Michael R. Green

Isolation of High-Molecular-Weight DNA Using Organic Solvents

Perhaps the most basic of all procedures in molecular cloning is the purification of nucleic acids. The key step, the removal of proteins, can often be performed simply by extracting aqueous solutions of nucleic acids with phenol:chloroform and chloroform.

Precipitation of DNA with Ethanol

DNA can be precipitated out of solution for the removal of salts and/or for resuspension in an alternative buffer. Either ethanol or isopropanol can be used to achieve this purpose; however, the use of ethanol is generally preferred. Cations, provided as salts, are typically included to neutralize the negative charge of the DNA phosphate backbone. This method describes ethanol precipitation of DNA in microcentrifuge tubes.

Isolation and Quantification of DNA

Purifying DNA is the key to successful cloning. The cleaner the final preparation of DNA, the more efficient will be the enzymatic reactions that use the DNA as a template or a substrate. In the 1930s and 1940s, the scientific literature began to accumulate methods to release DNA from cells and to remove cellular constituents that inhibit or act as competitors on enzymatically catalyzed reactions. Since then, thousands of protocols for purification of DNA from a wide variety of organisms, tissues, and bodily fluids have been published. This introduction provides an overview of methods for isolation and quantification of DNA.

Optimizing Primer and Probe Concentrations for Use in Real-Time Polymerase Chain Reaction (PCR) Assays

Once primers and probes have been designed and obtained, it is necessary to optimize their concentration for each real-time polymerase chain reaction (PCR) assay. A set of PCRs is assembled in which the concentrations of forward and reverse primers are varied independently. Following amplification of the template DNA, amplification plots are compared. A standard curve is generated to determine the efficiency, sensitivity, and reproducibility of the assay. If SYBR Green I is used as the probe, then the melting curves are also analyzed.

Constructing a Standard Curve for Real-Time Polymerase Chain Reaction (PCR) Experiments

It is essential to prepare a standard curve for every real-time polymerase chain reaction (PCR) experiment. This protocol is used to construct a standard curve in which the template concentration is unknown. Such a standard curve is suitable for optimization experiments and for performing relative quantification by the standard curve method. To construct a standard curve for absolute quantification, the same principles apply as those presented here, but the concentration of the standards must be determined by an independent method.

Analysis and Normalization of Real-Time Polymerase Chain Reaction (PCR) Experimental Data

In real-time polymerase chain reaction (PCR), also called quantitative real-time PCR [or simply quantitative PCR (qPCR)] or kinetic PCR, the amplification of DNA is monitored by the detection and quantitation of a fluorescent reporter signal, which increases in direct proportion to the amount of PCR product in the reaction. The fluorescent reporter is excited by light from the real-time PCR machine, a fluorescence-detecting thermocycler. By recording the amount of fluorescence emission at each cycle, the PCR can be monitored during the exponential phase when the first significant increase in the amount of PCR product correlates with the initial amount of target template. The ability to quantify the amplified DNA during the exponential phase of the PCR, when none of the components of the reaction is limiting, has resulted in dramatically improved precision in the quantitation of target sequences. In addition, because of the high sensitivity of fluorometric detection, real-time PCR is capable of measuring the initial concentration of target DNA over a vast dynamic range (up to eight or nine orders of magnitude) and with a high degree of sensitivity (as little as one copy of template DNA). Although it is a powerful technique, researchers often face challenges in reliability and reproducibility because of the lack of assay standardization. Therefore, it is critical to optimize the reagents and reaction conditions, include proper internal and external controls, and perform rigorous data analysis in order to generate accurate and reproducible results in real-time PCR experiments.

Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Minipreps

In this protocol, plasmid DNA is isolated from small-scale (1-2 mL) bacterial cultures. Yields vary between 100 and 5 µg of DNA, depending on the copy number of the plasmid. Miniprep DNA is sufficiently pure for use as a substrate or template in many in vitro enzymatic reactions. However, further purification is required if the plasmid DNA is used as the substrate in sequencing reactions.

Hot Start Polymerase Chain Reaction (PCR).

The purpose of hot start polymerase chain reaction (PCR) is to optimize the yield of the desired amplified product in PCRs and, simultaneously, to suppress nonspecific amplification and formation of primer dimers. This is achieved by withholding an essential component of the PCR-the DNA polymerase, or the primers, for example-until the reaction mixture has been heated to a temperature that inhibits hybridization of primers to one another or to nonspecific regions of the template.

Isolating DNA from Gram-Negative Bacteria

The isolation of DNA from bacteria, described in this protocol, relies upon the use of sodium dodecyl sulfate and proteinase K to lyse the cells. High-molecular-weight DNA is then sheared (to reduce its viscosity and make it more manageable), extracted with phenol:chloroform, and precipitated with isopropanol. DNA isolated according to this procedure ranges from 30 to 80 kb in length.

Precipitation of DNA with Isopropanol

DNA is less soluble in solutions containing isopropanol than in solutions containing ethanol. In contrast to precipitation with ethanol, which requires 2-3 volumes of alcohol, precipitation with isopropanol is performed with 0.6-0.7 volume of alcohol. Isopropanol is often the better choice when precipitating DNA from large volumes of solution. Precipitation with isopropanol, described here, is performed at room temperature to lessen the risk that solutes like sucrose or sodium chloride will be coprecipitated with the DNA.