Upon completion of this module topic, you will:
- be able to isolate plasmid DNA by harvesting bacterial cells through centrifugation.
- be able to lyse the bacterial cells and extract and purify the plasmid DNA using and following the protocol described in a Qiagen Miniprep or Maxiprep Kit depending upon the amount of plasmid product desired. You should be able to also explain the buffers and reactions that make up the Qiagen kits.
- be able to quantitate the concentration of DNA that has been purified through the Qiagen kit protocol using an Ultraviolet spectrophotometer.
- be able to explain which wavelengths are used to determine concentration and quality.
DNA Extraction & Purification
This is Part A, DNA Extraction & Purification, under the module topic, Preparation, Purification, and Quantitation of DNA & RNA. This topic part has two sections: Content Tutorial and Activities.
*Note: The tutorial sections for this module subset offer a variation from the common content-based scheme you have been exploring in the previous Nucleic Acid module subsets. This subset will provide you with the procedural information (protocols) you will need to learn to know how to appropriately prepare and purify DNA in the laboratory so that it can be used in the various techniques you have already explored in the previous subsets. You will also be provided with additional protocols at the end of the module subset (like the previous subsets) to further add to your protocol exposure and collection. As you are learning about the different steps involved in extracting and purifying DNA it is also important to understand the significance of each step and why it is being performed, what does each step do to the DNA?
A. Phenol/Chloroform extraction of DNA samples
Overview: Phenol extraction is a common technique used to purify a DNA sample. Generally, samples are extracted by addition of one volume of neutralized (with TE buffer, pH 7.5) phenol to the sample, followed by vigorous vortexing for a few seconds to form an emulsion (mixture of two or more “unblendable” liquids). The mixture is then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding the phenol interface and then is subjected to chloroform extractions to remove residual phenol. An equal volume of chloroform is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper, aqueous layer is recovered. After this extraction is repeated, the DNA is concentrated by ethanol precipitation.
Protocol Example: University of Calgary
1. Add an equal volume of TE-saturated phenol to the DNA sample contained in a 1.5 ml microcentrifuge tube and vortex for 15-30 seconds. 2. Centrifuge the sample for 5 minutes at room temperature to separate the phases. 3. Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding proteins at the aqueous:phenol interface. At this stage the aqueous phase can be extracted a second time with an equal volume of 1:1 TE-saturated phenol:chloroform, centrifuged and removed to a clean tube as above but this additional extraction usually is not necessary if care is taken during the first phenol extraction. 4. Add an equal volume of chloroform, vortex briefly, and centrifuge for 3 minutes at room temperature. Recover the upper, aqueous layer, taking care to not to remove chloroform at the chloroform:aqueous interface. 5. Ethanol precipitate the DNA by adding 2.5-3 volumes of ethanol-salt, as discussed below.
*Notes on phenol extraction of nucleic acids The standard and preferred way to remove proteins from nucleic acid solutions is by extraction with neutralized phenol or phenol/chloroform. If a heavy white precipitate layer is seen at the phenol:water interface, a second phenol extraction can be done. The heavy white precipitate suggests the phenol may be saturated with denatured proteins. A 25:24:1 mixture of phenol/chloroform/iso amyl alcohol also is useful for the removal of protein from nucleic acid samples where foaming may be an issue. Following extraction with phenol/chloroform/iso amyl alcohol, the sample should be extracted once with an equal volume of chloroform, and ethanol precipitated as described above. Reference: Modified from Roe BA.
Protocols For Phenol Extraction (userpages.umbc.edu, HTML Page)
B. Concentration of DNA by ethanol precipitation
Overview: Typically, 2.5 – 3 volumes of an ethanol/acetate solution is added to the DNA sample in a microcentrifuge tube, which is placed in an ice-water bath for at least 10 minutes. Frequently, this precipitation is performed by incubation at -20°C overnight (1). To recover the precipitated DNA, the tube is centrifuged, the supernatant discarded, and the DNA pellet is rinsed with a more dilute ethanol solution. After a second centrifugation, the supernatant again is discarded, and the DNA pellet is dried in a Speed-Vac.
