fits the requirements of the analytical technique to be used such as reverse transcription (RT) or PCR. Nucleic acid sample purification and quality assessment are important steps in experimental workflows since the quality of nucleic acids can affect the performance in downstream reactions. Common nucleic acid purification techniques, as well as characterization methods, are discussed in the sections below.
Related sections: Nucleic Acid Electrophoresis.
When working with cells or tissues as the starting material, the first step of the nucleic acid purification process is cell lysis or membrane permeabilization. This process breaks open the cell membranes and disrupts the cellular structure to create a cell lysate. This allows the nucleic acid of interest to be accessed and separated away from unwanted cellular components. Cell lysis can be achieved either by mechanical techniques such as grinding, douncing, and sonicating, or it can be performed chemically with a lysis buffer. Lysis buffers are designed to rupture cells by osmosis and their compositions can be tailored for specific applications. Lysis buffers typically contain a detergent such as NP-40 or Triton to disrupt membrane lipids. Chelating agents such as EDTA and EGTA may also be present in the lysis buffer to inhibit nuclease activity. Proteases such as proteinase K can also be used to facilitate cell lysis. The resulting cell lysate can then be subjected to a physical separation such as centrifugation, a chemical separation such as phenol/chloroform extraction, or a solid phase-based separation such as a column to isolate the nucleic acid of interest.
RNA Filtration Column Filter column purification can be used to purify RNA from mammalian cell cultures, bacteria, and yeast, as well as plant and animal tissue. By adjusting the pH and salt of the solution, RNA can be separated from the cellular debris or other contaminants by absorbing the RNA onto a silica membrane situated at the base of a filter column. This popular RNA isolation method is compatible with vacuum and centrifugation protocols. Typically, RNA molecules > 200 base pairs will absorb to the membrane. After the RNA is bound to the membrane, it is subjected to a low and a high stringency wash to remove contaminants such as DNA, proteins, and lipids. An optional DNase digest can be performed to minimize residual genomic DNA contamination. When preparing RNA samples for RT-qPCR, DNase-treatment may be necessary to prevent potential amplification of any contaminating genomic DNA, which could lead to overestimation of the copy number of an mRNA. The purified RNA is then eluted off the membrane with an elution buffer into a collection tube. The volume of the elution buffer used for elution can be varied according to the final concentration of RNA desired. For example, a smaller volume of elution buffer can be used to obtain a higher concentration of RNA.
RNA isolated using column purification is suitable for use in a variety of downstream applications including RT-PCR, real-time PCR, and northern blot analysis. Alterations of the column purification method can also be used to isolate small RNAs such as microRNAs. High throughput RNA purification formats such as 96-well plates are also available. See Bio-Rad's RNA isolation tools for a comprehensive list of available products.
Guanidinium Isothiocyanate Phenol Chloroform Extraction A variation of the phenol/chloroform DNA extraction method paired with guanidinium isothiocyanate can be used to obtain RNA of high purity. This method was originally developed by Chomczynski and Sacchi in 1987 and is intended for extraction of total RNA from animal and plant tissues, cultured mammalian cells, bacteri,a and yeast cells in under one hr. The method can also be used for the simultaneous extraction of RNA, DNA, and proteins from various samples. This reagent allows for processing of small amounts of starting material (50 cells or 5 mg of tissue), or can be scaled up to process larger amounts.
For this method, a sample of interest is treated with a monophasic phenol solution containing guanidinium isothiocyanate. Guanidinium isothiocyanate is a protein denaturant and effectively inactivates RNases. The sample is then subjected to an organic extraction with chloroform. Centrifugation is used to separate the homogenate into an organic phase and an aqueous phase. The RNA will partition into the upper, aqueous phase while the denatured proteins and lipds will partition into the interphase or the bottom, organic phase. The RNA can be recovered from the aqueous phase by the addition of ethanol or isopropyl alcohol, and subsequently solubilized in RNase-free water. Guanidinium isothiocyanate RNA isolation requires the use of phenol however it provides highly pure RNA and maintains RNA integrity. It can also be paired with column purification for difficult tissues such as fatty and fibrous samples.