Protocol Example: University of Calgary
- Add 2.5-3 volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water bath for at least 10 minutes. It is possible to place the sample at -20°C overnight at this stage.
- Centrifuge at 12,000 rpm in a microcentrifuge for 15 minutes at 4°C, decant the supernatant, and drain inverted on a paper towel.
- Add 80% ethanol (corresponding to about two volumes of the original sample), incubate at room temperature for 5-10 minutes and centrifuge again for 5 minutes, and decant and drain the tube, as above.
- Place the tube in a Speed-Vac and dry the DNA pellet for about 5-10 minutes, or until dry.
- Dissolve dried DNA in 10 mM Tris-HCl, pH 7.6-8.0, 0.1 mM EDTA (termed 10:0.1 TE buffer) or in molecular biology grade water depending on the downstream application.
- It is advisable to aliquot the DNA purified in large scale isolations (i.e. 100 ug or more) into several small (0.5 ml) microcentrifuge tubes for frozen storage because repeated freezing and thawing is not advisable.
*Notes on precipitation of nucleic acids: General rules Most nucleic acids may be precipitated by addition of monovalent cations (positively charged ions) and two to three volumes of cold 95% ethanol, followed by incubation at 0 to -70°C. The DNA or RNA then may be pelleted by centrifugation at 10 to 13,000 x g. for 15 minutes at 4°C. A subsequent wash with 70% ethanol, followed by brief centrifugation, removes residual salt and moisture.
The general procedure for precipitating DNA and RNA is: 1. Add one-tenth volume of 3M NaOAc, pH 5.2* to the nucleic acid solution to be precipitated. 2. Add two volumes of cold 95% ethanol. 3. Place at -70°C for at least 30 minutes, or at -20°C overnight.
Alternatively, 1. Combine 95 ml of 100% ethanol with 4 ml of 3 M NaOAc (pH 4.8) and 1ml of sterile water. Mix by inversion and store at -20°C. 2. Add 2.5 volumes of cold ethanol/acetate solution to the nucleic acid solution to be precipitated. 3. Place at at -70°C for at least 30 minutes or -20°C for two hours to overnight. * Note: 5M NH4OAc, pH 7.4, NaCl and LiCl may be used as alternatives to NaOAc. DNA also may be precipitated by addition of 0.6 volumes of isopropanol. Oligonucleotides Add one-tenth volume of 3M NaOAc, pH 6.5, and three volumes of cold 95% ethanol. Place at -70°C for at least one hour.
RNA Add one-tenth volume of 1M NaOAc, pH 4.5, and 2.5 volumes of cold 95% ethanol. Precipitate large volumes at -20°C overnight. Small volume samples may be precipitated by placing in powdered dry ice or dry ice-ethanol bath for five to 10 minutes.
Isobutanol concentration of DNA DNA samples may be concentrated by extraction with isobutanol. Add slightly more than one volume of isobutanol, vortex vigorously and centrifuge to separate the phases. Discard the isobutanol (upper) phase, and extract once with water-saturated diethyl ether to remove residual isobutanol. The nucleic acid then may be ethanol precipitated as described above. Reference: Modified from Roe BA.
C. Nucleic Acid Preparation from Samples (Bacteria, Tissues, Cells)
Miniprep double-stranded plasmid DNA isolation or Plasmid Mini Prep
Overview: Aliquots of antibiotic containing liquid media inoculated with plasmid-containing cell colonies are incubated in a 37°C shaker for 12-16 hours. After collecting the plasmid containing cells by centrifugation, the cell pellet is resuspended in a hypotonic sucrose buffer. The cells are successively incubated with an RNase-lysis buffer, alkaline detergent, and sodium acetate. The lysate is cleared of precipitated proteins and membranes by centrifugation, and the plasmid DNA is recovered from the supernatant by isopropanol precipitation. A typical yield for this method of DNA isolation is 10-15 ug of plasmid DNA from a 6 ml starting culture.
Protocol Example: University of Calgary Protocol
- 1. Pick a colony of bacteria harboring the plasmid DNA of interest into a 17 X 100 mm Falcon tube containing 6 ml of LB media supplemented with the appropriate antibiotic (typically ampicillin at 100 ug/ml) and incubate at 37°C 16-18 hours with shaking at 250 rpm. 2. Harvest the cells by centrifugation at 3000 rpm for 5 minutes in a 2 mL microfuge tuge in a microfuge (full speed, 1 min) and decant the supernatant. The cell pellets can be frozen at -70°C at this point. To increase yield, a second aliquoit of bacteria can be added to the same tube and centrifuged as above. 3. Resuspend the cell pellets in 0.2 ml of TE-RNase solution (50:10 TE buffer containing 40 ug/ml RNase A to a final concentration of 10 U/uL) by gentle vortexing or finger-flicking, add 0.2 ml of alkaline lysis solution, gently mix (DO NOT VORTEX), and incubate for 15 minutes at room temperature. 4. Add 0.2 ml of 3M NaOAc, pH 4.8, gently mix by swirling, and incubate at room temperature for 5 minutes. 5. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal DNA by centrifugation at 12,000 rpm for 15 minutes in a microcentrifuge. 6. Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube. Proceed to DNA purification by phenol extraction. In the kit methods, this will entail spinning the sample over a silica or other matrix. Other methods use similar materials to precipitate the DNA then wash it to remove proteins and other molecules.
*For standard alkaline lysis purification: Precipitate the DNA by adding 1 ml of 95% ethanol, and resuspend the dried DNA pellet in 100-200 ul 10:0.1 TE buffer.
Genomic DNA isolation from blood (or cells in culture)
Overview: Genomic DNA isolation from white blood cells is a common source of mammalian DNA. After the blood samples (stores at -70°C in EDTA vacutainer tubes ) are thawed, standard citrate buffer is added, mixed, and the tubes are centrifuged. The top portion of the supernatant is discarded and additional buffer is added, mixed, and again the tube is centrifuged. After the supernatant is discarded, the pellet is resuspended in a solution of SDS detergent and proteinase K, and the mixture is incubated at 55°C for one hour (this incubation may be longer for isolation of DNA from tissue samples). The sample then is phenol extracted once with a phenol/chloroform/isoamyl alcohol solution, and after centrifugation the aqueous layer is removed to a fresh microcentrifuge tube. The DNA is ethanol precipitated, resuspended in buffer, and then ethanol precipitated a second time. After the pellet is dried, buffer is added and the DNA is resuspended by incubation at room temperature to 55°C overnight. The DNA is appropriate for several downstream applications.
University of Calgary Sample Protocol
- Obtain the liquid blood samples in EDTA vacutainer tubes frozen at -70°C.
- Thaw the frozen samples, add 0.8 ml 1X SSC buffer, and mix. Centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.
- Remove 1 ml of the supernatant and discard into disinfectant (bleach).
- Add 1 ml of 1X SSC buffer, vortex, centrifuge as above for 1 minute, and remove all of the supernatant.
- Add 375 ul of 0.2M NaOAc to each pellet and vortex briefly. Then add 25 ul of 10% SDS and 5 ul of proteinase K (20 mg/ml H2O) (Sigma P-0390), vortex briefly and incubate for 1 hour at 55°C.
- Add an equal volume of phenol/chloroform/isoamyl alcohol and vortex for 30 seconds. Centrifuge the sample for 2 minutes at 12,000 rpm in a microcentrifuge tube.
- Carefully remove the aqueous layer to a new 1.5 ml microcentrifuge tube, add 1 ml of cold 100% ethanol, mix, and incubate for 15 minutes at -20°C.
- Centrifuge for 2 minutes at 12,000 rpm in a microcentrifuge. Decant the supernatant and drain.