RNA isolated with the guanidinium isothiocyanate phenol/chloroform extraction is generally DNA- and protein-free, and can be used for northern blot analysis, in vitro translation, poly (A)+ selection, RNase protection assays, RT-qPCR, and molecular cloning. For RT-qPCR analysis, a DNase treatment step is recommended to prevent the potential amplification of any contaminating genomic DNA, which could lead to overestimation of the copy number of an mRNA. When the starting material is limited, however, the DNase treatment may be inadvisable because the additional manipulation could result in the loss of the RNA. Bio-Rad offers PureZOL™ Isolation Reagent for isolating high yields of RNA from a variety of sources, including cultured cell, animal and plant tissue, yeast, virus, and bacterial samples.
RNA Isolation Selection Guide
* Removal not required. ** Total preparation time will vary depending on the tissue or cell type and on which format is used (vacuum or spin). For sample-specific yield infomration, please visit www.bio-rad.com/rna-isolation and check the RNA Isolation Selction Guide.
DNA Filtration Column Filter column purification can be used to purify DNA from mammalian cell cultures, bacteria, and yeast, as well as plant and animal tissue. By adjusting the pH and salt of the solution, DNA of interest can be separated from cellular debris or other unwanted contaminants by binding the DNA to a silica membrane situated at the bottom of a filter column. This is a popular method for genomic and plasmid DNA purification. Vacuum and centrifugation protocols are available. After the DNA is bound to the membrane, it is subjected to a low and a high stringency wash to remove contaminants such as RNA, proteins and lipids. The purified DNA is eluted off the membrane with an elution buffer into a collection tube. The volume of the elution buffer used can be varied depending on the final concentration of plasmid or DNA desired. Recovered DNA is suitable for use with PCR and Southern blot analysis.
Traditionally, cesium chloride gradients were used to purify plasmid DNA away from genomic DNA. While this method provides highly pure DNA, it is time consuming and requires removal of ethidium bromide and cesium chloride from the recovered plasmid. Plasmid purification kits provide a faster and more efficient means for the purification and concentration of DNA > 200 base pairs. DNA is adsorbed onto the silica-based membrane, and RNA, protein, and other cellular components are washed away. The purified DNA is then eluted using elution buffer and is recovered in a form immediately available for fluorescent sequencing, cell transfection, electroporation, and enzymatic restriction and modification. Plasmid purification kits are available from Bio-Rad for a varety of starting sample types.
Plasmid DNA Purification Selection Guide
* (OD600 of undiluted culture) x (culture volume in ml) = #of OD • ml. 1 OD600 is equivalent to approximately 8 x 108 cells/ml. ** Using the Aurum vacuum manifold.
Ion Exchange Resin As an alternative to filter column purification or phenol/chloroform extraction, a buffered ion exchange resin can be used for the removal of PCR contaminates from blood, cultured cells, and bacteria. InstaGene™ Matrix is a Chelex resin that removes PCR-inhibitors from a sample prior to PCR amplification. A single cell lysis step by boiling in the presence of the matrix is sufficient. This is possible because the matrix efficiently absorbs cell lysis products that interfere with the PCR amplification process. The matrix can then be pelleted out by centrifugation and the supernatant can be used for downstream PCR amplification.
Phenol/Chloroform Extraction and Ethanol Precipitation A traditional DNA purification method that can be used to obtain highly pure DNA is phenol/chloroform extraction followed by ethanol precipitation. This method is intended for the extraction of DNA from animal and plant tissues, cultured mammalian cells, bacteria and yeast cells in under one hour. The aqueous nucleic acid sample is combined with an equal volume of a phenol:chloroform mixture. Phenol dissociates proteins bound to DNA while chloroform denatures proteins and lipids. Three distinct phases will form: the aqueous phase, the interphase, and the organic phase. Of these, the aqueous phase contains the DNA, whereas the proteins and lipids remain in the other two phases. The aqueous phase can then be treated with ethanol to precipitate the DNA. The precipitated DNA can then be pelleted by centrifugation and dissolved in a buffer of choice for use in downstream reactions. DNA purified using the phenol/chloroform extraction and ethanol precipitation method is typically more pure than DNA recovered from filter column purification. The recovered DNA is suitable for use in PCR and Southern blot analysis.