- Add 180 ul 10:1 TE buffer, vortex, and incubate at 55°C for 10 minutes.
- Add 20 ul 2 M sodium acetate and mix. Add 500 ul of cold 100% ethanol, mix, and centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.
- Decant the supernatant and rinse the pellet with 1 ml of 80% ethanol. Centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.
- Decant the supernatant, and dry the pellet in a Speed-Vac for 10 minutes (or until dry).
- Resuspend the pellet by adding 200 ul of 10:1 TE buffer. Incubate overnight at room temperature to 55°C, mixing periodically to dissolve the genomic DNA. Store the samples at -20°C.
Reference: Modified from Roe BA.
D. Modern Preparation and Purification Kits (Example: Qiagen Kits)
DNA Extraction, Purification and Concentration (the new-fashioned kit methods)
Overview: There are so many ways now to extract DNA from different sources using kit and non-kit methods, that a complete review of them would be impossible. For specific applications, choose the best possible application and follow the manufacturer’s or other instructions. Interestingly, many of the kit methods use the basics described above, but use NA-binding matricies to precipitate the DNA/RNA/oligo and permit washing them to remove proteins and other molecules. From the above information regarding the different salts for NA precipitation, you should not be surprised that there are different matricies for each application and reagents and matricies are not interchangeable among kits.
Qiagen Kit Examples
Two examples of kits that have been designed by the Qiagen company and involve preparation and purification procedures for DNA are: 1. QIAprep Miniprep and Maxiprep Kits and 2. QIAquick Gel Extraction Kit. The QIAprep Kit allows you to extract and purify plasmid DNA from bacterial cells and the QIAquick Gel Extraction Kit allows you to excise a DNA fragment from an agarose gel upon completion of an agarose gel electrophoresis procedure and then purify the DNA fragment from the gel.
1. QIAprep Miniprep Kit: “The QIAprep Miniprep system provides a fast, simple, and cost-effective plasmid miniprep method for routine molecular biology applications including, restriction enzyme digestion, library screening, in vitro translation, sequencing, ligation and transformation, and transfection of cells. The QIAprep procedure is based on alkaline lysis of bacterial cells followed by adsorption of DNA onto silica in the presence of high salt. The unique silica membrane technology replaces glass or silica slurries for plasmid preps. Plasmid DNA purified with QIAprep kits is immediately ready for use. Phenol extraction and ethanol precipitation are not required, and high-quality plasmid DNA is eluted in a small volume of Tris buffer or water.” (Qiagen, QIAprep Miniprep Handbook, Second Edition November 2005)
- QIAprep (www.Qiagen.com, HTML Page)
*Note: As you can see from the description provided in the above excerpt, the methods implemented for the preparation and purification of DNA are very important for utilization of your DNA in a variety of molecular biology applications previously explored in module subsets II-a-d, and also mentioned in the QIAprep system descriptions above and below.
2. QIAquick Spin Handbook: “The QIAquick Gel Extraction Kit is designed for extraction of DNA fragments (70 base pairs to 10 kilobases) from standard, or low-melt agarose gels in TAE or TBE buffer and DNA cleanup from enzymatic reactions. QIAqck Kits provide high yields of pure nucleic acids for direct use in applications that include, fluorescent and radioactive sequencing, restriction, labeling, hybridization, ligation and transformation, amplification, in vitro transcription, and microinjection. The QIAquick system combines the convenience of spin-column technology with the selective binding properties of a uniquely designed silica membrane. Special buffers provided with each kit are optimized for efficient recovery of DNA and removal of contaminants in each specific application. DNA adsorbs to the silica membrane in the presence of high concentrations of salt while contaminants pass through the column. Impurities are efficiently washed away, and the pure DNA is eluted with Tris buffer or water.”(Qiagen, QIAquick Spin Handbook, March 2008)
- QIAquick Spin (www.Qiagen.com, HTML Page)
RNase Control: The Basics
Overview: To the average molecular biologist working with RNA, preventing, detecting and eliminating nuclease contamination is a constant challenge. The following discussion describes some of the sources of RNase contamination and highlights ways to eliminate contamination problems at various stages while working with RNA. RNases, especially those belonging to the RNase A family, are fairly small, compact proteins containing several cysteine residues that form numerous intramolecular disulfide bonds. As a result, denatured RNases tend to regain their native structure and partially function after being cooled to room temperature in the absence of a denaturant. Consequently, RNases can retain activity after freeze-thaw cycles and even autoclaving. The robust nature of these enzymes makes them refractory to many methods of decontamination. Indeed, drastic chemical methods are often required to eliminate RNases from surfaces and solutions.
Basic Precautions: Some basic precautions need to be taken when working with RNA. These include: wearing gloves throughout experiments to prevent contamination from RNases found on most human hands, changing gloves after touching skin, door knobs, and common surfaces, having a dedicated set of pipettes that are used solely for RNA work, using tips and tubes that are RNase-free, using RNase-free chemicals and reagents, and designating a “low-traffic” area of the lab that is away or shielded from air vents or open windows as an “RNase-free Zone”. These common sense precautions will go a long way towards minimizing RNase contamination problems.
Laboratory surfaces, such as benchtops, centrifuges and electrophoresis equipment, should be assumed to be contaminated with RNases, since they are usually exposed to the environment. Eliminating contamination from these surfaces is fairly easy and can be done rapidly. Prior to using any shared laboratory equipment or benchtops, decontaminate the surfaces by wiping it down using a mild solution of bleach or NaOH and then rinse it thoroughly with nuclease-free water. Glassware and metalware are usually baked to remove any RNase contaminations. These items are baked in an oven at 232 °C for 2 hours or more. Prior to baking, be sure to wrap the metalware items and the tops of beakers and flasks with aluminum foil to prevent contamination after baking. Tips and tubes are an easily overlooked source of RNase contamination and thus must also be baked. DNase and RNase free, pre-sterilized pipette tips are also readily available from commercial sources.
Buffers and solutions are a common source of RNase contamination. Using RNase-free buffers and solutions is always the best approach when working with RNA. As discussed earlier, merely autoclaving prepared solutions is not sufficient for eliminating RNase contamination. Solutions need to be treated with diethylpyrocarbonate (DEPC). DEPC treatment is the most commonly used method for eliminating RNase contamination from water, buffers, and other solutions. DEPC destroys enzymatic activity by modifying -NH, -SH and -OH groups in RNases and other proteins. The treatment typically involves incubating the solution at room temperature with 0.1% DEPC for a few hours, usually overnight, followed by autoclaving the solution to eliminate residual DEPC. A common concern that researchers have is the sweet, “fruity” aroma detected after autoclaving DEPC-treated solutions. When DEPC breaks down during autoclaving, a small amount of ethanol is produced. The ethanol can combine with trace amounts of carboxylic acid to produce volatile esters, which give off this characteristic smell. This is not a sign of incomplete DEPC removal and it will not interfere with any subsequent reactions. Reagents containing primary amine groups (e.g., Tris) and some reagents containing secondary or tertiary amines (e.g., HEPES) cannot be DEPC-treated. The amine groups tend to react with and absorb the DEPC, making it unavailable for inactivating RNases. Also, modification of the reagent’s amine groups could affect its buffering capability. Solutions that cannot withstand autoclaving and thus need to be filtered, such as MOPS, also cannot be DEPC-treated since autoclaving is essential for inactivating DEPC.