Gel electrophoresis is an effective method for separating DNA molecules based on their molecular weight. The extent of supercoiling, or DNA compaction, also affects how a DNA molecule will migrate through a gel. Smaller and more compact DNA molecules migrate more quickly through the gel matrix while larger or less compact molecules migrate more slowly. In this way, different size DNA molecules can be resolved on a gel. Once resolved, a DNA band of interest can be excised from the agarose gel and purified via filtration in a spin-column format. This allows the recovery of DNA over a wide range of fragment sizes (50 base pairs to 23k base pairs). The recovered DNA can be used for PCR, ligations, labeling, or other enzymatic reactions without further purification or sample preparation. Bio-Rad's Freeze 'N Squeeze™ DNA gel extraction spin columns provide a quick and effective means to purify double-stranded DNA fragments from TAE- or TBE-buffered agarose gels.
PCR Product Clean Up Spin column purification methods can be used for purifying PCR products and other DNA molecules >200 base pairs directly from PCR reaction mixtures. This effectively removes salts, nucleotides, enzymes, primers, and primer-dimers from the DNA sample of interest. Purified DNA fragments are eluted into a collection tube and are immediately available for secondary PCR, subcloning, restriction digests, ligations, sequencing, and other enzymatic manipulations. Bio-Rad's PCR Kleen spin columns purify PCR products and other DNA molecules >200 bp directly from reaction mixtures.
Radiolabled Probe Clean Up When generating radiolabeled DNA for molecular biology applications, the unincorporated radioactive nucleotides need to be removed from the reaction mixture. A spin column format using gel filtration can be used to separate molecules by size as well as to remove salts, nucleotides, dye terminators, and small molecules from the DNA probes. Bio-Rad's Micro Bio-Spin™ columns use specially-sized Bio-Gel polyacrylamide gels for purifying radiolabeled probes and PCR reaction mixtures.
PCR Product and DNA Fragment Purification Guide
Bio-Rad offers a variety of pre-PCR and post-PCR sample preparation and cleanup columns and prepacked columns; many are also suitable for DNA fragment purification.
Knowing the concentration, purity, and integrity of a nucleic acid sample is often important for downstream applications. Spectrophotometry and gel electrophoresis are common tools that can be used to measure and assess these characteristics.
Concentration The Beer-Lambert Law allows the concentration of nucleic acids in a sample to be calculated based on the amount of light absorbed at 260 nm:
where A is absorbance (also known as optical density), ε is the extinction coefficient, b is the path length of the sample cuvette, and c is the concentration of the compound in a solution.
Purity Spectrophotometry can also be used to estimate the purity of a given nucleic acid sample. Known contaminants such as protein and organic solvents absorb light at different wavelengths; thus, the absorbance ratio can be used to estimate sample purity. Since proteins absorb light at 280 nm, the ratio of 260 nm/280 nm is used to estimate protein contamination in a nucleic acid sample.
Contaminants such as phenol and other organic compounds will absorb at 230 nm. Therefore, another assessment of nucleic acid purity is the absorbance ratio of 260 nm/230 nm.
Integrity The integrity of a nucleic acid sample can be analyzed by gel electrophoresis. A sample of interest is typically run on a denaturing gel to eliminate its secondary structure. For pure total RNA, two discreet bands should appear in the gel corresponding to the 18S and 28S ribosomal RNA. If a smear is seen instead of the two bands, this indicates that the RNA in the sample is degraded. Commercially available microfluidic instruments now use a relatively small amount of input sample to determine the nucleic acid concentration, purity, and integrity in one assay. The Bio-Rad Experion™ automated electrophoresis station can be used to check the purity and integrity of RNA.
Chomczynski P and Sacchi N (1987). Single step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction. Anal Biochem 162, 156-9.
Sambrook J et al. (1989). Molecular Cloning: A Laboratory Manual. (New York: Cold Spring Harbor Press).
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