Storing RNA: Since RNA samples can be contaminated by small amounts of RNases carried over during RNA isolation or due to repeated sample use, proper storage can help minimize these problems. For short-term storage, RNA samples can be resuspended in water or buffer and stored at -80°C. If water is the preferred medium, be sure to use nuclease-free water. Using a buffer solution that contains a chelating (binding) agent is a better way to store RNA. Chelation of divalent cations such as Mg+2 and Ca+2 will prevent heat-induced strand scission. For long-term storage (more than a few weeks), RNA samples are best stored as a salt/ethanol slurry. To do this, take the RNA through all the steps of a regular precipitation with salt (e.g., 1/10 volume of 3 M NaOAc, pH 4.8) and ethanol (2 volumes of 100% ethanol) and store the mixture at -80°C without pelleting the RNA out of solution. The combination of low pH, low temperature and high alcohol content will stabilize the RNA and inhibit all enzymatic activity. Other alternatives for long-term storage are to store the RNA in formamide (Chomczynski, 1992) or to store the RNA in frozen aliquots at -20°C or below. If stored in formamide or ethanol, the RNA will need to be pelleted out of solution prior to quantitation or other manipulation.
Inhibiting RAases in Enzymatic Reactions : The traditional method for combating RNases in enzymatic reactions such as in vitro transcription, reverse transcription and translation is to use human placental ribonuclease inhibitor (also known as RNase Inhibitor Protein, RI or hPRI). This protein is an inhibitor only of the RNase A family of ribonucleases, which includes RNases A, B, and C. The mode of inhibition is non-competitive, i.e., the protein does not destroy these RNases but binds them in a 1:1 ratio.
RNases final words – Learn the difference between RNase-free and sterile. Something sterile may not be RNase-free and something RNase-free does not necessarily have to be sterile. Nowhere else in a laboratory is it critical to say as much as it is in molecular biology – “You wear gloves to protect your samples from you as much as you wear them to protect yourself from your samples.” RNase References: Chomczynski, P. (1992) Nucleic Acids Res. 20: 3791-3792. Some sections of above modified from Ambion web site:
- RNase III (www.ambion.com, HTML Page)
University of Calgary Biotechnology Training Centre
1. DNA Isolation: Indiana University-Purdue University Indianapolis The following website provides information and some illustrations for DNA Isolation from blood cells. When you have finished reading through the brief tutorial, respond to the four questions at the end and then click on “View Animation” to see a general depiction for extracting DNA from blood cells.
- Isolating DNA (www.iupui.edu, HTML Page)
Quantitation of DNA & RNA
This is Part A, DNA Extraction & Purification, under the module topic, Preparation, Purification, and Quantitation of DNA & RNA. This topic part has two sections: Content Tutorial and Activities.
Quantitation of DNA and RNA
Several commercial kits are now available for the quantitation DNA or RNA using fluorometers or luminometers. See manufacturer’s methodology or University of Southhampton’s Paper (www.molecular-beacons.org, HTML Page) for a good overview of the methods available if you are interested. Generally the simplest are to determine the OD at 260 nm or estimate against a known mass ladder in agarose gel after electrophoresis and ethidium bromide staining (refer to Module Subset II-a for further information).
For spectrophotemetric measurement (refer to Module I, Subset I-b for review), a UV spectrophotometer is required and quartz cuvettes in which to measure the sample. Quartz cuvettes have a 1 cm light path and can hold small volumes (for example 100 μl), are extremely expensive, should not be dropped and should only be touched on the surfaces that are not part of the light path. Clean them after use with bleach and ethanol.
To measure, the DNA or RNA sample is usually diluted in TE buffer (Tris EDTA) or high quality water (nanopure, autoclaved). The spec is blanked with the same diluent and the sample read. An OD (optical density, absorbance) of 1 corresponds to approximately 50 µg/ml for double stranded DNA, 37 μ/ml for ssDNA (single stranded), 40 μg/ml for ssRNA, and 30 μg/ml for oligonucleotides. Many methods will recommend also reading the OD280 and using the OD260/280 ratio of 1.8 as a measure of DNA purity (no protein). The latest recommendations suggest the ratio is a better measure of DNA contamination of protein samples rather than protein of DNA.
DNA/RNA Quantitation Reference: Sambrook J, MacCallum P, Russell D. Molecular Cloning: A Laboratory Manual, (Third Edition). 2001 Cold Spring Harbor Laboratory Press, NY.
Molecular Biology Grade Water/Reagents
One requirement for molecular biology is a good water source – often better than analytical grade. DNases, RNases and proteases that may contaminate the water from bacterial contamination will destroy the samples, break down your enzymes, or interfere with their reactions. Water systems that produce mili or nanopure water produce good water for molecular biology with a few additions – UV light for decontamination and breakdown of foreign DNA and degrading enzymes, filters for removal of particles. Finally, it should be autoclaved before use and aliquoted before use, such as in reagent preparation. Commercial reagents and water are available. From one such supplier, Cambrex, “Each reagent is prepared with 18 megOhm water, filtered using a 0.2 micron filter, and filled into sterile bottles. Reliable – Manufactured according to strict quality control standards to ensure lot-to-lot consistency. High Quality – No detectable DNase, RNase, or protease activities unless otherwise noted.”
Grade Water/Reagents (www.cambrex.com, HTML Page)
For RNase free work, water can also be treated with DEPC – see above.
Other Reagents: Where possible, buy molecular biology grade reagents and chemicals. Chemicals that are used for RNase-free work should be separate from those used for DNA work. Spatulas and measuring equipment should be baked as described above for measuring RNase-free chemicals. Obviously, after all these cautions – avoid contaminating your chemicals and reagents. HINT: Aliquot, aliquot, aliquot. Dispose after just a few uses.
Controls, Standards, Reference Standards, and Calibrators
Overview: DNA and RNA methods in research may not need more controls than a known template to show the reaction is working (positive) and a blank (negative) control to show you didn’t contaminate something. Other issues arise when looking at quantitative methods or more specialized testing from particular types of samples or tissues. The section below describes controls as recommended for molecular clinical diagnostic assays, but the lessons contained may be of value in research when you are consistently having problems and tried just about everything else.
1) Positive control or standard.
A good positive control will have a value at or near the lowest level of detection. The assay should be positive (qualitative) or able to detect that amount (quantitative) 100% of the time. For quantitative assays, standards or reference materials of known numbers may be very hard to find. Commonly, physical or biochemical means are used to quantitate the standard material. Control, standard or reference material can be natural: purified nucleic acid from sample (such as genomic DNA or bacterial plasmid), an organism (virus or bacteria) specimen. They can also be synthetic: plasmid from cloning, ssDNA or dsDNA or RNA fragments replicated or digested in vitro, synthesized DNA such as oligonucleotides, or of commercial origin. Synthetic controls have the advantage in ease of use and low cost, but are extremely likely sources of contamination of specimens.
2) Extraction control.
The questions answered by such a control – Does the method let the DNA/RNA out of the cell? Does it remove inhibitors? Does it not add anything else to the sample? Is it non-infectious?
This control can be combined with the first one, providing the right starting material is present in enough quantity. An example would be using plasma spiked with a known number of viral particles as a viral extraction control. If an assay doesn’t work though, this control would not be able to distinguish between a problem in the assay itself or in the extraction.
3) Nucleic Acid control.
This control will answer the question of enough DNA/RNA in the specimen itself. This can be addressed by quantitating a housekeeping gene.
A housekeeping gene is one that is highly conserved and constitutively expressed in the cell. Examples include GAP-DH, β-actin, 18S- or 28S rRNA (eukaryotic)/16S- or 23S rRNA (prokaryotic), etc.
*Note: There can be a wide variation in the terminology used for some of these controls. In some literature, the nucleic acid control just described is also called an internal control.
4) Inhibition controls.
Many substances in normal samples are inhibitory to molecular biology enzymes, particularly the polymerases. Examples include: heme, hemoglobin, bile, glycoproteins especially lactoferrin, urine crystals, heparin and even EDTA anticoagulants, and phenol (manual extraction protocols).
Reference: J Clin Microbiol. 2001 Feb;39(2):485-93 and J Clin Microbiol 2000 Dec;38(12):4463-70.
Inhibition of a molecular assay is sample specific, thus many of the controls are best done in the same tube as the test itself, although parallel testing is also done. Ways to do this include measuring a housekeeping gene (in the same tube or parallel tube) [internal control, as described above because the control is really a gene that is likely present in the sample- This is also known as an endogenous control] , spiked controls [also known as an internal control as it is done in the same tube as the test, but also sometimes known as an external control because the template is added to the specimen – This is also know as an exogenous control] .
Spiked controls (and calibrators) also come in two variations, competitive and non-competitive templates. A non-competitive control includes a template/primers/probe that is not related to the target sequence, but should have the same amplification conditions and nucleotide composition as the target. The non-competitive template should amplify regardless of the target. If it does not, inhibitors are a likely cause, thus this type of control is good for qualitative assays.
Competitive templates are designed to: a) be amplified using the same primers as the desired target sequence, b) have the same amplification efficiency and nucleotide composition as the target sequence, c) be of a significantly different size from the target to allow differentiation of the two products on an agarose gel, and d) control for variations in the reverse transcriptase reaction in RT-PCR when included prior to that step. Competitive templates are common in quantitative PCR or RT-PCR thus they are also known as internal calibrators because they are in the same tube as the target (External calibrators are run in parallel to the sample). Calibrators can also be used to spike the sample prior to DNA/RNA extraction to determine the efficiency or the extraction protocol. Therefore, the nucleic acid control (number 3 above) and the inhibitor control (number 4) may also be combined into one control.
*Note: Calibrators may not be the same as a standard unless the calibrator was quantified as the standards described above.
5) Contamination control.
The same characteristics of amplification procedures that make them powerful, also make them susceptible to contamination by controls, standards, other specimens, even airborne organisms. Contamination is largely avoided by controlling the environment, but controls should also be included in the assay run. Negative controls are most often reagent blanks: all the reagents with a) sterile water and b) extraction reagent used instead of a sample. Amplification reagents must be prepared with the highest quality of water, particularly for bacterial 16S- or 23S rRNA assays. While the reagents may be sterile, any contaminating bacterial DNA can cause a negative control to be positive. Other negative controls that can be used include multiple negative controls to screen for sporadic contamination from equipment, and wipe tests of the environment to detect aerosol contamination.
University of Calgary Biotechnology Training Centre
DNA Extraction Virtual Lab
“DNA is extracted from human cells for a variety of reasons. With a pure sample of DNA you can test a newborn for a genetic disease, analyze forensic evidence, or study a gene involved in cancer. In the virtual laboratory you will perform a cheek swab and extract DNA from human cells.DNA extraction is typically the first step in a longer laboratory process. DNA extraction is an important part of that process because the DNA first needs to be purified away from proteins and other cellular contaminant.”
Reference: Genetic Science Learning Center, University of Utah
- Genetic Science Learning Center (learn.genetics.utah.edu, Multimedia Page)
Task: Complete the DNA Extraction Virtual Lab. In the virtual lab you will participate in an introductory animated tutorial that will provide and reinforce the background information that applies to the lab. You will conduct the following four main steps as you complete the lab: Collect cheek cells (your sample) Burst cells open to release DNA (lysis) Separate DNA from proteins and debris Isolate concentrated DNA
All of the required laboratory equipment and solutions will be provided to you at your virtual laboratory bench. In preparation for the lab assessment it is suggested that you maintain a laboratory notebook of the materials you are using, the procedure you are following, and any data/results that you generate during the lab. This information will be helpful later for conducting this type of experiment in an authentic laboratory setting.
- Virtual Lab (learn.genetics.utah.edu, Multimedia Page)
Virtual Lab Assignment: Upon completion of the virtual lab, provide a detailed procedure of the specific steps involved in performing DNA extraction. In addition, provide a description that explains the significance behind each step and what is specifically happening to the sample/DNA at each one of those steps. For example: What are the two important ingredients in the lysis solution that you added to your sample and what is the function of each ingredient?, What do these solutions do to the DNA?
Virtual Lab – Genetic Science Learning Center, University of Utah